Fungal glutaminases: Production, optimization, and purification with antimicrobial activities of L- glutaminase from novel isolate Aspergillus tamarii AUMC 10198 under solid-state fermentation

Fungal glutaminases: Production, optimization, and purification with antimicrobial activities of L- glutaminase from novel isolate Aspergillus tamarii AUMC 10198 under solid-state fermentation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Fungal glutaminases: Production, optimization, and purification with antimicrobial activities of L- glutaminase from novel isolate Aspergillus tamarii AUMC 10198 under solid-state fermentation Ghada A. Youssef, Maii S. Zaid, Amany S. Youssef, Samy El-Aassar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5423591/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Aug, 2025 Read the published version in Microbial Cell Factories → Version 1 posted 18 You are reading this latest preprint version Abstract Introduction Fungal L-glutaminase has garnered significant attention in recent times with respect to its possible applications in the field of medical therapy and biotechnology. The principal aim of this research was to pick out microbial strains that can efficiently produce L-glutaminase from agricultural by-products under solid-state fermentation (SSF). Various fungal isolates were screened for extracellular L-glutaminase production. During the fermentation process, numerous parameters were adjusted one variable at a time (OVAT) to increase L-glutaminase production. The L-glutaminase of Aspergillus tamarii AUMC 10198 was purified in three sequential stages. The properties of the purified enzyme and the antimicrobial efficiency were also fulfilled. Results The potentiality of four out of fourteen fungal isolates served as indicators of the enzyme's productivity. The fungus Aspergillus tamarii AUMC 10198, designated with the GenBank accession number OQ976977, was determined to be the potent for estimating L-glutaminase synthesis, under SSF using wheat bran as a solid substrate. The solid-state yield of L-glutaminase exhibited a 3.20-fold increase in comparison to the unoptimized state. The Aspergillus tamarii AUMC 10198 L-glutaminase underwent three stages of purification, resulting in an increase in enzyme productivity by 12.90 times. Following these steps, the ultimate enzyme recovery was 18.45%. The isolated L-glutaminase exhibited optimal activity at a pH of 8, a temperature of 45 °C, and partial stability up to 60 °C, as determined by characterization. The purified L-glutaminase exhibited a Vmax of 10.10 U/ml and a Km of 0.28 mg/ml when glutamine was used as the substrate. The metal ions Fe 2+ , Ca 2+ , K + , Mg 2+ , and Na + demonstrated significant enzyme-activating properties at a concentration of 0.01 M, resulting in an enhancement of L-glutaminase productivity. The antimicrobial activity indicates its capability for various therapeutic and pharmaceutical applications. Conclusion The present investigation revealed that the local fungal strain of Aspergillus tamarii AUMC10198 could potentially be utilized in the production of L-glutaminase for industrial applications from agricultural by-products. L-glutaminase Aspergillus tamarii AUMC10198 Solid-state fermentation Optimization Purification and characterization Antimicrobial activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background An enzyme that catalyzes the deamination of L-glutamine to L-glutamic acid and ammonia is glutamine amidohydrolase (EC 3.5.1.2), also referred to as glutaminase [ 1 , 2 ]. The significance of L-glutaminases as enzymes with substantial commercial applications in the pharmaceutical and agricultural sectors has been underscored in recent publications [ 3 , 4 ]. L-glutaminase, a green chemotherapeutic agent that inhibits the proliferation of numerous carcinoma cell lines and thereby impedes their development by depriving them of their essential amino acid (glutamine), may find prospective application as an anti-cancer therapy in the medical field [ 5 – 9 ]. Moreover, L-glutaminases have been involved in the biocontrol of microbial pathogens [ 10 , 11 ]. Glutamic acid is produced through the hydrolysis of L-glutamine, the most essential amino acid in food production, owing to its flavorful and aromatic attributes [ 12 ]. This process obviates the necessity for monosodium glutamate (MSG) in food production. A considerable number of fermented foods, including Eastern condiments and soy sauce, acquire a robust, palatable, and umami taste due to the accumulation of L-glutamic acid [ 13 , 14 ]. Furthermore, L-glutaminase has been employed in the development of glutamine biosensors to detect the glutamine concentrations in mammalian cell cultures, the biosynthesis of numerous nitrogenous metabolic intermediates at the cellular level, and the production of the nutraceutical theanine [ 15 – 19 ]. Microbial organisms that generate glutaminases include actinomycetes, fungi, yeasts, and bacteria [ 20 , 21 ]. Fungi are the primary producers of extra-cellular hydrolytic enzymes. The environmentally friendly and sustainable properties of fungal enzymes have been the subject of extensive investigation and implementation in numerous industries, including agriculture, medicine, bioremediation, and industrial processing. These fungal enzymes have high productivity, stability, and low extraction and purification costs [ 22 , 23 ]. The significance of fungal glutaminases is due to the potential of their biological functions in different industrial and agricultural applications. One of the most important aspect is the production of a variety of fungicides with potential antifungal activities that play an impact role in crop protection [ 24 , 25 ]. Specific fungal species, including Candida, Saccharomyces, Trichoderma, Aspergillus, Penicillium, Fusarium , and a few endophytic species, are capable of producing glutaminase [ 26 , 27 ]. The efficient production of L-glutaminase was identified in marine filamentous fungi ( Aspergillus, Penicillium ) and yeasts ( Pichia sp.) [ 4 ]. Two distinct fermentation processes are utilized for enzyme synthesis: solid-state fermentation (SSF) and submerged fermentation (SmF) [ 28 , 29 ]. SSF has higher production, lower energy usage, simpler operations, and better product consistency than traditional SmF [ 30 , 31 ]. Agricultural by-products have significant potential for use as sustainable carbon and energy sources, which generates economic and environmental benefits [ 32 ]. In addition, the vicinity of solid fermentation systems to the natural habitats of microorganisms enables them to facilitate the secretion and synthesis of a vast array of metabolites and enzymes [ 27 , 33 , 34 ]. The objective of this study is to provide a comprehensive explanation of the SSF process employed in the production of L-glutaminase from a novel fungal strain with high L-glutaminase productivity. The synthesis of L-glutaminase was accelerated and optimized through the adjustment of process parameters. Additionally, purification aspects were detailed and the characterization of L-glutaminase was also fulfilled, followed by determination of its antimicrobial activity. Materials and methods Screening and qualitative isolation of L-glutaminase-producing fungi Wheat bran as a degrading lignocellulosic by-product possesses a high nutritional value. Samples of wheat bran were purchased from commercial marketplaces in the Alexandria, Egypt. The materials were air-dried, pulverized to an 80-micron powder, and stored at 4–5°C prior to processing. The serial dilutions technique (One g of each specimen) was used to isolate fungi on modified Czapek Dox’s (CZD) agar medium [ 35 ]. Each screened fungal colony observed after five days of incubation period at 28°C was purified and subsequently preserved on Potato Dextrose Agar (PDA) medium at a temperature of 4°C pending further analysis. The rapid plate assay was employed to approximate the qualitative evaluation of L-glutaminase production. The experiment was performed utilizing modified CZD solid medium, comprising the subsequent components in g/L: sucrose 2.0 g, KCl 0.5 g, MgSO 4 . 7H 2 O 0.5 g, KH 2 PO 4 1.0 g, FeSO 4 . 7H 2 O 0.1 g, ZnSO 4 0.1 g, L-glutamine 10.0 g, and agar 20.0 g at pH of 6.0. The medium was supplemented with 0.009% (v/v) phenol red dye (Sigma-Aldrich, Saint Louis, Missouri (USA)) as an indicator. A control medium devoid of dye was employed [ 36 ]. The selection process centered on the most expansive and profound pink color zone encircling the colonies that were chosen for additional quantitative analysis due to their positive production of L-glutaminase. The mean value of the zone diameters was calculated. Quantitative estimation of L-glutaminase A qualitative evaluation was conducted to select the four fungal isolates (F-S1, F-S2, F-S3, and F-S4) that exhibited the greatest ability of L-glutaminase production in the qualitative assay. The enzyme productivity was quantified using submerged culture technique. One milliliter of spore suspension containing 1.5 × 10 7 spores/mL was inoculated into 250 mL Erlenmeyer flask. The suspension was obtained from newly made PDA slants that were seven days old and cultured in 50 mL of modified Czapek Dox’s Broth containing L-glutamine as a carbon source. The containers underwent incubation at 30°C for 5 days using a rotary agitator adjusted to 150 rpm under SmF. After fermentation, centrifuge the mélange at 4°C for 15 minutes at 5000 revolutions per minute (Chilspin made in England). The clear supernatant was considered as a crude enzyme and stored at a temperature of 4°C until it was required. Assay of L-glutaminase In accordance with the methodology described by Imada et al., the L-glutaminase activity was assessed utilizing the direct Nesslerization technique Imada et al. method [ 37 , 38 ]. One milliliter of the crude enzyme was mixed with one milliliter of 40 mM L-glutamine, which was utilized as the substrate, in a citrate-phosphate buffer (0.1 M, pH 7). After incubating the mixture at 30°C for one hour, the reaction was halted by adding 0.5 milliliters of 1.5 M trichloroacetic acid. In order to obtain the precipitated protein, a solution was prepared by combining 0.1 ml of the solution mentioned above with 3.7 ml of distilled water. The mixture was subsequently adjusted with 0.2 ml of Nessler's reagent (Himedia, India). Following this, a 5-minute centrifugation at 5,000 rpm was conducted. At 450 nanometers, the absorbance was quantified with a spectrophotometer (Alpha 1102, Laxco, USA) following 15 minutes. A unit of L-glutaminase (U) was designated to represent the quantity of enzyme required to produce 1 µmol of ammonia per minute under ideal assay conditions. Protein estimation The crude enzyme's protein content was determined calorimetrically in accordance with the method described by Lowry et al., with bovine serum albumin serving as a standard [ 39 ]. The protein concentration was expressed in milligrams per milliliter of crude enzyme. Morphological characterization and molecular identification At the Mycological Center, Assiut University, Assiut, Egypt (A.U.M.C.), the efficient L-glutaminase-producing fungal isolate was identified by combining taxonomic keys with morphological and reproductive characteristics (Diba et al., 2007). The specimen under investigation's genomic DNA was isolated, purified, and analyzed for molecular identification in accordance with the methodology described by Moubasher et al. [ 40 ]. Using universal primers ITS 4 (5'- TCC TCC GCT TAT TGA TAT GC- 3') and ITS 1 (5'- TCC GTA GGT GAA CCT GCG G- 3') produced by SolGent Co. in Yuseong-Gu, Daejeon, South Korea, the internal transcribed spacer (ITS) was amplified. To examine the obtained sequences, the National Center for Biotechnology (NCBI) BLAST research tool was used (blast.ncbi.nlm.nih.gov). In order to construct the phylogenetic tree, 35 sequences from closely related Aspergillus (section Flavi) species in the GenBank database were aligned. Maximum likelihood (ML) models and maximum parsimony (MP) analyses were performed using version 10.2.6 of MEGA X [ 41 ]. The robustness of the sparsest-packed trees was assessed through the utilization of 1000 bootstrap replications [ 42 ]. Solid-state fermentation and culture conditions The quantitative analysis of L-glutaminase was performed utilizing the SSF method. A comprehensive examination was conducted of the commercial marketplaces in Alexandria, Egypt, in search of a variety of agricultural by-products, including wheat bran, soybean, sugarcane bagasse, and graved corn grains. An investigation was conducted into the effectiveness of agro-industrial residues to maximize the production of L-glutaminase by Aspergillus tamarii . Following the mechanical drying, grinding, and sieving of ten grams of each substrate into 250 milliliter Erlenmeyer conical flasks, twenty milliliters of 0.01 M phosphate buffer pH 7.4 were added to moisten each flask [ 43 ]. After autoclaving at 121°C for 20 minutes the flasks were inoculated with 2 ml of the spore suspension obtained from new slants (7-day old culture) of Aspergillus tamarii . The inoculated containers were incubated at a temperature of 30°C for five days under static condition. In accordance with the modified methodology described by Kashyap et al. [ 44 ], crude L-glutaminase was extracted from the fermented solid culture medium by subjecting it to 20 minutes of rotary stirring at 200 rpm, followed by 20 minutes of centrifugation at 1500 rpm at 4°C. A 50 ml volume of 0.1 M phosphate buffer with a pH of 7.0 was utilized as the solvent. In the cell-free supernatant, the protein content and enzyme activity were evaluated. The mean ± standard deviation values were reported following the three repetitions of each experiment. Optimization of various parameters for L-glutaminase production under solid-state fermentation The impact of various culture factors on the L-glutaminase production of the most potent strain Aspergillus tamarii during SSF was investigated by researchers employing a one-variable-at-a-time approach (OVAT). The research investigated the effects of different culture parameters on L-glutaminase synthesis. These parameters comprised inoculum size (1.0–5.0% v/v), temperature (25–40°C), incubation time (three to nine days), moisture content (0.25–4%), and pH's ranging from 4.0–9.0 (1N HCl or 1N NaOH). Three replicates were conducted for each experiment. Purification of L-glutaminase The impure-optimized L-glutaminase was subjected to three consecutive phases of purification following its extraction under optimal solid-state conditions: ethanol precipitation, ion exchange, and gel filtration. Partial purification of L-glutaminase The partial purification of L-glutaminase was achieved through fractional precipitation using extremely absolute ethanol (4°C). Combine cold crude enzyme and cold ethanol in equal parts while agitating gently; allow to remain at 4°C for 20 minutes. The precipitated fraction was obtained through 15 minutes of centrifugation at 5,000 rpm and 4°C. The supernatant was then saturated to 90% by adding additional ethanol. Diverse precipitate fractions, including 30%, 50%, 70%, and 90%, were produced by this