Immobilization of hydroaminase-expressing recombinant Escherichia coli whole-cell biocatalysts for the production of β-alanine

Immobilization of hydroaminase-expressing recombinant Escherichia coli whole-cell biocatalysts for the production of β-alanine | 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 Immobilization of hydroaminase-expressing recombinant Escherichia coli whole-cell biocatalysts for the production of β-alanine Li Ma, Yuyu Wang, Ruiqi Liu, Jingjia Hu, Xiaobing Zheng, Xiaoyang Yue, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7412633/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Mar, 2026 Read the published version in Biotechnology Letters → Version 1 posted 5 You are reading this latest preprint version Abstract Developing stable and easy-to-operate biocatalysts is crucial for their use as industrial catalysts. Here immobilized whole-cell catalysts were used for β-alanine production by immobilizing recombinant Escherichia coli cells (containing hydroaminase) with diatomite. E. coli BL21 (DE3)/pET-30a (+)-HAMase was genetically engineered for the efficient synthesis of β-alanine from acrylic acid and aqueous ammonia. Using glutaraldehyde as a cross-linking agent, polyethyleneimine (PEI) as a flocculant, and diatomite as the immobilization carrier, optimal immobilization was achieved with 8 % (w/v) PEI solution, 5 % (w/v) glutaraldehyde, and 100 mg wet cell/mL cell suspension, along with a PEI flocculation time of 2.5 h and glutaraldehyde cross-linking time of 1.5 h. The enzyme activity recovery rate reached 70.72 %. Remarkably, the immobilized whole-cell catalysts exhibited excellent stability, retaining over 90 % of initial enzyme activity after 12 h incubation at 45 °C and maintaining over 72 % enzyme activity after storage for 60 days at 4 °C. Additionally, the immobilized cells demonstrated enhanced reusability, maintaining consistent β-alanine yield even after ten consecutive reaction batches with an average yield of approximately 80 %. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction β-Alanine, the only naturally occurring β-amino acid, plays pivotal physiological roles in living organisms and serves as a key precursor for synthesizing nitrogen-containing bioactive compounds. β-Alanine serves as a crucial precursor for the production of diverse nitrogen-containing chemicals and plays an essential role as an intermediate in synthesizing compounds such as acrylamide, acrylonitrile, pantothenic acid, myostatin, pamidronate, and balsalazide. These compounds have extensive applications in various fields including drug synthesis, fine chemicals, environmental applications, food, and feed additives (M. Könst 2009, Coxon et al. 2005) . Due to its versatility, β-alanine is considered one of the 12 most promising three-carbon compounds for future development (Jang et al. 2012) . The current industrial production of β-alanine primarily involves the ammonolytic hydrolysis of acrylonitrile and the β-aminopropionitrile hydrolysis. However, the production method faces several limitations: the use of strong acids and bases necessitates corrosion-resistant equipment. High temperature and pressure conditions lead to increased energy consumption and production costs, the production of nitrile, among other by-products, poses potential toxic effects on organisms and the environment (Ford et al. 1947, Ji and Luo 2005) .The biosynthetic routes of β-alanine has attracting growing interest in recent years due to its environmental friendliness, mild reaction conditions, and minimal by-product formation. Nitrile hydrolases, primarily sourced from (Liang et al. 2008) Compared with chemical synthesis, the conversion method of nitrile hydrolase is milder, but Substrate inhibition at elevated concentrations of β-aminopropionitrile can inhibit nitrile hydrolase, thereby affecting the synthesis efficiency of the product. The production of by-products also brings inconvenience to separation and purification, which cannot meet the requirements of industrial production. The aspartic acid decarboxylase conversion method utilizes aspartic acid-α-decarboxylase, or aspartate lyase combined with PanD, to catalyze the production of β-alanine from aspartic acid. PanD is a key enzyme in the synthesis of β-alanine in organisms, which can efficiently catalyze the cleavage of the C α carboxyl group in L-aspartic acid (L-Asp) to generate β-alanine.At present, researchers mostly screen for PanD in prokaryotes such as Escherichia coli (Zhang et al. 2018) , Bacillus subtilis , Corynebacterium glutamicum , as well as some pathogenic bacteria such as Mycobacterium tuberculosis and Helicobacter pylori . The synthesis of β-alanine by aspartate decarboxylase conversion