method. Fractional separation was the initial step, which was followed by dialysis of the precipitated fractions against 0.01 M phosphate buffer (pH 8) overnight at 4°C. The collected enzyme fractions were analyzed for total protein content and L-glutaminase activity. DEAE Sephadex A-50 ion-exchange chromatography Partially active ethanol fraction (70%) was loaded onto a DEAE Sephadex A-50 (2.5 cm × 30 cm) ion-exchange column pre-equilibrated with 0.1 M NaCl in a 0.05 M acetate buffer (pH 5.2). The enzyme elution was conducted at a flow rate of 30 ml/h. The entire quantity was collected at a temperature of 4°C. The total protein content and L-glutaminase activity of the active fractions were evaluated subsequent to their collection, pooling, and concentration. Gel filtration chromatography on Sephadex G-100 Sephadex G-100 (2 cm × 28 cm) was applied to load the active and concentrated fractions generated in the previous stage. At a flow rate of 60 mL/h, the substance was eluted and brought to equilibrium utilizing the identical buffer. Ultimately, the absorbance of the protein was determined at 280 nanometers, and 5 ml of the active fractions containing the most significant amount of L-glutaminase activity were extracted, concentrated by dialyzing against distilled water, lyophilized, and stored at -20°C for the characterization of the purified form of the enzyme. Properties of the purified L-glutaminase Effect of substrate concentration on glutaminase activity The optimal substrate concentration for the experiment was determined by performing separate incubations with purified L-glutaminase and different concentrations of L-glutamine (as a substrate) in the assay mixture (ranging from 0.2 to 2.0 mg/ml) under ideal conditions. Kinetics parameters To determine the kinetic parameters (maximal velocity (Vmax) and Michaelis-Menten constant (Km)) of the purified L-glutaminase, standard assay conditions were used to measure the reaction velocities at various concentrations of L-glutamine (0.2-2.0 mg/ml). The apparent Km and Vmax values were obtained from the Lineweaver-Burk plot, which establishes a relationship between 1/Vmax and 1/S (reciprocal values), using the Michaelis-Menten equation, V 0 = V max [S] / K m +[S] [ 45 ]. In contrast, where V 0 represents the initial velocity of the reaction, Vmax denotes the maximal velocity, S is the concentration of the substrate, and Km represents the constant. Effect of pH, temperature, and thermal stability on glutaminase activity By subjecting the purified enzyme to different pH values ranging from 3.6–9.6 using acetate buffer (0.05 M; pH 3.6–5.4), phosphate buffer (0.05 M; pH 5.6-8.0), and sodium carbonate buffer (0.05 M; pH 8.6–10). Incubation was carried out under optimal assay conditions to establish the optimal pH for L-glutaminase activity. To investigate the effect of temperature on enzyme activity, the reaction mixture was incubated at temperatures spanning from 30 to 60°C for 30 minutes at the optimal pH. To ascertain the thermostability of L-glutaminase, the enzyme was pre-incubated for 15, 30, and 60 minutes, respectively, at temperatures of 50, 60, and 70°C, in the absence of a substrate. The remaining enzyme activity was assessed subsequent to chilling. Effect of metal ions (activators/inhibitors) on glutaminase activity The activity of purified L-glutaminase was investigated using a variety of metal ions at a concentration of 0.01 M: Na + (NaCl), K + (KCl), Ca 2+ (CaCl 2 ), Mg 2+ (MgSO 4 .7H 2 O), Ba 2+ (Bacl 2 ), Cd 2 + (CdCl 2 ), Cu 2+ (CuSO 4 ), Zn 2+ (Zn (CH 3 COO) 2 ), and Fe 2+ (FeSO 4 ∙7 H 2 O). In the test mixture, metal ions were allowed to incubate each enzyme solution for 30 minutes at room temperature in accordance with the recommended assay protocol. The estimated residual activity was compared to the 100% activity of the control group, which received no additives. Antimicrobial assay for the purified L-glutaminase The antimicrobial efficacy of the purified L-glutaminase was determined using the agar well diffusion technique [ 46 ] against six microbial pathogens kindly provided by the Microbiological lab at Department of Microbiology, Faculty of Science, Helwan University, Egypt. Bacillus subtilis NRRL B-543, Staphylococcus aureus ATCC 25923 as Gram + ve bacterial indicators and Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 as Gram -ve bacterial indicators. Moreover, Candida albicans ATCC 20231 and Aspergillus flavus as fungal indicators. Bacterial pathogens were grown in Mueller Hinton Agar (MHA) at 35°C for 1 day and fungal strains were grown in potato dextrose agar at 27°C for 3 days. Muller Hinton Agar plates were swabbed with 100 µL (0.5 McFarland standards) of each strain of pathogenic organisms. Approximately 6-mm diameters of wells were punctured aseptically in solid agar with a cork borer. One hundred µL of the produced L-glutaminase was injected into each well. The injected plates were refrigerated for 2 h to permit the diffusion of enzyme. Subsequently, the plates were incubated for 24 h at 37 ºC for bacteria and 5 days at 30 ºC for fungi. Gentamicin (10 µg/mL) was used as a positive control for bacteria and nystatin (100 U) for fungi [ 47 ]. Evaluation of the antimicrobial potentiality was detected by monitoring the mean diameter of the growth inhibition zones (mm) in triplicate. Statistical analysis The data were reported in the form of the mean value ± standard deviation. Utilizing information from three distinct investigations (n = 3), the means were calculated. The ANOVA test was employed to compare the various groups that were the subject of the statistical investigation. The predetermined significance levels were p ≤ 0.05. Results and Discussion Qualitative Screening and Quantitative estimation of L-glutaminase-producing fungi The competency of fourteen fungal isolates to produce L-glutaminase was verified utilizing the rapid-plate assay method. Agro-industrial by-product (wheat bran) was utilized as a substrate for this screening. Four specific fungal isolates F-S1, F-S2, F-S3, and F-S4 have the capability of secreting L-glutaminase after 72 h of incubation with measurable pink zone diameter ranged from 8 to 32 mm. The mean and standard deviation were estimated to show the results in Table 1 . As determined qualitatively by the greatest mean diameter of the zone encircling the colonies on the plated agar, fungal isolate F-S1 showed the largest diameter zone (32.30 mm) and the lowest zone (7.88 mm) was detected by fungal isolate F-S3. Quantitative estimation of L-glutaminase producing isolates was assessed in triplicate using the agitated method with a submerged culture medium. The mean and standard deviation were utilized to estimate the results in Table 1 . The observed qualitative and quantitative values exhibited statistical dissimilarity when p ≤ 0.05. The L-glutaminase activity of the fungal isolates exhibited a broad spectrum, with values varying from 0.45 ± 0.0 d to 2.32 ± 0.2 a U/ml. The discernible discrepancy is illustrated by the small letters. Based on a substantially distinct estimate (p < 0.05), the F-S1 isolate exhibited the highest enzyme productivity (2.32 ± 0.2 a U/ml) as a glutamine hydrolyzer. The L-glutaminase activities of F-S2, F-S4, and F-S3 were arranged as follows: 1.25 ± 0.1 b , 0.83 ± 0.1 c , and 0.45 ± 0.0 d U/ml, respectively. Following a meticulous evaluation employing molecular and morphological techniques for the potent isolate (F-S1) exhibiting the greatest glutaminolytic activity that was selected for subsequent investigations. Table 1 Qualitative and Quantitative screening of L-glutaminase producing fungal isolates Isolate number Qualitative screening Mean pink zone diameter (mm) Protein content (mg/ml) Quantitative estimation Enzyme activity (U/ml) Dry weight (g/50 ml) F-S1 32.30 ± 3.55 a 1.14 ± 0.11 a 2.32 ± 0.2 a 0.56 ± 0.1 a F-S2 15.55 ± 2.18 b 0.623 ± 0.1 b 1.25 ± 0.1 b 0.39 ± 0.0 b F-S3 7.88 ± 0.87 c 0.35 ± 0.0 c 0.45 ± 0.0 d 0.21 ± 0.0 c F-S4 9.02 ± 1.26 c 0.43 ± 0.0 c 0.83 ± 0.1 c 0.36 ± 0.0 b ANOVA test p value 16.88 0.0013* 10.41 0.008* 15.85 0.001* 8.98 0.0136* Small different letters ( a,b,c,d ) indicate that there was a significant variation with different studied groups. ANOVA test was performed for comparing between different groups. Values are the means ± standard deviations (n = 3), P value* was significant if ≤ 0.05. Morphological properties and molecular identification of potent L- glutaminase-producing isolate The morphological identification of the potent glutaminase-producing fungal isolate (F-S1) Aspergillus tamarii AUMC 10198 was accomplished through phenotypic characterizations, reproductive structures, and comparisons with authentic isolates at the Mycological Center, Assiut University (AUMC), Assiut, Egypt. At cultural level, colonies of Aspergillus tamarii (AUMC 10198) grown on Czapek's agar ultimately grew to a diameter of 5–6 centimeters after seven days at a temperature of 25°C. The conidial region exhibited a subdued greenish-yellow hue with a white edge; the reverse lacked any exudates or pigmentation (Fig. 1 A). Regarding to microscopic examination (Fig. 1 B), the lactophenol cotton blue-stained conidiophores were observed to be colorless, measuring a maximum of 1–2 mm in length and 8 µm in width. Additionally, they exhibited a grainy surface with an abrupt constriction of the wall at the base of the vehicle. The conidial head exhibits a loose radiating pattern and harbors sizable vesicles ranging in diameter from 30 µm. These vesicles are globose to sub-globose in shape and have thin walls. When adhered to phialides that are loosely packed and measure 8.5–11.5 x 5.0–6.0 µm. Large heads generally possess metulae measuring 10–15 x 4–8 µm in dimension, while small heads lack such features. Mature conidia are globose, bounded by chains that are conspicuously roughened, and range in diameter from 4.2 to 6.6 µm. In contrast, young conidia are cylindrical to pyriform. A BLAST analysis was conducted to compare the extracted ITS sequences of the target strain with those that were previously archived in the NCBI Nucleotide Sequence Database. Aspergillus tamarii CBS 104.13, which had a GenBank accession number of MH854614 and 591 out of 597 identities (98.99%), was the most closely related match. The sequence, designated with accession number OQ976977, has been successfully submitted to the GenBank database. As shown in Fig. 2 , the species has been identified as Aspergillus tamarii AUMC 10198.Fig. 1 Morphological identification: Aspergillus tamarii (AUMC 10198) was cultured for seven days at 25°C on Czapek's agar medium (A). A microscopic examination was performed using lactophenol cotton blue stain at X 1000 magnification (B) Solid-state fermentation (SSF) for L- glutaminase production SSF is a sustainable and environmentally beneficial method that utilizes substantial by-products from the agro-industrial sector as inexpensive raw materials [ 48 ]. The selection of an optimal solid substrate is critical during the fermentation process. SSF effectively synthesized glutaminase from a variety of agro-industrial by-products, including wheat bran, soybean, sugarcane bagasse, and graved corn grains. The values presented in Fig. 3 ( n = 3) are the mean plus or minus the standard deviation. L-glutaminase output varied in response to the substrate type that was utilized. The outcomes demonstrated that wheat bran, in comparison to sugarcane bagasse, soybean, and graves corn grains functioned as the most effective solid substrate, exhibiting the highest specific activity of 2.61 ± 0.26 U/mg protein and enzyme activity of 3.85 ± 0.4 U/mL, respectively. Based on the current findings, there were significant differences in the specific activities of wheat bran, sugarcane bagasse, and graved corn grains (2.61 ± 0.26 a , 2.17 ± 0.18 b , and 1.44 ± 0.13 c U/mg protein), respectively. Our findings corroborate those of Soren et al. [ 49 ], that wheat bran served as the optimal substrate for the maximal production of L-glutaminase. Wheat bran was the most likely of seven solid by-products for Fusarium solani AUMC 8615 that increase L-asparaginase activity [ 50 ]. The supplementation of wheat bran resulted in the greatest L-glutaminase output. Wheat bran's strong nutritional content may promote spore formation and fungus growth. A resistance to aggregation enhances the mechanical effectiveness of wheat bran particles [ 44 , 51 , 52 ]. Optimization of SSF parameters for L-glutaminase production by OVAT approach In order to maximize the yield of L-glutaminase via solid fermentation process, numerous parameters were optimized (Fig. 4 ). Incubation time The enzyme yield is notably impacted by the duration of fermentation, as illustrated in Fig. 4 A. The maximum synthesis of L-glutaminase was observed on the fifth day of incubation at 3.85 ± 0.3 a U/mL, following a significant increase. The activity of the enzyme remained relatively constant with no significant difference at 1.74 ± 0.1 c and 1.85 ± 0.2 c U/mL for three and nine incubation periods, respectively. An increase in the fermentation period beyond five days resulted in a substantial reduction in enzyme production due to the degradation of enzymes [ 53 , 54 ]. The optimal time to detect L-glutaminase synthesis for the majority of fungal strains was between the fifth and seventh day of incubation [ 55 , 56 ]. Inoculum size The effects of different inoculum concentrations (1–5% v/v) on L-glutaminase synthesis during the current fermentation process were investigated in a study, as depicted in Fig. 4 B. The enzyme production was most significant at 4% (2 ml/flask) of inoculum size with 3.85 ± 0.4 a U/ml. Trichoderma koningii strain demonstrated its highest L-glutaminase activity at a volume of 2 ml, which is consistent with our findings [ 56 ]. The enzyme activity was notably perturbed for all values that deviated from the optimal range. According to Abdel-Hamid et al., the Fusarium oxysporum strain exhibited highest efficiency of enzyme activity at 3% of inoculum level under SSF [ 57 ]. A diminished enzyme output and insufficient biomass result from a limited inoculum size. A significantly increased inoculum volume results in both the depletion of vital nutrients and the suppression of enzyme activity [ 44 ]. Moisture content Moisture content is a known variable that influences indoor fungal growth, regulates and enhances metabolic activity, and ultimately affects product production [ 58 , 59 ]. A significant discrepancy in L-glutaminase production was observed among the letters ranging from a to e (p ≤ 0.05). The peak activity of L-glutaminase at 2% initial moisture content was 5.52 ± 0.5a U/mL with specific activity 2.77±0.25 a U/mg protein, as illustrated in Fig. 4 C. In excess of the optimal moisture content, enzyme production decreased significantly. Liquid in motion hinders gaseous exchange, restricts aeration, and promotes the clustering of substrate particles, all while reducing porosity [ 60 ]. These findings are consistent with prior investigations that documented the production of L-glutaminase by Zygosaccharomyces rouxii and L-glucoamylase by Aspergillus niger [ 44 , 61 ]. Hydrogen ion concentration An important factor that affects development, metabolic activity, and enzyme production is the concentration of hydrogen ion in the fermentation medium [ 62 , 63 ]. The activity of L-glutaminase improved over time when the starting pH was raised, reaching a maximum of 8.04 ± 0.7 a U/mL in a slightly alkaline medium at pH 8.0 (Fig. 4 D). The findings align with Mostafa et al. and Dueramae et al. who found that marine Halomonas meridiana and halophilic Tetragenococcus muriaticus FF5302 produced the optimal L-glutaminase at 8.0 pH [ 64 , 20 ]. When compared to the optimal pH value, the enzyme activity was declined to be 66.67% at an acidic pH of 5.0 and 36.32% at an alkaline pH of 9.0. It has been shown that Aspergillus tamarii can grow over a broad range of pH values (6–9), as there is no discernible difference between pH 6 (5.52 ± 0.6 c ) and pH 9 (5.12 ± 0.5 c ). At pH 8 in SSF, it was found that Aspergillus terreus MTCC 1782 produced the most L-asparaginase [ 65 ]. However, Beauveria sp. showed two pH optimum values for L-glutaminase production under SSF: 6.0 and 9.0 [ 29 ]. Incubation temperature The maximum L-glutaminase productivity (12.33 ± 1.4 a U/mL) was detected at 35°C incubation temperature in solid state cultural medium (Fig. 4 E). Gomaa, 2022b detected that the maximum glutaminase activity (40.80 U/mL) by halophilic Bacillus sp. DV2-37 at 37°C [ 7 ]. Whereas, maximum L-glutaminase productivity was achieved by Trichoderma koningii at 30°C [ 55 ]. A noteworthy reduction of enzyme activity was observed at 40°C (7.3 ± 0.7 b U/mL). This may be attributed to the denaturation of the enzyme molecules at lower and higher temperatures [ 54 ]. According to the net results of Fig. 4 , L-glutaminase production was obviously increased by 3.20-fold of that before optimization under solid-state condition. It was estimated that L-glutaminase activity was multiplied by 3.23-fold as compared to unoptimized medium by Aspergillus terreus ZHG2 [ 48 ]. Furthermore, optimization of L-glutaminase productivity was exhibited maximum yield of 703.8 U/gds with 3.8-fold increase under SSF [ 66 ]. L-glutaminase purification Aspergillus tamarii AUMC 10198 L-glutaminase was purified through three distinct procedures: ethanol fractionation (for partial purification), ion-exchange chromatography using DEAE (Sephadex A-50), and gel filtration utilizing a Sephadex G-100 column. Everything was completed in a sequential sequence. Ethanol fractionation L-glutaminase was partially purified by ethanol precipitation at 70% saturation Table 2 . It exhibited a total activity of 303.98 U, a specific activity of 13.10 U/mg protein, enzyme recovery of 24.65%, and a purification fold of 3.23. Table 2 Ethanol precipitation of Aspergillus tamarii AUMC 10198 L-glutaminase Ethanol Concentration (%) Protein content (mg/ml) Total protein (mg) Recovered protein (%) Glutaminase activity (U/ml) Total activity unit (U) Recovered activity (%) Specific activity (U/mg protein) Purification (fold) Culture extract 3.04 304 100 12.33 1233 100 4.06 1 30 1.31 13.1 4.31 13.98 139.8 11.33 10.67 2.63 50 1.69 16.95 5.58 19.83 198.3 16.09 11.70 2.88 70 2.32 23.20 7.63 30.39 303.98 24.65 13.10 3.23 90 0.53 5.30 1.74 4.12 41.2 3.34 7.77 1.91 Total 19.26 55.41 Ion-exchange chromatography The DEAE (Sephadex A-50) column was employed for the subsequent purification phase, resulting in the acquisition of three protein peaks, which corresponded with three peaks of L-glutaminase at fractions 6–11, 25–30, and 38–42 (Fig. 5 A). Maximum activity was observed during the second apex surge. The specific activity increased by 6.89-fold at a yield of 22.57%, from 13.10 to 27.99 U/mg protein as shown in Table 3 . Gel filtration chromatography The most active fractions obtained from an ion-exchange chromatography system (Sephadex-50) were executed using a gel filtration column composed of Sephadex G-100. The eluent exhibited two protein peaks, along with a sharp and distinct peak indicative of L-glutaminase activity (fractions 13, 14, 15, 16, and 17), as depicted in Fig. 5 B. The active fractions (5 ml) were stored at -20°C following pooling and concentration. They were then utilized as purified L-glutaminase to satisfy additional purity standards. The results of the purification procedure are briefly outlined in Table 3 . The calculated values are as follows: 54.17 U/mg of protein for specific activity, 227.50 (U) for total activity, 18.45% for final yield, and 12.90 purification fold comparable to the crude enzyme. The L-glutaminase of Aspergillus flavus exhibited a specific activity of 613.30 U/mg and a yield of 51.11%; the purification procedure was multiplied by 12.47 [ 51 ]. The L-glutaminase obtained from Aspergillus versicolor Faesay4 was found to have a purification fold of 2.10 ± 3.18 and a specific activity of 398.79 ± 9.81 U/mg protein with overall activity 13.16 ± 22.76 unit [ 38 ]. In contrast, Penicillium brevicompactum NRC 829 demonstrated 869.08 U/mg of specific activity, 321.6 U of total activity, 162.75-fold purification, and 48.21% yield on Sephadex G-200 [ 67 ]. Based on the findings of Ali et al., the Penicillium politans NRC510 L-glutaminase exhibited the following values: a yield of 25%, a specific activity of 133 U/mg, and a purification level of 230-fold [ 68 ]. Table 3 Summary of purification stages of Aspergillus tamarii AUMC 10198 L-glutaminase Purification stage Total Protein (mg) Total activity unit (U) Specific activity (U/mg protein) Yield (%) Purification (fold) • Crude extract 304 1233 4.06 100 1 • Ethanol (70%) 23.20 303.98 13.10 24.65 3.23 • Ion exchange on DEAE-Sephadex A-50 9.94 278.25 27.99 22.57 6.89 • Gel filtration on Sephadex G-100 4.2 227.50 54.17 18.45 12.90 Characterization of purified Aspergillus tamarii AUMC10198 L-glutaminase A number of parameters were considered when determining the L-glutaminase activity of Aspergillus tamarii AUMC10198 under test conditions (Fig. 6 ), including substrate concentration, kinetic parameters Km and Vmax, pH level, reaction temperature, thermal stability, and metal ions. Effect of substrate concentration and estimation of the kinetic parameters (Km and Vmax) The impact of varying amounts of L-glutamine added to the reaction mixture on the activity of purified L-glutaminase is illustrated in Fig. 6 A. A range of concentrations between 0.2 and 2.0 mg/ml were utilized. The enzyme activity increased progressively as the substrate concentration was increased to 1.4 mg/ml of L-glutamine; ultimately, it reached an optimal relative activity of 115.38%. Nevertheless, further substrate additions resulted in a decline in enzyme activity. Ahmed et al., reported the most significant quantity of marine Aspergillus sp. ALAA-2000 L-glutaminase at a substrate concentration of 4.38 mg/ml [ 15 ]. The optimal activity of Brevundimonas diminuta MTCC 8486 L-glutaminase was observed at 1% glutamine, according to a study of Jayabalan et al. [ 69 ]. The strong substrate affinity of L-glutaminase for L-glutamine was predicted by its low Km of 0.28 mg/ml and high Vmax of 10.10 U/ml, as illustrated in Fig. 6 A. In agreement with the results, Singh and Banik reported that the great affinity of purified L-glutaminase to L-glutamine was detected at small Km (0.129 mmol) by Bacillus cereus MTCC 1305 [ 70 ]. Penicillium brevicompactum NRC829 demonstrated the highest affinity for L-glutaminase activity at a concentration of 1.66 mM L-glutamine with km value of 0.13 mmol, as determined by Elshafei et al. [ 67 ]. Effect of pH, temperature, and thermal stability on glutaminase activity The data presented in Fig. 6 B, the maximal relative activity of 119.73% was achieved at a pH of 8. This finding suggests that the optimal pH range for L-glutaminase activity was between neutral and mildly alkaline (7–8). At an alkaline pH 9 and an acidic pH 4, a noticeable decrease was observed. The optimal pH values for glutaminase activity were determined to be 8.0 and 8.5, respectively [ 67 , 71 ]. It has been demonstrated that the ideal pH range for glutaminase activity is between 5.0 and 9.0 [ 72 – 74 ]. There is a relationship between incubation temperature and glutaminase activity climax. The enzyme exhibited its highest activity at 45°C and retained over 85% at 60°C ( Fig. 6 C ). The results reported by Farag et al. [ 75 ], which indicated that the highest activity of purified L-glutaminase was observed by marine Aspergillus terreus , corroborated this ideal temperature. The thermostability of the purified enzyme was demonstrated by its retention of approximately 88.59% and 66.29% of its activity, respectively, after preincubation at 50 ºC for 60 minutes and 60 ºC for 60 minutes (Fig. 6 D). The initial activity of purified L-glutaminase, was reduced by approximately 38.11% subsequent to a 15-minute heating period at 70 ºC followed by a 30-minute total inactivation period at 70°C. According to a study by Elshafei et al. [ 67 ], L-glutaminase purified from Penicillium brevicompactum NRC829 exhibited stability across a temperature range of 50 to 60°C. At 45°C, Aspergillus oryzae L-glutaminase remained stable; however, at 55°C, it ceased to function [ 52 ]. Effect of metal ions (activators/inhibitors) on the activity of purified L-glutaminase Metal ions play a vital role in enzyme activity by either donating or receiving electrons. The enzyme maintained over 100% of its initial activity when activated with Fe 2+ , Ca 2+ , K + , Mg 2+ , and Na + ions as shown in Fig. 6 E. This suggests that these ions engage in activatory mechanisms with L-glutaminase. The fold values of purified L-glutaminase for each of these metal ions were as follows: 1.80, 1.58, 1.29, 1.12, and 1.03. On the contrary, it was demonstrated that Cd 2+ , Cu 2+ and Ba 2+ significantly impeded the activity of the enzyme. However, the activity of L-glutaminase was only slightly inhibited by Zn 2+ . An additional 75% reduction in enzyme activity was observed in the presence of Cd 2+ . Aspergillus sp. ALAA-2000 and A. flavus were capable of eliciting the activity of purified L-glutaminase when subjected to Na + and Mn 2+ ions [ 15 , 51 ]. Conversely, Fe 2+ exhibits moderate inhibitory activity against L-glutaminase from marine Bacillus subtilis [ 76 ]. Antimicrobial activity of purified Aspergillus tamarii AUMC10198 L-glutaminase The data detected in Table 4 recorded the antimicrobial efficiency of purified Aspergillus tamarii AUMC10198 L-glutaminase against some pathogenic indicators. The enzyme showed the highest antibacterial activity against Staphylococcus aureus ATCC 25923 followed by Bacillus subtilis NRRL B-543 with mean inhibition zone diameter 36.80 ± 1.20 mm and 30.40 ± 0.60 mm, respectively. The lowest zone of inhibition (12.80 ± 1.20 mm) was achieved against Pseudomonas aeruginosa ATCC 27853. Marine Streptomyces griseorubens NAHE L-glutaminase proved to have potential antibacterial activity against Gram-positive comparable to Gram-ve bacteria [ 10 ]. The antifungal activity of Aspergillus tamarii AUMC10198 L-glutaminase showed moderate inhibition zone diameters. The antimicrobial impact of Pseudomonas sp. RAS123 L-glutaminase, which showed no activity against fungal pathogens Aspergillus fumigatus (RCMB 002008) and Candida albicans RCMB 005003 (1) ATCC 10231[ 72 ]. Table 4 Antimicrobial efficiency of purified Aspergillus tamarii AUMC10198 L-glutaminase against various pathogenic indicators Agent Mean diameter of the growth inhibition zones (mm) Bacterial indicators Fungal indicators Gram + ve Gram -ve S. aureus ATCC 25923 B. subtilis NRRL B-543 E. coli ATCC 25922 P. aeruginosa ATCC 27853 C. albicans ATCC 20231 A. flavus Purified L-glutaminase 36.80 ± 1.20 30.40 ± 0.60 15.30 ± 1.50 12.80 ± 1.20 22.40 ± 0.50 26.09 ± 1.20 Gentamicin (10 µg/mL) 28.20 ± 1.60 26.20 ± 0.30 20.20 ± 0.50 18.70 ± 0.10 0.0 0.0 Nystatin (100 U) 0.0 0.0 0.0 0.0 23.00 ± 1.40 18.06 ± 0.90 Conclusion Glutaminase, a crucial enzyme found in agriculture, industry, and medicine, possesses significant biotechnological and therapeutic value. L-glutaminase production from wheat bran as an agro-industrial residue is a prospective process for the biotechnology industry. The findings indicated that Aspergillus tamarii AUMC10198 could potentially utilize wheat bran, an economically and ecologically sustainable substrate, in the synthesis of L-glutaminase. The implementation of solid-state culture to optimize the fermentation process resulted in a 31.22% increase in L-glutaminase output. The final yield of the purified L-glutaminase was 18.45%, the total activity was 227.50 U, and the purification fold was 12.90 comparable to those of the crude enzyme. Furthermore, Aspergillus tamarii AUMC10198 L-glutaminase investigated promising antimicrobial properties against various pathogenic indicators. Future research will focus on elucidating the molecular pathways underlying the enzyme's chemotherapeutic activity against malignancy. Declarations Disclosures and statements Author Contributions: All authors participated in conceptualization and methodology. M Z. Performed all experiments. Gh. Y. & M. Z. 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Cite Share Download PDF Status: Published Journal Publication published 04 Aug, 2025 Read the published version in Microbial Cell Factories → Version 1 posted Editorial decision: Revision requested 29 Apr, 2025 Reviews received at journal 29 Apr, 2025 Reviews received at journal 28 Apr, 2025 Reviews received at journal 27 Apr, 2025 Reviews received at journal 18 Apr, 2025 Reviews received at journal 17 Apr, 2025 Reviewers agreed at journal 08 Apr, 2025 Reviewers agreed at journal 08 Apr, 2025 Reviewers agreed at journal 06 Apr, 2025 Reviewers agreed at journal 05 Apr, 2025 Reviewers agreed at journal 05 Apr, 2025 Reviewers agreed at journal 03 Apr, 2025 Reviewers agreed at journal 03 Apr, 2025 Reviewers agreed at journal 03 Apr, 2025 Reviewers invited by journal 03 Apr, 2025 Editor assigned by journal 11 Nov, 2024 Submission checks completed at journal 11 Nov, 2024 First submitted to journal 09 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5423591","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":376792957,"identity":"cab081ad-22e8-46fc-9bf8-a96054ca516c","order_by":0,"name":"Ghada A. Youssef","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYFACxgYIYm8++ADI5eEjXgvPsWQDkBY24i2S8FGTAHEIauGfdrjt4dcddrL9M3jYKr/m2MmwMTA/fHQDjxaJ24ntxrJnko1n3O49dlt2WzLQYWzGxjn4rLmd2CYt2cac2HDnXNptyW3MQC08bNL4tMhDtNQnzr+RY1Ysua2esBYDoBbJj22HEzcAtTB+3HaYsBZDkC2MbceNN545lizNuO04DxszAb/I3U5/JvmzrVp23vHmgx9/bqu252dvfvgYr/eBgJkHhcFMQDkIMP5AZ4yCUTAKRsEoQAYAo+FOYc0wD3sAAAAASUVORK5CYII=","orcid":"","institution":"Alexandria University","correspondingAuthor":true,"prefix":"","firstName":"Ghada","middleName":"A.","lastName":"Youssef","suffix":""},{"id":376792958,"identity":"871765d6-e364-4272-8e8c-cd432f54a999","order_by":1,"name":"Maii S. Zaid","email":"","orcid":"","institution":"Alexandria University","correspondingAuthor":false,"prefix":"","firstName":"Maii","middleName":"S.","lastName":"Zaid","suffix":""},{"id":376792959,"identity":"8baa1a16-34c4-432c-9019-a97c440798bf","order_by":2,"name":"Amany S. Youssef","email":"","orcid":"","institution":"Alexandria University","correspondingAuthor":false,"prefix":"","firstName":"Amany","middleName":"S.","lastName":"Youssef","suffix":""},{"id":376792960,"identity":"335a3a2d-3e4c-4aa3-b035-8f423bf38c86","order_by":3,"name":"Samy El-Aassar","email":"","orcid":"","institution":"Alexandria University","correspondingAuthor":false,"prefix":"","firstName":"Samy","middleName":"","lastName":"El-Aassar","suffix":""}],"badges":[],"createdAt":"2024-11-09 21:38:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5423591/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5423591/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12934-025-02802-0","type":"published","date":"2025-08-04T15:57:10+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":70200716,"identity":"79338fbc-b563-4ebb-a4f5-eb0de1a8b1bb","added_by":"auto","created_at":"2024-11-29 12:26:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":804910,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological identification: \u003cem\u003eAspergillus tamarii\u003c/em\u003e (AUMC 10198) was cultured for seven days at 25 °C on Czapek's agar medium (A). A microscopic examination was performed using lactophenol cotton blue stain at X 1000 magnification (B)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5423591/v1/8c156d0d22a8f510675956f3.png"},{"id":70200712,"identity":"dc09ec71-154f-48b6-b80a-5edc9ebda50b","added_by":"auto","created_at":"2024-11-29 12:26:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":282385,"visible":true,"origin":"","legend":"\u003cp\u003eA phylogenetic tree of \u003cem\u003eAspergillus tamarii\u003c/em\u003e AUMC 10198 constructed from the ITS sequence in comparison to GenBank belonging to Aspergillus: section Flavi sequences that are closely related. This is the \u003cem\u003eAspergillus tamarii\u003c/em\u003e blue sequence, designated with the accession number OQ976977. The Bootstrap support values (calculated from 1000 replications) for ML/MP that are equal to or greater than 50% are displayed in close proximity to each node. In ML/MP bootstraps, a symbol (*) denotes a significance level below 50%. The red \u003cem\u003eAspergillus alliaceus\u003c/em\u003e NRRL 315 constitutes the root system of the tree.