method has a simple reaction mechanism and high yield, but the purification of AspA and PanD requires expensive costs and is not suitable for large-scale production. Therefore, the synthesis of β-alanine by biotransformation mainly relies on whole cell transformation. In addition to using L-aspartic acid as a substrate, Lou Jian (Li et al. 2022) used acrylic acid as a substrate and utilized an acrylic acid aminase to catalyze the synthesis of β-alanine from acrylic acid. Acrylic acid is relatively inexpensive and structurally similar to β-alanine. Using acrylic acid as a substrate to produce β-alanine can complete the reaction in a short period of time, reducing the loss of raw materials. Theoretically, it does not produce harmful substances, is environmentally friendly, and has significant economic benefits. It is worth further exploration. The one-step generation of β-alanine from acrylic acid and ammonia belongs to the hydroamination reaction, which is the β-enantioselective addition of ammonia to unsaturated acids. performed molecular redesign of aspartate decarboxylase from Bacillus subtilis and successfully incorporated it into Escherichia coli , resulting in the development of an engineered strain capable of synthesizing β-amino acids efficiently. The biocatalytic system facilitates the synthesis of β-amino acids under mild conditions, utilizing cost-effective substrates and aqueous ammonia in a single-step process (Li et al. 2018) catalyst preparation process (Norman et al. 2021, Cerón-Carrasco and Jacquemin 2021, Liu et al. 2022, Kumar et al. 2020, Jayasena et al. 2015) . The selection of the carrier significantly impacts catalytic performance. Consequently, a wide range of polymeric, organic, and inorganic materials has been investigated as carriers. In particular, inorganic materials have garnered attention due to their mechanical stability, resistance to microbial corrosion, modifiability and reusability (Milioni et al. 2019) . Diatomite (DE), composed of amorphous silica with abundant siloxanes, features a highly porous surface and exhibits high mechanical strength, chemical stability, and biocompatibility. Additionally, it is cost-effective and readily available (Zhang et al. 2018) . However, direct cell adsorption onto diatomite is susceptible to shedding. Polyethyleneimine is synthesized through the polymerization of cyclic ethylamine, resulting in a polymer with repeating -CH 2 CH 2 NH 2 units (Marques et al. 2020) . Cross-linking agents react with the primary amine groups present in polyethyleneimine (PEI) to enhance the stability of immobilized cells. Jin et al. prepared immobilized E. coli by modifying diatomite with PEI and utilizing glutaraldehyde (GLU) as a cross-linking agent (Jin et al. 2021) . The reusability of the immobilized cells showed significant improvement. Hydroxymethylphosphine (THP) employs a nonessential NH 2 group to mitigate enzyme inactivation during immobilization while also forming a stable P-CH 2 -N linkage. (Zhang et al. 2018) immobilized recombinant E. coli cells using DE/PEI/THP. The immobilized cells exhibited superior thermal stability and reusability compared to free cells, making them suitable for the industrial production of R-mandelic acid. In this study, We demonstrate that hydroaminase (HAMase), a recombinant enzyme, can effectively catalyze the production of β-alanine. Diatomite was employed as a carrier to immobilize recombinant Escherichia coli whole cells, resulting in immobilized cells that Enhanced operational stability, mechanical robustness, and reusability. Fig. 1 illustrates preparation process of immobilized E. coli BL21 (DE3) /pET-30a (+) -AspB whole-cells.This method facilitated the immobilization of whole cells through a streamlined synthetic procedure while preserving high enzymatic activity. Finally, we assessed the production capacity for the continuous synthesis of β-alanine using immobilized cells in a fixed-bed bioreactor. This approach presents a promising candidate for whole-cell immobilization in various applications. Results and Discussion Cell immobilization and characterization The scanning electron microscopy (SEM) micrographs in Fig. 2a demonstrate that E. coli particles exhibit a rod-like morphology with smooth surfaces, measuring between 0.5 μm and 2 μm in length. Fig. 2b present SEM images of DE/PEI/GLU-immobilized E. coli , where the immobilized E. coli cells exhibit uniform particle sizes, characterized by membrane-coated surfaces and tightly interconnected cellular junctions, with no noticeable structural damage. This observation suggests that following diatomite adsorption, glutaraldehyde crosslinked with PEI, leading to the changes in cell structure. FT-IR analysis was performed