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5423591/v1/3ef050448d9b0a536810cd0c.png"},{"id":70200715,"identity":"6c4a71b7-e3fc-48e2-a386-c9deb3aea406","added_by":"auto","created_at":"2024-11-29 12:26:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":66152,"visible":true,"origin":"","legend":"\u003cp\u003eScreening of different solid substrates for the production of L-glutaminase by \u003cem\u003eAspergillus tamarii\u003c/em\u003e (F-S1) under solid-state fermentation\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5423591/v1/bef85eba5f6eae8930ccfae8.png"},{"id":70200951,"identity":"76475f2a-47f2-4f7e-9dcb-36d586649f51","added_by":"auto","created_at":"2024-11-29 12:34:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":230218,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of various parameters on L-glutaminase production under solid-state fermentation by \u003cem\u003eAspergillus tamarii\u003c/em\u003e AUMC 10198: Effect of incubation period (A), inoculum size (B), moisture content (C), initial pH (D), and incubation temperature (E). Mean ± standard error (n = 3), different letters are significantly different at p ≤ 0.05\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5423591/v1/b01420754b9e9d34ef997cdb.png"},{"id":70200713,"identity":"eb1e2a75-3d9c-4b91-b809-030c055e0c49","added_by":"auto","created_at":"2024-11-29 12:26:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":105749,"visible":true,"origin":"","legend":"\u003cp\u003ePurification stages of \u003cem\u003eAspergillus tamarii\u003c/em\u003e AUMC10198 L-glutaminase: A 0.1M NaCl solution was introduced into a 0.05M acetate buffer with a pH of 5.2 and a flow rate of 30 ml/h to calibrate a 2.5 cm × 30 cm DEAE (Sephadex A-50) ion-exchange chromatography column (A). The 5 mL fractions were subsequently obtained by means of gel filtration through a Sephadex G-100 column measuring 2 cm × 28 cm with identical buffer and flow rate of 60 mL/h (B)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5423591/v1/9f584226a4863d769dd34c2d.png"},{"id":70200717,"identity":"64888366-4676-47d6-9e8e-c5e9aa23d3f5","added_by":"auto","created_at":"2024-11-29 12:26:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":171521,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of \u003cem\u003eAspergillus tamarii\u003c/em\u003e AUMC10198 L-glutaminase: \u0026nbsp;Lineweaver-Burk plot evaluation of the kinetic parameters (Km and Vmax) with respect to substrate concentration (A), pH level (B), temperature of reaction (C), thermal stability (D), and metal ions (E)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5423591/v1/134a2871e7589ad23588afde.png"},{"id":88814104,"identity":"f97f4060-acf3-4049-8984-2ecbcf2eefb4","added_by":"auto","created_at":"2025-08-11 16:06:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3692593,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5423591/v1/c096a6df-01eb-4d8f-b37d-81fbe90c81e7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Fungal glutaminases: Production, optimization, and purification with antimicrobial activities of L- glutaminase from novel isolate Aspergillus tamarii AUMC 10198 under solid-state fermentation","fulltext":[{"header":"Background","content":"\u003cp\u003eAn enzyme that catalyzes the deamination of L-glutamine to L-glutamic acid and ammonia is glutamine amidohydrolase (EC 3.5.1.2), also referred to as glutaminase [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The significance of L-glutaminases as enzymes with substantial commercial applications in the pharmaceutical and agricultural sectors has been underscored in recent publications [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. L-glutaminase, a green chemotherapeutic agent that inhibits the proliferation of numerous carcinoma cell lines and thereby impedes their development by depriving them of their essential amino acid (glutamine), may find prospective application as an anti-cancer therapy in the medical field [\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, L-glutaminases have been involved in the biocontrol of microbial pathogens [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Glutamic acid is produced through the hydrolysis of L-glutamine, the most essential amino acid in food production, owing to its flavorful and aromatic attributes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This process obviates the necessity for monosodium glutamate (MSG) in food production. A considerable number of fermented foods, including Eastern condiments and soy sauce, acquire a robust, palatable, and umami taste due to the accumulation of L-glutamic acid [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthermore, L-glutaminase has been employed in the development of glutamine biosensors to detect the glutamine concentrations in mammalian cell cultures, the biosynthesis of numerous nitrogenous metabolic intermediates at the cellular level, and the production of the nutraceutical theanine [\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMicrobial organisms that generate glutaminases include actinomycetes, fungi, yeasts, and bacteria [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Fungi are the primary producers of extra-cellular hydrolytic enzymes. The environmentally friendly and sustainable properties of fungal enzymes have been the subject of extensive investigation and implementation in numerous industries, including agriculture, medicine, bioremediation, and industrial processing. These fungal enzymes have high productivity, stability, and low extraction and purification costs [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The significance of fungal glutaminases is due to the potential of their biological functions in different industrial and agricultural applications. One of the most important aspect is the production of a variety of fungicides with potential antifungal activities that play an impact role in crop protection [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Specific fungal species, including \u003cem\u003eCandida, Saccharomyces, Trichoderma, Aspergillus, Penicillium, Fusarium\u003c/em\u003e, and a few endophytic species, are capable of producing glutaminase [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The efficient production of L-glutaminase was identified in marine filamentous fungi (\u003cem\u003eAspergillus, Penicillium\u003c/em\u003e) and yeasts (\u003cem\u003ePichia\u003c/em\u003e sp.) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTwo distinct fermentation processes are utilized for enzyme synthesis: solid-state fermentation (SSF) and submerged fermentation (SmF) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. SSF has higher production, lower energy usage, simpler operations, and better product consistency than traditional SmF [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Agricultural by-products have significant potential for use as sustainable carbon and energy sources, which generates economic and environmental benefits [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In addition, the vicinity of solid fermentation systems to the natural habitats of microorganisms enables them to facilitate the secretion and synthesis of a vast array of metabolites and enzymes [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The objective of this study is to provide a comprehensive explanation of the SSF process employed in the production of L-glutaminase from a novel fungal strain with high L-glutaminase productivity. The synthesis of L-glutaminase was accelerated and optimized through the adjustment of process parameters. Additionally, purification aspects were detailed and the characterization of L-glutaminase was also fulfilled, followed by determination of its antimicrobial activity.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eScreening and qualitative isolation of L-glutaminase-producing fungi\u003c/h2\u003e \u003cp\u003eWheat bran as a degrading lignocellulosic by-product possesses a high nutritional value. Samples of wheat bran were purchased from commercial marketplaces in the Alexandria, Egypt. The materials were air-dried, pulverized to an 80-micron powder, and stored at 4\u0026ndash;5\u0026deg;C prior to processing. The serial dilutions technique (One g of each specimen) was used to isolate fungi on modified Czapek Dox\u0026rsquo;s (CZD) agar medium [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Each screened fungal colony observed after five days of incubation period at 28\u0026deg;C was purified and subsequently preserved on Potato Dextrose Agar (PDA) medium at a temperature of 4\u0026deg;C pending further analysis. The rapid plate assay was employed to approximate the qualitative evaluation of L-glutaminase production. The experiment was performed utilizing modified CZD solid medium, comprising the subsequent components in g/L: sucrose 2.0 g, KCl 0.5 g, MgSO\u003csub\u003e4\u003c/sub\u003e. 7H\u003csub\u003e2\u003c/sub\u003eO 0.5 g, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 1.0 g, FeSO\u003csub\u003e4\u003c/sub\u003e. 7H\u003csub\u003e2\u003c/sub\u003eO 0.1 g, ZnSO\u003csub\u003e4\u003c/sub\u003e 0.1 g, L-glutamine 10.0 g, and agar 20.0 g at pH of 6.0. The medium was supplemented with 0.009% (v/v) phenol red dye (Sigma-Aldrich, Saint Louis, Missouri (USA)) as an indicator. A control medium devoid of dye was employed [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The selection process centered on the most expansive and profound pink color zone encircling the colonies that were chosen for additional quantitative analysis due to their positive production of L-glutaminase. The mean value of the zone diameters was calculated.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eQuantitative estimation of L-glutaminase\u003c/h3\u003e\n\u003cp\u003eA qualitative evaluation was conducted to select the four fungal isolates (F-S1, F-S2, F-S3, and F-S4) that exhibited the greatest ability of L-glutaminase production in the qualitative assay. The enzyme productivity was quantified using submerged culture technique. One milliliter of spore suspension containing 1.5 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e spores/mL was inoculated into 250 mL Erlenmeyer flask. The suspension was obtained from newly made PDA slants that were seven days old and cultured in 50 mL of modified Czapek Dox\u0026rsquo;s Broth containing L-glutamine as a carbon source. The containers underwent incubation at 30\u0026deg;C for 5 days using a rotary agitator adjusted to 150 rpm under SmF. After fermentation, centrifuge the m\u0026eacute;lange at 4\u0026deg;C for 15 minutes at 5000 revolutions per minute (Chilspin made in England). The clear supernatant was considered as a crude enzyme and stored at a temperature of 4\u0026deg;C until it was required.\u003c/p\u003e\n\u003ch3\u003eAssay of L-glutaminase\u003c/h3\u003e\n\u003cp\u003eIn accordance with the methodology described by Imada et al., the L-glutaminase activity was assessed utilizing the direct Nesslerization technique Imada et al. method [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. One milliliter of the crude enzyme was mixed with one milliliter of 40 mM L-glutamine, which was utilized as the substrate, in a citrate-phosphate buffer (0.1 M, pH 7). After incubating the mixture at 30\u0026deg;C for one hour, the reaction was halted by adding 0.5 milliliters of 1.5 M trichloroacetic acid. In order to obtain the precipitated protein, a solution was prepared by combining 0.1 ml of the solution mentioned above with 3.7 ml of distilled water. The mixture was subsequently adjusted with 0.2 ml of Nessler's reagent (Himedia, India). Following this, a 5-minute centrifugation at 5,000 rpm was conducted. At 450 nanometers, the absorbance was quantified with a spectrophotometer (Alpha 1102, Laxco, USA) following 15 minutes. A unit of L-glutaminase (U) was designated to represent the quantity of enzyme required to produce 1 \u0026micro;mol of ammonia per minute under ideal assay conditions.\u003c/p\u003e\n\u003ch3\u003eProtein estimation\u003c/h3\u003e\n\u003cp\u003eThe crude enzyme's protein content was determined calorimetrically in accordance with the method described by Lowry et al., with bovine serum albumin serving as a standard [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The protein concentration was expressed in milligrams per milliliter of crude enzyme.\u003c/p\u003e\n\u003ch3\u003eMorphological characterization and molecular identification\u003c/h3\u003e\n\u003cp\u003eAt the Mycological Center, Assiut University, Assiut, Egypt (A.U.M.C.), the efficient L-glutaminase-producing fungal isolate was identified by combining taxonomic keys with morphological and reproductive characteristics (Diba et al., 2007). The specimen under investigation's genomic DNA was isolated, purified, and analyzed for molecular identification in accordance with the methodology described by Moubasher et al. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Using universal primers ITS 4 (5'- TCC TCC GCT TAT TGA TAT GC- 3') and ITS 1 (5'- TCC GTA GGT GAA CCT GCG G- 3') produced by SolGent Co. in Yuseong-Gu, Daejeon, South Korea, the internal transcribed spacer (ITS) was amplified. To examine the obtained sequences, the National Center for Biotechnology (NCBI) BLAST research tool was used (blast.ncbi.nlm.nih.gov). In order to construct the phylogenetic tree, 35 sequences from closely related \u003cem\u003eAspergillus\u003c/em\u003e (section Flavi) species in the GenBank database were aligned. Maximum likelihood (ML) models and maximum parsimony (MP) analyses were performed using version 10.2.6 of MEGA X [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The robustness of the sparsest-packed trees was assessed through the utilization of 1000 bootstrap replications [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSolid-state fermentation and culture conditions\u003c/h2\u003e \u003cp\u003eThe quantitative analysis of L-glutaminase was performed utilizing the SSF method. A comprehensive examination was conducted of the commercial marketplaces in Alexandria, Egypt, in search of a variety of agricultural by-products, including wheat bran, soybean, sugarcane bagasse, and graved corn grains. An investigation was conducted into the effectiveness of agro-industrial residues to maximize the production of L-glutaminase by \u003cem\u003eAspergillus tamarii\u003c/em\u003e. Following the mechanical drying, grinding, and sieving of ten grams of each substrate into 250 milliliter Erlenmeyer conical flasks, twenty milliliters of 0.01 M phosphate buffer pH 7.4 were added to moisten each flask [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. After autoclaving at 121\u0026deg;C for 20 minutes the flasks were inoculated with 2 ml of the spore suspension obtained from new slants (7-day old culture) of \u003cem\u003eAspergillus tamarii\u003c/em\u003e. The inoculated containers were incubated at a temperature of 30\u0026deg;C for five days under static condition. In accordance with the modified methodology described by Kashyap et al. [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], crude L-glutaminase was extracted from the fermented solid culture medium by subjecting it to 20 minutes of rotary stirring at 200 rpm, followed by 20 minutes of centrifugation at 1500 rpm at 4\u0026deg;C. A 50 ml volume of 0.1 M phosphate buffer with a pH of 7.0 was utilized as the solvent. In the cell-free supernatant, the protein content and enzyme activity were evaluated. The mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation values were reported following the three repetitions of each experiment.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOptimization of various parameters for L-glutaminase production under solid-state fermentation\u003c/h3\u003e\n\u003cp\u003eThe impact of various culture factors on the L-glutaminase production of the most potent strain \u003cem\u003eAspergillus tamarii\u003c/em\u003e during SSF was investigated by researchers employing a one-variable-at-a-time approach (OVAT). The research investigated the effects of different culture parameters on L-glutaminase synthesis. These parameters comprised inoculum size (1.0\u0026ndash;5.0% v/v), temperature (25\u0026ndash;40\u0026deg;C), incubation time (three to nine days), moisture content (0.25\u0026ndash;4%), and pH's ranging from 4.0\u0026ndash;9.0 (1N HCl or 1N NaOH). Three replicates were conducted for each experiment.\u003c/p\u003e\n\u003ch3\u003ePurification of L-glutaminase\u003c/h3\u003e\n\u003cp\u003eThe impure-optimized L-glutaminase was subjected to three consecutive phases of purification following its extraction under optimal solid-state conditions: ethanol precipitation, ion exchange, and gel filtration.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePartial purification of L-glutaminase\u003c/h2\u003e \u003cp\u003eThe partial purification of L-glutaminase was achieved through fractional precipitation using extremely absolute ethanol (4\u0026deg;C). Combine cold crude enzyme and cold ethanol in equal parts while agitating gently; allow to remain at 4\u0026deg;C for 20 minutes. The precipitated fraction was obtained through 15 minutes of centrifugation at 5,000 rpm and 4\u0026deg;C. The supernatant was then saturated to 90% by adding additional ethanol. Diverse precipitate fractions, including 30%, 50%, 70%, and 90%, were produced by this method. Fractional separation was the initial step, which was followed by dialysis of the precipitated fractions against 0.01 M phosphate buffer (pH 8) overnight at 4\u0026deg;C. The collected enzyme fractions were analyzed for total protein content and L-glutaminase activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDEAE Sephadex A-50 ion-exchange chromatography\u003c/h2\u003e \u003cp\u003ePartially active ethanol fraction (70%) was loaded onto a DEAE Sephadex A-50 (2.5 cm \u0026times; 30 cm) ion-exchange column pre-equilibrated with 0.1 M NaCl in a 0.05 M acetate buffer (pH 5.2). The enzyme elution was conducted at a flow rate of 30 ml/h. The entire quantity was collected at a temperature of 4\u0026deg;C. The total protein content and L-glutaminase activity of the active fractions were evaluated subsequent to their collection, pooling, and concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGel filtration chromatography on Sephadex G-100\u003c/h2\u003e \u003cp\u003eSephadex G-100 (2 cm \u0026times; 28 cm) was applied to load the active and concentrated fractions generated in the previous stage. At a flow rate of 60 mL/h, the substance was eluted and brought to equilibrium utilizing the identical buffer. Ultimately, the absorbance of the protein was determined at 280 nanometers, and 5 ml of the active fractions containing the most significant amount of L-glutaminase activity were extracted, concentrated by dialyzing against distilled water, lyophilized, and stored at -20\u0026deg;C for the characterization of the purified form of the enzyme.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eProperties of the purified L-glutaminase\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003eEffect of substrate concentration on glutaminase activity\u003c/h2\u003e \u003cp\u003eThe optimal substrate concentration for the experiment was determined by performing separate incubations with purified L-glutaminase and different concentrations of L-glutamine (as a substrate) in the assay mixture (ranging from 0.2 to 2.0 mg/ml) under ideal conditions.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eKinetics parameters\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo determine the kinetic parameters (maximal velocity (Vmax) and Michaelis-Menten constant (Km)) of the purified L-glutaminase, standard assay conditions were used to measure the reaction velocities at various concentrations of L-glutamine (0.2-2.0 mg/ml). The apparent Km and Vmax values were obtained from the Lineweaver-Burk plot, which establishes a relationship between 1/Vmax and 1/S (reciprocal values), using the Michaelis-Menten equation, V\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;V\u003csub\u003emax\u003c/sub\u003e[S] / K\u003csub\u003em\u003c/sub\u003e+[S] [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn contrast, where V\u003csub\u003e0\u003c/sub\u003e represents the initial velocity of the reaction, Vmax denotes the maximal velocity, S is the concentration of the substrate, and Km represents the constant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffect of pH, temperature, and thermal stability on glutaminase activity\u003c/h2\u003e \u003cp\u003eBy subjecting the purified enzyme to different pH values ranging from 3.6\u0026ndash;9.6 using acetate buffer (0.05 M; pH 3.6\u0026ndash;5.4), phosphate buffer (0.05 M; pH 5.6-8.0), and sodium carbonate buffer (0.05 M; pH 8.6\u0026ndash;10). Incubation was carried out under optimal assay conditions to establish the optimal pH for L-glutaminase activity. To investigate the effect of temperature on enzyme activity, the reaction mixture was incubated at temperatures spanning from 30 to 60\u0026deg;C for 30 minutes at the optimal pH. To ascertain the thermostability of L-glutaminase, the enzyme was pre-incubated for 15, 30, and 60 minutes, respectively, at temperatures of 50, 60, and 70\u0026deg;C, in the absence of a substrate. The remaining enzyme activity was assessed subsequent to chilling.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEffect of metal ions (activators/inhibitors) on glutaminase activity\u003c/h2\u003e \u003cp\u003eThe activity of purified L-glutaminase was investigated using a variety of metal ions at a concentration of 0.01 M: Na\u003csup\u003e+\u003c/sup\u003e (NaCl), K\u003csup\u003e+\u003c/sup\u003e (KCl), Ca\u003csup\u003e2+\u003c/sup\u003e (CaCl\u003csub\u003e2\u003c/sub\u003e), Mg\u003csup\u003e2+\u003c/sup\u003e (MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO), Ba\u003csup\u003e2+\u003c/sup\u003e (Bacl\u003csub\u003e2\u003c/sub\u003e), Cd\u003csup\u003e2 +\u003c/sup\u003e (CdCl\u003csub\u003e2\u003c/sub\u003e), Cu\u003csup\u003e2+\u003c/sup\u003e (CuSO\u003csub\u003e4\u003c/sub\u003e), Zn\u003csup\u003e2+\u003c/sup\u003e (Zn (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003e), and Fe\u003csup\u003e2+\u003c/sup\u003e (FeSO\u003csub\u003e4\u003c/sub\u003e∙7 H\u003csub\u003e2\u003c/sub\u003eO). In the test mixture, metal ions were allowed to incubate each enzyme solution for 30 minutes at room temperature in accordance with the recommended assay protocol. The estimated residual activity was compared to the 100% activity of the control group, which received no additives.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAntimicrobial assay for the purified L-glutaminase\u003c/h2\u003e \u003cp\u003eThe antimicrobial efficacy of the purified L-glutaminase was determined using the agar well diffusion technique [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] against six microbial pathogens kindly provided by the Microbiological lab at Department of Microbiology, Faculty of Science, Helwan University, Egypt. \u003cem\u003eBacillus subtilis\u003c/em\u003e NRRL B-543, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923 as Gram\u0026thinsp;+\u0026thinsp;ve bacterial indicators and \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e ATCC 27853 as Gram -ve bacterial indicators. Moreover, \u003cem\u003eCandida albicans\u003c/em\u003e ATCC 20231 and \u003cem\u003eAspergillus flavus\u003c/em\u003e as fungal indicators. Bacterial pathogens were grown in Mueller Hinton Agar (MHA) at 35\u0026deg;C for 1 day and fungal strains were grown in potato dextrose agar at 27\u0026deg;C for 3 days. Muller Hinton Agar plates were swabbed with 100 \u0026micro;L (0.5 McFarland standards) of each strain of pathogenic organisms. Approximately 6-mm diameters of wells were punctured aseptically in solid agar with a cork borer. One hundred \u0026micro;L of the produced L-glutaminase was injected into each well. The injected plates were refrigerated for 2 h to permit the diffusion of enzyme. Subsequently, the plates were incubated for 24 h at 37 \u0026ordm;C for bacteria and 5 days at 30 \u0026ordm;C for fungi. Gentamicin (10 \u0026micro;g/mL) was used as a positive control for bacteria and nystatin (100 U) for fungi [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Evaluation of the antimicrobial potentiality was detected by monitoring the mean diameter of the growth inhibition zones (mm) in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data were reported in the form of the mean value\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Utilizing information from three distinct investigations (n\u0026thinsp;=\u0026thinsp;3), the means were calculated. The ANOVA test was employed to compare the various groups that were the subject of the statistical investigation. The predetermined significance levels were p\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eQualitative Screening and Quantitative estimation of L-glutaminase-producing fungi\u003c/h2\u003e\n \u003cp\u003eThe competency of fourteen fungal isolates to produce L-glutaminase was verified utilizing the rapid-plate assay method. Agro-industrial by-product (wheat bran) was utilized as a substrate for this screening. Four specific fungal isolates F-S1, F-S2, F-S3, and F-S4 have the capability of secreting L-glutaminase after 72 h of incubation with measurable pink zone diameter ranged from 8 to 32 mm. The mean and standard deviation were estimated to show the results in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. As determined qualitatively by the greatest mean diameter of the zone encircling the colonies on the plated agar, fungal isolate F-S1 showed the largest diameter zone (32.30 mm) and the lowest zone (7.88 mm) was detected by fungal isolate F-S3.\u003c/p\u003e\n \u003cp\u003eQuantitative estimation of L-glutaminase producing isolates was assessed in triplicate using the agitated method with a submerged culture medium. The mean and standard deviation were utilized to estimate the results in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The observed qualitative and quantitative values exhibited statistical dissimilarity when p\u0026thinsp;\u0026le;\u0026thinsp;0.05. The L-glutaminase activity of the fungal isolates exhibited a broad spectrum, with values varying from 0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ed\u003c/sup\u003e to 2.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003ea\u003c/sup\u003e U/ml. The discernible discrepancy is illustrated by the small letters. Based on a substantially distinct estimate (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), the F-S1 isolate exhibited the highest enzyme productivity (2.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003ea\u003c/sup\u003e U/ml) as a glutamine hydrolyzer. The L-glutaminase activities of F-S2, F-S4, and F-S3 were arranged as follows: 1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003eb\u003c/sup\u003e, 0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ec\u003c/sup\u003e, and 0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ed\u003c/sup\u003e U/ml, respectively. Following a meticulous evaluation employing molecular and morphological techniques for the potent isolate (F-S1) exhibiting the greatest glutaminolytic activity that was selected for subsequent investigations.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eQualitative and Quantitative screening of L-glutaminase producing fungal isolates\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIsolate number\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eQualitative screening\u003c/p\u003e\n \u003cp\u003eMean pink zone diameter (mm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtein content (mg/ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eQuantitative estimation\u003c/p\u003e\n \u003cp\u003eEnzyme activity\u003c/p\u003e\n \u003cp\u003e(U/ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDry weight\u003c/p\u003e\n \u003cp\u003e(g/50 ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eF-S1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.30\u0026thinsp;\u0026plusmn;\u0026thinsp;3.55\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eF-S2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.55\u0026thinsp;\u0026plusmn;\u0026thinsp;2.18\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.623\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eF-S3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eF-S4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eANOVA test\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ep\u003c/strong\u003e \u003cstrong\u003evalue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.88\u003c/p\u003e\n \u003cp\u003e0.0013*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.41\u003c/p\u003e\n \u003cp\u003e0.008*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.85\u003c/p\u003e\n \u003cp\u003e0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.98\u003c/p\u003e\n \u003cp\u003e0.0136*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cem\u003eSmall different letters (\u003c/em\u003e \u003csup\u003e\u0026nbsp;\u003cem\u003ea,b,c,d\u003c/em\u003e\u0026nbsp;\u003c/sup\u003e \u003cem\u003e) indicate that there was a significant variation with different studied groups.