to confirm the successful immobilization of diatomite and cells using GLU as a crosslinking agent. Fig. 3 illustrates the spectra of free cells, diatomite, and diatomite-immobilized cells. Upon cell immobilization, several distinct peaks emerged in the spectrum, differentiating it from those of E. coli and diatomite alone. Specifically, peaks at 792, 617, 471, and 1090 cm -1 were attributed to Si-O stretching vibrations, Si-O-Si symmetric stretching vibrations, Si-O bending vibrations, and the binding vibration of calcite to CO₃ 2- in diatomite, respectively. Furthermore the peak at 3305 cm -1 corresponded to the stretching vibrations of -OH and -NH, indicating the presence of these functional groups (Hu et al. 2017, Benayache et al. 2018) . Additionally, the peaks at 1656 cm -1 and 1537 cm -1 represented the bending vibrations within the N-H plane of primary and secondary amines. Upon treatment with GLU and PEI as cross-linking agent, the stretching vibrations of the C-H bond at 2927 cm -1 , bending vibrational peak of CH 2 at 1393 cm -1 , in-plane rocking absorption peak for CH 2 in diatomite immobilized cells at 1230 cm -1 , which can be attributed to PEI and GLU (Hu et al. 2017) . Additionally, a new stretch at 1453 cm −1 can be assigned to stretching vibration absorption peaks of -C=N-. These peaks in the spectra (1230, 1393, 1453 and 2937 cm -1 ) indicate the successful immobilization of diatomite and cells using GLU as a cross-linking agent. Optimization of parameters for cell immobilization by diatomite The use of diatomite as an immobilization matrix presents several advantages, including its high resistance to dissolution and exceptional adsorption capacity. However, relying solely on its adsorption properties for cell immobilization can result in challenges such as cell dislodgement. To improve the stability of immobilized cells, we investigated the efficacy of various cross-linking agents in forming covalent bonds with the cells. The effects of different types of cross-linker on enzyme activity recovery is presented in Table 1, while their impact on reusability is illustrated in Fig. 4a. The enzyme activity recoveries of the immobilized cells were found to be similar across all four cross-linkers. However, when polyvinyl alcohol and p-phenylenedial were employed as cross-linking agents, the reusability of the immobilized cells was considerably poor, with yields of β-alanine amounting to only 25.15 % and 17.55 % after three recycling cycles, respectively. In contrast, the use of GLU as a cross-linking agent resulted in a yield of 75.88 % after six recycling cycles. This enhanced reusability can be attributed to the utilization of free amino groups by GLU for cross-linking, which minimizes the impact on the activity. Consequently, GLU was selected as the preferred cross-linking agent for subsequent experiments. Table 1 Effect of crosslinking agents on enzyme activity. PEI serves as a flocculant for the immobilization of cells onto diatomite. The primary amine groups in PEI can cross-link with glutaraldehyde, resulting in the formation of a stable mesh structure. Variation in the relative molecular mass of PEI lead to changes in its viscosity and the proportion of secondary to tertiary amines, thereby influencing the immobilization effectiveness. Consequently, we conducted an investigation on the impact of PEI molecular weight on the immobilization effect, as illustrated in Fig. 4b.The viability of immobilized cells exhibited a gradual increase with the molecular weight of PEI up to 10 kDa, followed by a decline upon reaching 70 kDa. This phenomenon can be attributed to the increased viscosity and greater branching of the polymer when the molecular weight of PEI reaches 10 kDa. Consequently, a denser polymer film forms on the cell surface, leading to an enhanced effective specific surface area during interactions with the cross-linking agent, thereby improving the efficiency of immobilization (Zhao et al. 2021) . An increase in the molecular weight of PEI to 70 kDa may result in a reduction in the proportion of secondary amine groups within the amine group, potentially decreasing the efficiency of immobilization. Ultimately, PEI with a molecular mass of 10 kDa was chosen as the optimal flocculant. Diatomite serves as a carrier for immobilized cells, and its particle size significantly influences the effectiveness of immobilization. Fig. 4c demonstrates that the viability of immobilized cells initially increases and then decreases as the diatomite particle size increases. This phenomenon can be attributed to the enhancement of the pore structure, total pore volume, and micropore volume of diatomite with increasing particle size. As a result, both the loading capacity and loading efficiency of cells are improved. Consequently, diatomite with a particle size of 36.8 μm was chosen as the optimal immobilization carrier. A quantity of 0.3 g of diatomite was measured and added to varying masses of recombinant E. coli cells, aiming to investigate the impact of cell loading on the viability of immobilized cells, as depicted in Fig. 4d. The viability of immobilized cells exhibited an increase as the cell loading was raised to 5 g, but declined beyond this threshold. This phenomenon can be attributed to the accumulation of a high concentration of bacteria within the immobilized cells, which results in the blockage of channels and a reduced probability of substrate-cell contact. Consequently, the viability of the immobilized cells decreases. Therefore, a cell loading of 5 g was selected. The optimal volume fraction of PEI for immobilization was determined, as shown in Fig. 4e. The viability of immobilized cells exhibited a gradual increase with an increase as the PEI volume fraction continues to increase, the viability of immobilized cells began to decline. At low concentrations of PEI, the structure formed by the cross-linking of GLU and cells is loose, making the cells prone to detachment (Chen et al. 2008) . Conversely, as the concentration of PEI in the system increases, the molecules of PEI cross-link, resulting in heightened mass transfer resistance and a subsequent decrease in the viability of the immobilized cells (HOU et al. 2016) . Consequently, a volume fraction of 8 % PEI was chosen as the optimal flocculant. The selection of flocculation time for PEI is illustrated in Fig. 4f. Initially, the viability of immobilized cells increased with longer PEI flocculation time increased, peaking at 2.5 h. However, further increase in flocculation time resulted in a decrease in cell viability. This phenomenon may be attributed to the tightening of the crosslinked structure of the immobilized cells as the flocculation time is extended, which leads to excessive mass transfer resistance and ultimately reducing cell viability. Therefore, a flocculation time of 2.5 h was determined to be optimal. When immobilizing cells using cross-linking, the choice of crosslinker volume fraction significantly and irreversibly influences cell structure. Therefore, optimal immobilization is often achieved through precise control of the concentration and duration. The efffect of GLU on the viability of immobilized cells is depicted in Fig. 4g, revealing a positive correlation between GLU concentrations and cell viability. Optimal viability was observed at a GLU volume fraction of 5 %. This maximum viability is attributed to the toxicity of GLU to recombinant E. coli ; excessive GLU concentration can reduce the viability of immobilized cells. Consequently, a GLU the concentration of 5 % was selected. The impact of GLU crosslinking duration on the activity of immobilized is illustrated in Fig. 4h. The viability of the immobilized cells increased as the crosslinking time extended, peaking at 1.5 h (The specific activity was 310.73 U/g, with an activity recovery yield of 70.71%.). This enhancement is attributed to the low molecular weight of GLU and the limited availability of aldehyde groups for crosslinking with PEI, a process that is completed within 1.5 h. Beyond this duration, structural changes in the immobilized cells remained minimal, but any residual GLU in the system began to affect viability, resulting in a decline in cell activity. Therefore, a final crosslinking time of 1.5 h for GLU was established. Properties of immobilized cells In biocatalysis, the rate of catalytic reactions typically increases with increasing temperatures. However, excessively high temperatures can lead to a decline in cellular activity. The impact of temperature on the specific activity of both immobilized and free cells is illustrated in Fig. 5a. Viability for both types of cells showed an upward trend with increasing temperature, peaking at 50 ℃. Beyond this point, viability for both immobilized and free cells began to decrease. The optimal temperature for cell activity, whether immobilized or free, was determined to be 50 ℃. The effect of temperature on the stability of free and immobilized cells is depicted in Fig. 5b. Following a 12-hour incubation at 45 ℃ , the activities of immobilized cells and free cells were 90.15 % and 82.50 % of their initial activities, respectively, indicating minimal activity loss and good stability. After the same duration at 50 ℃ , the activities decreased to 81.29 % and 65.59 % for immobilized and free cells, respectively. Subsequent incubation at 55 ℃ resulted in further activity decline, with immobilized and free cells reaching 