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eANOVA test was performed for comparing between different groups.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eValues are the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (n\u0026thinsp;=\u0026thinsp;3), P value* was significant if\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/em\u003e\u003c/p\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003eMorphological properties and molecular identification of potent L- glutaminase-producing isolate\u003c/h2\u003e\n \u003cp\u003eThe morphological identification of the potent glutaminase-producing fungal isolate (F-S1) \u003cem\u003eAspergillus tamarii\u003c/em\u003e AUMC 10198 was accomplished through phenotypic characterizations, reproductive structures, and comparisons with authentic isolates at the Mycological Center, Assiut University (AUMC), Assiut, Egypt. At cultural level, colonies of \u003cem\u003eAspergillus tamarii\u003c/em\u003e (AUMC 10198) grown on Czapek\u0026apos;s agar ultimately grew to a diameter of 5\u0026ndash;6 centimeters after seven days at a temperature of 25\u0026deg;C. The conidial region exhibited a subdued greenish-yellow hue with a white edge; the reverse lacked any exudates or pigmentation (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Regarding to microscopic examination (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB), the lactophenol cotton blue-stained conidiophores were observed to be colorless, measuring a maximum of 1\u0026ndash;2 mm in length and 8 \u0026micro;m in width. Additionally, they exhibited a grainy surface with an abrupt constriction of the wall at the base of the vehicle. The conidial head exhibits a loose radiating pattern and harbors sizable vesicles ranging in diameter from 30 \u0026micro;m. These vesicles are globose to sub-globose in shape and have thin walls. When adhered to phialides that are loosely packed and measure 8.5\u0026ndash;11.5 x 5.0\u0026ndash;6.0 \u0026micro;m. Large heads generally possess metulae measuring 10\u0026ndash;15 x 4\u0026ndash;8 \u0026micro;m in dimension, while small heads lack such features. Mature conidia are globose, bounded by chains that are conspicuously roughened, and range in diameter from 4.2 to 6.6 \u0026micro;m. In contrast, young conidia are cylindrical to pyriform.\u003c/p\u003e\n \u003cp\u003eA BLAST analysis was conducted to compare the extracted ITS sequences of the target strain with those that were previously archived in the NCBI Nucleotide Sequence Database. \u003cem\u003eAspergillus tamarii\u003c/em\u003e CBS 104.13, which had a GenBank accession number of MH854614 and 591 out of 597 identities (98.99%), was the most closely related match. The sequence, designated with accession number OQ976977, has been successfully submitted to the GenBank database. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, the species has been identified as \u003cem\u003eAspergillus tamarii\u003c/em\u003e AUMC 10198.Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e Morphological identification: \u003cem\u003eAspergillus tamarii\u003c/em\u003e (AUMC 10198) was cultured for seven days at 25\u0026deg;C on Czapek\u0026apos;s agar medium (A). A microscopic examination was performed using lactophenol cotton blue stain at X 1000 magnification (B)\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003eSolid-state fermentation (SSF) for L- glutaminase production\u003c/h2\u003e\n \u003cp\u003eSSF is a sustainable and environmentally beneficial method that utilizes substantial by-products from the agro-industrial sector as inexpensive raw materials [\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e]. The selection of an optimal solid substrate is critical during the fermentation process. SSF effectively synthesized glutaminase from a variety of agro-industrial by-products, including wheat bran, soybean, sugarcane bagasse, and graved corn grains. The values presented in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3) are the mean plus or minus the standard deviation. L-glutaminase output varied in response to the substrate type that was utilized. The outcomes demonstrated that wheat bran, in comparison to sugarcane bagasse, soybean, and graves corn grains functioned as the most effective solid substrate, exhibiting the highest specific activity of 2.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 U/mg protein and enzyme activity of 3.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 U/mL, respectively. Based on the current findings, there were significant differences in the specific activities of wheat bran, sugarcane bagasse, and graved corn grains (2.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003csup\u003ea\u003c/sup\u003e, 2.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003eb\u003c/sup\u003e, and 1.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ec\u003c/sup\u003e U/mg protein), respectively.\u003c/p\u003e\n \u003cp\u003eOur findings corroborate those of Soren et al. [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e], that wheat bran served as the optimal substrate for the maximal production of L-glutaminase. Wheat bran was the most likely of seven solid by-products for \u003cem\u003eFusarium solani\u003c/em\u003e AUMC 8615 that increase L-asparaginase activity [\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e]. The supplementation of wheat bran resulted in the greatest L-glutaminase output. Wheat bran\u0026apos;s strong nutritional content may promote spore formation and fungus growth. A resistance to aggregation enhances the mechanical effectiveness of wheat bran particles [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003eOptimization of SSF parameters for L-glutaminase production by OVAT approach\u003c/h2\u003e\n \u003cp\u003eIn order to maximize the yield of L-glutaminase \u003cem\u003evia\u003c/em\u003e solid fermentation process, numerous parameters were optimized (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n \u003ch2\u003eIncubation time\u003c/h2\u003e\n \u003cp\u003eThe enzyme yield is notably impacted by the duration of fermentation, as illustrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA. The maximum synthesis of L-glutaminase was observed on the fifth day of incubation at 3.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003ea\u003c/sup\u003e U/mL, following a significant increase. The activity of the enzyme remained relatively constant with no significant difference at 1.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ec\u003c/sup\u003e and 1.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003ec\u003c/sup\u003e U/mL for three and nine incubation periods, respectively. An increase in the fermentation period beyond five days resulted in a substantial reduction in enzyme production due to the degradation of enzymes [\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e]. The optimal time to detect L-glutaminase synthesis for the majority of fungal strains was between the fifth and seventh day of incubation [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e]. \u003cstrong\u003eInoculum size\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe effects of different inoculum concentrations (1\u0026ndash;5% v/v) on L-glutaminase synthesis during the current fermentation process were investigated in a study, as depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB. The enzyme production was most significant at 4% (2 ml/flask) of inoculum size with 3.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003csup\u003ea\u003c/sup\u003e U/ml. \u003cem\u003eTrichoderma koningii\u003c/em\u003e strain demonstrated its highest L-glutaminase activity at a volume of 2 ml, which is consistent with our findings [\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e]. The enzyme activity was notably perturbed for all values that deviated from the optimal range. According to Abdel-Hamid et al., the \u003cem\u003eFusarium oxysporum\u003c/em\u003e strain exhibited highest efficiency of enzyme activity at 3% of inoculum level under SSF [\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e]. A diminished enzyme output and insufficient biomass result from a limited inoculum size. A significantly increased inoculum volume results in both the depletion of vital nutrients and the suppression of enzyme activity [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n \u003ch2\u003eMoisture content\u003c/h2\u003e\n \u003cp\u003eMoisture content is a known variable that influences indoor fungal growth, regulates and enhances metabolic activity, and ultimately affects product production [\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e]. A significant discrepancy in L-glutaminase production was observed among the letters ranging from a to e (p\u0026thinsp;\u0026le;\u0026thinsp;0.05). The peak activity of L-glutaminase at 2% initial moisture content was 5.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5a U/mL with specific activity 2.77\u0026plusmn;0.25\u003csup\u003ea\u003c/sup\u003e U/mg protein, as illustrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC. In excess of the optimal moisture content, enzyme production decreased significantly. Liquid in motion hinders gaseous exchange, restricts aeration, and promotes the clustering of substrate particles, all while reducing porosity [\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e]. These findings are consistent with prior investigations that documented the production of L-glutaminase by \u003cem\u003eZygosaccharomyces rouxii\u003c/em\u003e and L-glucoamylase by \u003cem\u003eAspergillus niger\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\n \u003ch2\u003eHydrogen ion concentration\u003c/h2\u003e\n \u003cp\u003eAn important factor that affects development, metabolic activity, and enzyme production is the concentration of hydrogen ion in the fermentation medium [\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e]. The activity of L-glutaminase improved over time when the starting pH was raised, reaching a maximum of 8.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003csup\u003ea\u003c/sup\u003e U/mL in a slightly alkaline medium at pH 8.0 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). The findings align with Mostafa et al. and Dueramae et al. who found that marine \u003cem\u003eHalomonas meridiana\u003c/em\u003e and halophilic \u003cem\u003eTetragenococcus muriaticus\u003c/em\u003e FF5302 produced the optimal L-glutaminase at 8.0 pH [\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. When compared to the optimal pH value, the enzyme activity was declined to be 66.67% at an acidic pH of 5.0 and 36.32% at an alkaline pH of 9.0. It has been shown that \u003cem\u003eAspergillus tamarii\u003c/em\u003e can grow over a broad range of pH values (6\u0026ndash;9), as there is no discernible difference between pH 6 (5.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003csup\u003ec\u003c/sup\u003e) and pH 9 (5.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003csup\u003ec\u003c/sup\u003e). At pH 8 in SSF, it was found that \u003cem\u003eAspergillus terreus\u003c/em\u003e MTCC 1782 produced the most L-asparaginase [\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e]. However, \u003cem\u003eBeauveria\u003c/em\u003e sp. showed two pH optimum values for L-glutaminase production under SSF: 6.0 and 9.0 [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\n \u003ch2\u003eIncubation temperature\u003c/h2\u003e\n \u003cp\u003eThe maximum L-glutaminase productivity (12.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003csup\u003ea\u003c/sup\u003e U/mL) was detected at 35\u0026deg;C incubation temperature in solid state cultural medium (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE). Gomaa, 2022b detected that the maximum glutaminase activity (40.80 U/mL) by halophilic \u003cem\u003eBacillus\u003c/em\u003e sp. DV2-37 at 37\u0026deg;C [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. Whereas, maximum L-glutaminase productivity was achieved by \u003cem\u003eTrichoderma koningii\u003c/em\u003e at 30\u0026deg;C [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e]. A noteworthy reduction of enzyme activity was observed at 40\u0026deg;C (7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003csup\u003eb\u003c/sup\u003e U/mL). This may be attributed to the denaturation of the enzyme molecules at lower and higher temperatures [\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e]. According to the net results of Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, L-glutaminase production was obviously increased by 3.20-fold of that before optimization under solid-state condition. It was estimated that L-glutaminase activity was multiplied by 3.23-fold as compared to unoptimized medium by \u003cem\u003eAspergillus terreus\u003c/em\u003e ZHG2 [\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e]. Furthermore, optimization of L-glutaminase productivity was exhibited maximum yield of 703.8 U/gds with 3.8-fold increase under SSF [\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eL-glutaminase purification\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eAspergillus tamarii\u003c/em\u003e AUMC 10198 L-glutaminase was purified through three distinct procedures: ethanol fractionation (for partial purification), ion-exchange chromatography using DEAE (Sephadex A-50), and gel filtration utilizing a Sephadex G-100 column. Everything was completed in a sequential sequence.\u003c/p\u003e\n\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\n \u003ch2\u003eEthanol fractionation\u003c/h2\u003e\n \u003cp\u003eL-glutaminase was partially purified by ethanol precipitation at 70% saturation Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. It exhibited a total activity of 303.98 U, a specific activity of 13.10 U/mg protein, enzyme recovery of 24.65%, and a purification fold of 3.23.