26.60 % and 12.56 % of their initial activities. Immobilization enhanced thermal stability, with diatomite providing additional cellular protection. Optimal stability for both cell types was observed at 45 ℃ , which was consequently chosen as the preferred reaction temperature. The effect of temperature on the stability of free and immobilized cells is depicted in Fig. 5b. After a 12 h incubation at 45 ℃ , the activities of immobilized cells and free cells were 90.15 % cells achieving 26.60 % and 12.56 % of their initial activities. Immobilization enhanced thermal stability, with diatomite providing and 82.50 % of their initial activities, respectively, indicating minimal activity loss and good stability. Following the same duration at 50 ℃ , the activities decreased to 81.29 % and 65.59 % for immobilized and free cells, respectively. Subsequent incubation at 55℃ resulted in a further decline in activity, with immobilized and free cells additional cellular protection. Optimal stability for both cell types was observed at 45 ℃ , which was subsequently chosen as the preferred reaction temperature. In biocatalytic reactions, variations in the pH of the reaction solution lead to the dissociation of specific groups in aspartate lyase to varying degrees, which affects their substrate binding capacities and results in differing activities at different pH levels. The impact of pH on the specific activity of both immobilized and free cells is illustrated in Fig. 5c. Viability for both types of cells exhibited a gradual increase with rising pH. The peak viability for immobilized cells was observed at pH 9.5, followed by a decline with further increases in pH. Conversely, free cell activity reached its maximum at pH 10.0, decreasing beyond this point. The optimal pH was determined to be 9.5 for immobilized cells and 10.0 for free cells. The effect of pH on cell stability before and after immobilization is shown in Fig. 5d. Following a 12 h storage in Tris-HCl buffer at pH 8.0, both immobilized and free cells exhibited higher viability compared to those stored in other buffers, with free cells demonstrating superior pH stability. Immobilized cells displayed robust stability within the pH range of 8.0 to 9.5, retaining over 85 % of their activity. Overall, pH stability increased following immobilization likely due to the protective cross-linking network formed by PEI and GLU, which shielded the cells and minimized environmental influences. Kinetic Properties Investigating kinetic parameters is essential for assessing the efficiency of an immobilization process. The kinetics of the catalytic reaction were analyzed, and the Michaelis-Menten parameters were determined using a Lineweaver-Burk plot. Fig. 6 depicts the resultant fitted curves, revealing a K m of 636.93 mmol/L and V max of 99.66 U/g for free cells, and a K m of 748.13 mmol/L and V max of 43.55 U/g for immobilized cells. The K m value for immobilized cells increased by a factor of 1.17, primarily due to the heightened mass transfer resistance induced by the mesh structure within the immobilized cells. Storage stability and reusability Immobilized and free cells were stored at 4 °C for 60 days to evaluate changes in activity (Fig. 7a). Immobilized cells remained above 93 % for the first 30 days, demonstrating a 64 % higher residual viability compared to free cells. By day 60, immobilized cells retained a viability of 72.29 %, while free cells only reached 14.26 %. Immobilized cells exhibit significantly enhanced storage stability, effectively preserving cell activity during storage. Systematic evaluation of biocatalyst recyclability revealed that the immobilized cell system maintained excellent operational stability during repeated-batch β-alanine synthesis in Fig. 7b. The initial 6-hour conversion cycle achieved near-quantitative yield (96.6 ± 1.2%), with subsequent cycles showing minimal biocatalyst deactivation (0.89 ± 0.15% per cycle), ultimately retaining 80.06% production yield and 86.26% initial activity after 10 consecutive batches. These results demonstrate exceptional sustainability for potential industrial implementation. In contrast, batches using free cells achieved only around a 10 % yield after four consecutive reactions. Diatomite-immobilized cells demonstrate superior reusability, offering promising prospects for industrial applications. β-alanine synthesis in fixed bed bioreactor To achieve continuous production, the immobilized cells were placed in a fixed bed reactor. This method eliminates the need for mechanical stirring and prevents direct contact between the enzyme and acidoid, thereby ensuring the overall stability of the whole cell. The effects of acrylic acid concentration and volumetric flow rate