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEthanol precipitation of \u003cem\u003eAspergillus tamarii\u003c/em\u003e AUMC 10198 L-glutaminase\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"9\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEthanol\u003c/p\u003e\n \u003cp\u003eConcentration\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtein content\u003c/p\u003e\n \u003cp\u003e(mg/ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal protein\u003c/p\u003e\n \u003cp\u003e(mg)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRecovered protein (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGlutaminase activity (U/ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal activity unit\u003c/p\u003e\n \u003cp\u003e(U)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRecovered activity (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpecific activity\u003c/p\u003e\n \u003cp\u003e(U/mg protein)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePurification (fold)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCulture extract\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e304\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1233\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e139.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e198.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.88\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e303.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\n \u003ch2\u003eIon-exchange chromatography\u003c/h2\u003e\n \u003cp\u003eThe DEAE (Sephadex A-50) column was employed for the subsequent purification phase, resulting in the acquisition of three protein peaks, which corresponded with three peaks of L-glutaminase at fractions 6\u0026ndash;11, 25\u0026ndash;30, and 38\u0026ndash;42 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Maximum activity was observed during the second apex surge. The specific activity increased by 6.89-fold at a yield of 22.57%, from 13.10 to 27.99 U/mg protein as shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e\n \u003ch2\u003eGel filtration chromatography\u003c/h2\u003e\n \u003cp\u003eThe most active fractions obtained from an ion-exchange chromatography system (Sephadex-50) were executed using a gel filtration column composed of Sephadex G-100. The eluent exhibited two protein peaks, along with a sharp and distinct peak indicative of L-glutaminase activity (fractions 13, 14, 15, 16, and 17), as depicted in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB. The active fractions (5 ml) were stored at -20\u0026deg;C following pooling and concentration. They were then utilized as purified L-glutaminase to satisfy additional purity standards.\u003c/p\u003e\n \u003cp\u003eThe results of the purification procedure are briefly outlined in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. The calculated values are as follows: 54.17 U/mg of protein for specific activity, 227.50 (U) for total activity, 18.45% for final yield, and 12.90 purification fold comparable to the crude enzyme. The L-glutaminase of \u003cem\u003eAspergillus flavus\u003c/em\u003e exhibited a specific activity of 613.30 U/mg and a yield of 51.11%; the purification procedure was multiplied by 12.47 [\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e]. The L-glutaminase obtained from \u003cem\u003eAspergillus versicolor Faesay4\u003c/em\u003e was found to have a purification fold of 2.10\u0026thinsp;\u0026plusmn;\u0026thinsp;3.18 and a specific activity of 398.79\u0026thinsp;\u0026plusmn;\u0026thinsp;9.81 U/mg protein with overall activity 13.16\u0026thinsp;\u0026plusmn;\u0026thinsp;22.76 unit [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]. In contrast, \u003cem\u003ePenicillium brevicompactum\u003c/em\u003e NRC 829 demonstrated 869.08 U/mg of specific activity, 321.6 U of total activity, 162.75-fold purification, and 48.21% yield on Sephadex G-200 [\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e]. Based on the findings of Ali et al., the \u003cem\u003ePenicillium politans\u003c/em\u003e NRC510 L-glutaminase exhibited the following values: a yield of 25%, a specific activity of 133 U/mg, and a purification level of 230-fold [\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSummary of purification stages of \u003cem\u003eAspergillus tamarii\u003c/em\u003e AUMC 10198 L-glutaminase\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePurification stage\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal Protein\u003c/p\u003e\n \u003cp\u003e(mg)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal activity unit\u003c/p\u003e\n \u003cp\u003e(U)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpecific activity\u003c/p\u003e\n \u003cp\u003e(U/mg protein)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eYield\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePurification (fold)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026bull; Crude extract\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e304\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1233\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026bull; Ethanol (70%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e303.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026bull; Ion exchange on DEAE-Sephadex A-50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e278.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.89\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026bull; Gel filtration on Sephadex G-100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e227.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e54.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cstrong\u003eCharacterization of purified\u003c/strong\u003e \u003cstrong\u003eAspergillus tamarii\u003c/strong\u003e \u003cstrong\u003eAUMC10198 L-glutaminase\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eA number of parameters were considered when determining the L-glutaminase activity of \u003cem\u003eAspergillus tamarii\u003c/em\u003e AUMC10198 under test conditions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e), including substrate concentration, kinetic parameters Km and Vmax, pH level, reaction temperature, thermal stability, and metal ions.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e\n \u003ch2\u003eEffect of substrate concentration and estimation of the kinetic parameters (Km and Vmax)\u003c/h2\u003e\n \u003cp\u003eThe impact of varying amounts of L-glutamine added to the reaction mixture on the activity of purified L-glutaminase is illustrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA. A range of concentrations between 0.2 and 2.0 mg/ml were utilized. The enzyme activity increased progressively as the substrate concentration was increased to 1.4 mg/ml of L-glutamine; ultimately, it reached an optimal relative activity of 115.38%. Nevertheless, further substrate additions resulted in a decline in enzyme activity. Ahmed et al., reported the most significant quantity of marine \u003cem\u003eAspergillus\u003c/em\u003e sp. ALAA-2000 L-glutaminase at a substrate concentration of 4.38 mg/ml [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]. The optimal activity of \u003cem\u003eBrevundimonas diminuta\u003c/em\u003e MTCC 8486 L-glutaminase was observed at 1% glutamine, according to a study of Jayabalan et al. [\u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e]. The strong substrate affinity of L-glutaminase for L-glutamine was predicted by its low Km of 0.28 mg/ml and high Vmax of 10.10 U/ml, as illustrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA. In agreement with the results, Singh and Banik reported that the great affinity of purified L-glutaminase to L-glutamine was detected at small Km (0.129 mmol) by \u003cem\u003eBacillus cereus\u003c/em\u003e MTCC 1305 [\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e]. \u003cem\u003ePenicillium brevicompactum\u003c/em\u003e NRC829 demonstrated the highest affinity for L-glutaminase activity at a concentration of 1.66 mM L-glutamine with km value of 0.13 mmol, as determined by Elshafei et al. [\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003ch3\u003eEffect of pH, temperature, and thermal stability on glutaminase activity\u003c/h3\u003e\n\u003cp\u003eThe data presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB, the maximal relative activity of 119.73% was achieved at a pH of 8. This finding suggests that the optimal pH range for L-glutaminase activity was between neutral and mildly alkaline (7\u0026ndash;8). At an alkaline pH 9 and an acidic pH 4, a noticeable decrease was observed. The optimal pH values for glutaminase activity were determined to be 8.0 and 8.5, respectively [\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e71\u003c/span\u003e]. It has been demonstrated that the ideal pH range for glutaminase activity is between 5.0 and 9.0 [\u003cspan class=\"CitationRef\"\u003e72\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e74\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThere is a relationship between incubation temperature and glutaminase activity climax. The enzyme exhibited its highest activity at 45\u0026deg;C and retained over 85% at 60\u0026deg;C \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC\u003cstrong\u003e).\u003c/strong\u003e The results reported by Farag et al. [\u003cspan class=\"CitationRef\"\u003e75\u003c/span\u003e], which indicated that the highest activity of purified L-glutaminase was observed by marine \u003cem\u003eAspergillus terreus\u003c/em\u003e, corroborated this ideal temperature. The thermostability of the purified enzyme was demonstrated by its retention of approximately 88.59% and 66.29% of its activity, respectively, after preincubation at 50 \u0026ordm;C for 60 minutes and 60 \u0026ordm;C for 60 minutes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD). The initial activity of purified L-glutaminase, was reduced by approximately 38.11% subsequent to a 15-minute heating period at 70 \u0026ordm;C followed by a 30-minute total inactivation period at 70\u0026deg;C. According to a study by Elshafei et al. [\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e], L-glutaminase purified from \u003cem\u003ePenicillium brevicompactum\u003c/em\u003e NRC829 exhibited stability across a temperature range of 50 to 60\u0026deg;C. At 45\u0026deg;C, \u003cem\u003eAspergillus oryzae\u003c/em\u003e L-glutaminase remained stable; however, at 55\u0026deg;C, it ceased to function [\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eEffect of metal ions (activators/inhibitors) on the activity of purified L-glutaminase\u003c/h3\u003e\n\u003cp\u003eMetal ions play a vital role in enzyme activity by either donating or receiving electrons. The enzyme maintained over 100% of its initial activity when activated with Fe\u003csup\u003e2+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, and Na\u003csup\u003e+\u003c/sup\u003e ions as shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE. This suggests that these ions engage in activatory mechanisms with L-glutaminase. The fold values of purified L-glutaminase for each of these metal ions were as follows: 1.80, 1.58, 1.29, 1.12, and 1.03. On the contrary, it was demonstrated that Cd\u003csup\u003e2+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e and Ba\u003csup\u003e2+\u003c/sup\u003e significantly impeded the activity of the enzyme. However, the activity of L-glutaminase was only slightly inhibited by Zn\u003csup\u003e2+\u003c/sup\u003e. An additional 75% reduction in enzyme activity was observed in the presence of Cd\u003csup\u003e2+\u003c/sup\u003e. \u003cem\u003eAspergillus\u003c/em\u003e sp. ALAA-2000 and \u003cem\u003eA. flavus\u003c/em\u003e were capable of eliciting the activity of purified L-glutaminase when subjected to Na\u003csup\u003e+\u003c/sup\u003e and Mn\u003csup\u003e2+\u003c/sup\u003e ions [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e]. Conversely, Fe\u003csup\u003e2+\u003c/sup\u003e exhibits moderate inhibitory activity against L-glutaminase from marine \u003cem\u003eBacillus subtilis\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e76\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntimicrobial activity of purified\u003c/strong\u003e \u003cstrong\u003eAspergillus tamarii\u003c/strong\u003e \u003cstrong\u003eAUMC10198 L-glutaminase\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data detected in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e recorded the antimicrobial efficiency of purified \u003cem\u003eAspergillus tamarii\u003c/em\u003e AUMC10198 L-glutaminase against some pathogenic indicators. The enzyme showed the highest antibacterial activity against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923 followed by \u003cem\u003eBacillus subtilis\u003c/em\u003e NRRL B-543 with mean inhibition zone diameter 36.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20 mm and 30.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60 mm, respectively. The lowest zone of inhibition (12.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20 mm) was achieved against \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e ATCC 27853. Marine \u003cem\u003eStreptomyces griseorubens\u003c/em\u003e NAHE L-glutaminase proved to have potential antibacterial activity against Gram-positive comparable to Gram-ve bacteria [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]. The antifungal activity of \u003cem\u003eAspergillus tamarii\u003c/em\u003e AUMC10198 L-glutaminase showed moderate inhibition zone diameters. The antimicrobial impact of \u003cem\u003ePseudomonas\u003c/em\u003e sp. RAS123 L-glutaminase, which showed no activity against fungal pathogens \u003cem\u003eAspergillus fumigatus\u003c/em\u003e (RCMB 002008) and \u003cem\u003eCandida albicans\u003c/em\u003e RCMB 005003 (1) ATCC 10231[\u003cspan class=\"CitationRef\"\u003e72\u003c/span\u003e].\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAntimicrobial efficiency of purified \u003cem\u003eAspergillus tamarii\u003c/em\u003e AUMC10198 L-glutaminase against various pathogenic indicators\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003eAgent\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003eMean diameter of the growth inhibition zones (mm)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eBacterial indicators\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" rowspan=\"2\"\u003e\n \u003cp\u003eFungal indicators\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eGram\u0026thinsp;+\u0026thinsp;ve\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eGram -ve\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eATCC 25923\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eB. subtilis\u003c/strong\u003e \u003cstrong\u003eNRRL B-543\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e \u003cstrong\u003eATCC 25922\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eP. aeruginosa\u003c/strong\u003e \u003cstrong\u003eATCC 27853\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eC. albicans\u003c/strong\u003e \u003cstrong\u003eATCC 20231\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eA. flavus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePurified\u003c/p\u003e\n \u003cp\u003eL-glutaminase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.09\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGentamicin\u003c/p\u003e\n \u003cp\u003e(10 \u0026micro;g/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNystatin\u003c/p\u003e\n \u003cp\u003e(100 U)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eGlutaminase, a crucial enzyme found in agriculture, industry, and medicine, possesses significant biotechnological and therapeutic value. L-glutaminase production from wheat bran as an agro-industrial residue is a prospective process for the biotechnology industry. \u0026nbsp;The findings indicated that \u003cem\u003eAspergillus tamarii\u003c/em\u003e AUMC10198 could potentially utilize wheat bran, an economically and ecologically sustainable substrate, in the synthesis of L-glutaminase. The implementation of solid-state culture to optimize the fermentation process resulted in a 31.22% increase in L-glutaminase output. The final yield of the purified L-glutaminase was 18.45%, the total activity was 227.50 U, and the purification fold was 12.90 comparable to those of the crude enzyme. Furthermore, \u003cem\u003eAspergillus tamarii\u003c/em\u003eAUMC10198 L-glutaminase investigated promising antimicrobial properties against various pathogenic indicators. Future research will focus on elucidating the molecular pathways underlying the enzyme's chemotherapeutic activity against malignancy.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDisclosures and statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e All authors participated in conceptualization and methodology. M Z. Performed all experiments. Gh. Y. \u0026amp; M. Z. Designed all experiments, participated in methodology \u0026amp; data curation. A. Y. \u0026amp; S. E. General administration. Gh.Y. Investigation, writing original draft, review and editing. All authors have approved the final submitted version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e Open access funding provided by The Science, Technology \u0026amp; Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003eAll data generated or analyzed during this study are included in this article and be available on reasonable request. No datasets were generated or analyzed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Ethics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e: The authors declare no competing interests. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDura M, Flores M, Toldr\u0026aacute; F. 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Exploitation of agricultural waste as sole substrate for production of bacterial L-glutaminase under submerged fermentation and the proficient application of fermented hydrolysate as growth promoting agent for probiotic organisms. Waste and Biomass Valorization. 2020;11:4245-4257.\u003c/li\u003e\n\u003cli\u003eIsaac G, Abu-Tahon M. Production of extracellular anti-leukemic enzyme L-asparaginase from \u003cem\u003eFusarium solani \u003c/em\u003eAUMC 8615 grown under solid-state fermentation conditions: purification and characterization of the free and immobilized enzyme. Egypt J Bot. 2016;56:799-816.\u003c/li\u003e\n\u003cli\u003eAbu-Tahon MA, Isaac GS. Purification, characterization and anticancer efficiency of L-glutaminase from \u003cem\u003eAspergillus flavus.\u003c/em\u003e The Journal of General and Applied Microbiology. 2019;65(6):284-292.\u003c/li\u003e\n\u003cli\u003eKoibuchi K, Nagasaki H, Yuasa A, Kataoka J, Kitamoto K. Molecular cloning and characterization of a gene encoding glutaminase from \u003cem\u003eAspergillus oryzae\u003c/em\u003e. Applied Microbiology and Biotechnology. 2000;54:59-68. \u003c/li\u003e\n\u003cli\u003e53. Khalil M, Moubasher M, El-Zawahry M, Miche M. Evaluation of antitumor activity of fungal L-glutaminase produced by Egyptian isolates. Letters in Applied NanoBioScience. 2020;9:924-930.\u003c/li\u003e\n\u003cli\u003eOlarewaju MO, Nzelibe HC. Statistical optimization of Lglutaminase production by \u003cem\u003eTrichoderma\u003c/em\u003e species under solid state fermentation using African locust beans as substrate. African Journal of Biochemistry Research. 2019;13(6):73-81.\u003c/li\u003e\n\u003cli\u003eEl-Sayed AS. L-glutaminase production by \u003cem\u003eTrichoderma koningii\u003c/em\u003e under solid-state fermentation. Indian Journal of Microbiology. 2009;49:243-250.\u003c/li\u003e\n\u003cli\u003ePallem C, Manipati S, Somalanka SR. Process optimization of L-glutaminase production by \u003cem\u003eTrichoderma koningii\u003c/em\u003e under solid state fermentation (SSF). Int J Appl Biol Pharm Technol. 2010;1:1168-1174.\u003c/li\u003e\n\u003cli\u003eAbdel-Hamid NS, Abdel-Khalek HH, Ramadan E, Mattar ZA, Abou-Taleb KA. Optimization of L-Asparaginase Production from \u003cem\u003eFusarium oxysporum\u003c/em\u003e F-S3 using Irradiated Pomegranate Peel under Solid-State Fermentation. Egyptian Journal of Chemistry. 2022;65(6):381-397.\u003c/li\u003e\n\u003cli\u003eAdan OCG, Huinink HP, Bekker M. Water relations of indoor fungi. In Fundamentals of Mold Growth in Indoor Environments and Strategies for Healthy Living. O.C.G. Adan, and R.A. Samson (eds). Wageningen: Wageningen Academic Publishers, 2011: pp. 41\u0026ndash;65.\u003c/li\u003e\n\u003cli\u003eJohansson P, Bok G, Ekstrand-Tobin A. The effect of cyclic moisture and temperature on mould growth on wood compared to steady state conditions. Build Environ. 2013;65:178\u0026ndash;184.\u003c/li\u003e\n\u003cli\u003eHan BZ, Nout RM. Effects of temperature, water activity and gas atmosphere on mycelial growth of tempe fungi \u003cem\u003eRhizopus microsporus\u003c/em\u003e var. \u003cem\u003emicrosporus\u003c/em\u003e and \u003cem\u003eR. microsporus\u003c/em\u003e var. \u003cem\u003eoligosporus.\u003c/em\u003e World Journal of Microbiology and Biotechnology. 2000;16:853-858.\u003c/li\u003e\n\u003cli\u003ePandey A, Ashakumary L, Selvakumar P, Vijayalakshmi K. Influence of water activity on growth and activity of \u003cem\u003eAspergillus niger\u003c/em\u003e for glycoamylase production in solid-state fermentation. World Journal of Microbiology and Biotechnology.1994;10:485-486.\u003c/li\u003e\n\u003cli\u003eKrishna C. Solid-state fermentation systems\u0026mdash;an overview. Critical reviews in biotechnology. 2005;25(1-2):1-30.\u003c/li\u003e\n\u003cli\u003eMurad H, Salem M. Utilization of uf-permeate for producing exopolysaccharides from lactic acid bacteria. Mansoura University Journal of Agricultural Sciences (Egypt). 2001;26(4):2167-2175.\u003c/li\u003e\n\u003cli\u003eMostafa YS, Alamri SA, Alfaifi MY, Alrumman SA, Elbehairi SEI, Taha TH, Hashem M. L-glutaminase synthesis by marine \u003cem\u003eHalomonas meridiana\u003c/em\u003e isolated from the red sea and its efficiency against colorectal cancer cell lines. Molecules. 2021;26(7):1963.\u003c/li\u003e\n\u003cli\u003eVaralakshmi V, Raju KJ. Optimization of l-asparaginase production by \u003cem\u003eAspergillus terreus\u003c/em\u003e mtcc 1782 using bajra seed flour under solid state fermentation. Int J Res Eng Technol. 2013;2(09):121-129.\u003c/li\u003e\n\u003cli\u003eKumari D, Raju KJ. Production and optimization of L-glutaminase with mixed substrate using \u003cem\u003eAspergillus wentii\u003c/em\u003e MTCC 1901 by solid state fermentation. Int J Eng Res Technol. 2016;5:10-18.\u003c/li\u003e\n\u003cli\u003eElshafei AM, Hassan MM, Abouzeid MA-E, Mahmoud DA, Elghonemy DH. Purification, characterization and antitumor activity of L-asparaginase from \u003cem\u003ePenicillium brevicompactum\u003c/em\u003e NRC 829. British Microbiology Research Journal. 2012;2(3):158.\u003c/li\u003e\n\u003cli\u003eAli TH, Ali NH, Mohamed LA. Glutamine amidohydrolase from \u003cem\u003ePenicillinum politans\u003c/em\u003e NRC 510. Polish Journal of Food and Nutrition Sciences. 2009;59(3).\u003c/li\u003e\n\u003cli\u003eJayabalan R, Jeeva S, Sasikumar A, Inbakandan D, Swaminathan K,Yun S. Extracellular L-glutaminase production by marine \u003cem\u003eBrevundimonas diminuta\u003c/em\u003e MTCC 8486. Int J Appl Bioeng. 2010;4:19-24.\u003c/li\u003e\n\u003cli\u003eSingh P, Banik R. Biochemical characterization and antitumor study of L-glutaminase from \u003cem\u003eBacillus cereus\u003c/em\u003e MTCC 1305. Applied Biochemistry and Biotechnology;2013;171:522-531.\u003c/li\u003e\n\u003cli\u003eDutta S, Ghosh S, Pramanik S. L-asparaginase and L-glutaminase from \u003cem\u003eAspergillus fumigatus\u003c/em\u003e WL002: Production and some physicochemical properties. Applied Biochemistry and Microbiology. 2015;51:425-431.\u003c/li\u003e\n\u003cli\u003eElborai A, Sayed R, Farag A, Elassar S. A Highly Purified L-Glutaminase from Immobilized \u003cem\u003ePseudomonas\u003c/em\u003e Sp. Ras123 Cultures with Antitumor and Antibacterial Activities. Journal of Microbiology, Biotechnology and Food Sciences. 2023; 13(1):e5637-e5637.\u003c/li\u003e\n\u003cli\u003eOhshima M, Yamamoto T, Soda K. Further characterization of glutaminase isozymes from\u003cem\u003e Pseudomonas aeruginosa\u003c/em\u003e. Agricultural and Biological Chemistry.1976;40(11):2251-2256.\u003c/li\u003e\n\u003cli\u003eSaleem R, Ahmed S. Characterization of a New L-Glutaminase Produced by \u003cem\u003eAchromobacter xylosoxidans\u003c/em\u003e RSHG1, Isolated from an Expired Hydrolyzed L-Glutamine Sample. Catalysts. 2021;11(11):1262.\u003c/li\u003e\n\u003cli\u003eFarag AM., Abd-Elnabey HM, Ibrahim HA, El-Shenawy M. Purification, characterization and antimicrobial activity of chitinase from marine-derived \u003cem\u003eAspergillus terreus\u003c/em\u003e. The Egyptian Journal of Aquatic Rsearch. 2016;42(2):185-192.\u003c/li\u003e\n\u003cli\u003eKiruthika J, Swathi S. Purification and characterisation of a novel broad spectrum anti-tumor L-glutaminase enzyme from marine \u003cem\u003eBacillus subtilis\u003c/em\u003e strain JK-79. African Journal of Microbiology Research. 2019;13(12):232-244.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"L-glutaminase, Aspergillus tamarii AUMC10198, Solid-state fermentation, Optimization, Purification and characterization, Antimicrobial activity","lastPublishedDoi":"10.21203/rs.3.rs-5423591/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5423591/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction\u003c/strong\u003e Fungal L-glutaminase has garnered significant attention in recent times with respect to its possible applications in the field of medical therapy and biotechnology. The principal aim of this research was to pick out microbial strains that can\u0026nbsp;efficiently produce L-glutaminase from agricultural by-products under solid-state fermentation (SSF). Various fungal isolates were screened for extracellular\u0026nbsp;L-glutaminase production. During the fermentation process, numerous parameters were adjusted one variable at a time (OVAT) to increase L-glutaminase production. The L-glutaminase of \u003cem\u003eAspergillus tamarii\u003c/em\u003e AUMC 10198 was purified in three sequential stages. The properties of the purified enzyme and the antimicrobial efficiency were also fulfilled.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eThe potentiality of four out of fourteen fungal isolates served as indicators of the enzyme's productivity. The fungus \u003cem\u003eAspergillus tamarii\u003c/em\u003e AUMC 10198, designated with the GenBank accession number OQ976977, was determined to be the potent for estimating L-glutaminase synthesis, under SSF using wheat bran as a solid substrate. The solid-state yield of L-glutaminase exhibited a 3.20-fold increase in comparison to the unoptimized state. The \u003cem\u003eAspergillus tamarii\u003c/em\u003e AUMC 10198 L-glutaminase underwent three stages of purification, resulting in an increase in enzyme productivity by 12.90 times. Following these steps, the ultimate enzyme recovery was 18.45%. The isolated L-glutaminase exhibited optimal activity at a pH of 8, a temperature of 45 °C, and partial stability up to 60 °C, as determined by characterization. The purified L-glutaminase exhibited a Vmax of 10.10 U/ml and a Km of 0.28 mg/ml when glutamine was used as the substrate. The metal ions Fe\u003csup\u003e2+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, and Na\u003csup\u003e+\u003c/sup\u003e demonstrated significant enzyme-activating properties at a concentration of 0.01 M, resulting in an enhancement of L-glutaminase productivity. The antimicrobial activity indicates its capability for various therapeutic and pharmaceutical applications.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003cstrong\u003eConclusion\u003c/strong\u003e The present investigation revealed that the local fungal strain of \u003cem\u003eAspergillus tamarii\u003c/em\u003e AUMC10198 could potentially be utilized in the production of L-glutaminase for industrial applications from agricultural by-products.\u003c/p\u003e","manuscriptTitle":"Fungal glutaminases: Production, optimization, and purification with antimicrobial activities of L- glutaminase from novel isolate Aspergillus tamarii AUMC 10198 under solid-state fermentation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-29 12:25:57","doi":"10.21203/rs.3.rs-5423591/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-29T20:10:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-29T11:46:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-28T21:42:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-27T18:53:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-18T19:23:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-17T23:25:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171161656597940809251252135285192071201","date":"2025-04-08T20:48:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"218909101396605161704763324039462885067","date":"2025-04-08T20:46:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"165336283771734522434101973547635876871","date":"2025-04-06T04:24:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"162331306013503950967285775327215492350","date":"2025-04-05T23:21:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"229438347929586603210740153507993601819","date":"2025-04-05T20:41:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"108454506223372078761012121012452081656","date":"2025-04-03T21:18:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32620621415226578042327880558058843882","date":"2025-04-03T20:52:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"152890443658396860032762417866781340345","date":"2025-04-03T19:59:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-03T19:54:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-11T11:03:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-11T11:01:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Cell Factories","date":"2024-11-09T21:30:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f5f0ec03-a306-4975-9314-241a5753818c","owner":[],"postedDate":"November 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-11T15:59:47+00:00","versionOfRecord":{"articleIdentity":"rs-5423591","link":"https://doi.org/10.1186/s12934-025-02802-0","journal":{"identity":"microbial-cell-factories","isVorOnly":false,"title":"Microbial Cell Factories"},"publishedOn":"2025-08-04 15:57:10","publishedOnDateReadable":"August 4th, 2025"},"versionCreatedAt":"2024-11-29 12:25:57","video":"","vorDoi":"10.1186/s12934-025-02802-0","vorDoiUrl":"https://doi.org/10.1186/s12934-025-02802-0","workflowStages":[]},"version":"v1","identity":"rs-5423591","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5423591","identity":"rs-5423591","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}