on the continuous production yield of β-alanine were systematically evaluated in a fixed-bed bioreactor packed with immobilized E. coli cells. The experimental results are shown in Fig. 8a. When the concentration of acrylic acid is 2 mol/L, the optimal flow rate is 0.03 mL/min, resulting in a space-time yield of 193.08 g·L -1 ·h -1 . At acrylic acid concentrations of 1.5 mol/L and 2 mol/L, the space-time yield remians the same at the optimal flow rate. Higher concentrations of acrylic acid and aqueous ammonia can cause damage to the cells. Therefore, the subsequent synthesis of β-alanine was carried out in a fixed-bed reactor under the conditions of 1.5 mol/L acrylic acid concentration and a flow rate of 0.04 mL/min. The ability of immobilized cells to continuously synthesize β-alanine was investigated in the fixed-bed reactor under optimal conditions for 360 h, as shown in Fig. 8b. In the fixed-bed reactor, the immobilized cells demonstrated stable synthesis of β-alanine, achieving a yield of over 71.30 % after 360 h of continuous reaction. Conclusion In this study, a straightforward method for preparing immobilized cells was devised. Diatomite served as the carrier, facilitating the crosslinking of whole-cell catalysts with PEI and glutaraldehyde. The immobilized cells enhanced thermal and pH stability, while retaining retained high enzyme activity. Furthermore, the immobilized cells have shown favourable reusability , which is a prerequisite for applications in industrial continuous production. The performance of the immobilized cells was investigated in both batch reactor and fixed bed bioreactors. The immobilized cells demonstrated stable synthesis of β-alanine, with a yield of over 71.30 % after 360 h of continuous reaction. An assessment of the immobilized whole-cell catalyst highlighted its exceptional reusability and robust storage stability. This method presents a viable and economical approach to preparing whole-cell catalysts for industrial applications of β-alanine. Experimental Section Materials and Strain The experimental strain E. coli BL21 (DE3)/pET-30a (+)-HAMase was obtained from Anhui Pengpai Biological Co., Ltd. (Anhui, China). Yeast maceration powder and tryptone were purchased from Beijing Auboxing Biotechnology Co. Glycerol and isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from Tianjin Dingguo Biological Co. Dipotassium hydrogen phosphate and potassium dihydrogen phosphate were purchased from Tianjin Komeo Chemical Reagent Co. Ammonia, acrylic acid, acetonitrile, ammonium persulfate, sodium dodecyl sulfate (SDS), tris (hydroxymethyl) aminomethane (Tris), diatomite, polyethyleneimine, glutaraldehyde , potassium hydroxide, Tetrakis(hydroxymethyl) phosphonium chloride (THPC), poly(vinyl alcohol) (PVA), p-phenylene dichalcogenide (p-phenylene dichalcogenide) were purchased from Aladdin Technology (Shanghai) Co. Concentrated hydrochloric acid was purchased from Huanghua Century Kobo Technology Development Co. Among these, acetonitrile was chromatographically pure, diatomite consisted of calcined products, and the others were analytically pure. Preparation of whole-cell biocatalysts The preserved recombinant strains were cultured in 10 mL of LB medium for 14 h using a volume of 10 microliters. Subsequently, the cultures were transformed by transferring 500 μL into 50 mL of TB and cultivated at 37 ℃. Upon reaching an OD 600 of 0.6-0.8, protein overexpression was induced by adding IPTG to achieve a final concentration of 0.3 mM. Strains were cultured at 25 ℃ for another 20 h. After the cultivation and induction, the cells were collected by centrifugation at 8000 g and 4 ℃ for 6 min. The cells were washed once with 50 mM Tris-HCl buffer (pH 7.5). Whole-cell catalysts were obtained. The cells were homogenized using a high-pressure homogenizer, and the resulting supernatant was centrifuged at 4 ℃ and 12000 r/min for 10 min to obtain the crude enzyme solution. The crude enzyme solution was subsequently analyzed using SDS-PAGE. Whole-cell catalyst activity assay The reaction system was supplemented with 2 mL of 600 mM acrylic acid and 600 mM ammonia. The pH was then adjusted to 10.0 using HCl. The appropriate whole-cell catalyst was added, and the reaction was conducted at 50 ℃ for 30 min. The reaction was terminated by heating at 95 ℃ for 5 min. β-Alanine was analyzed using HPLC. One unit of activity is defined as the amount of enzyme that produces 1 μmol of β-alanine per minute. The unit of activity per gram of wet cells is defined as the specific activity (U/g cell ). In a 20 mL reaction system, 1 mol/L acrylic acid and 1.5 mol/L ammonia were added, and the pH of the reaction system was adjusted to 9.5 with concentrated hydrochloric acid recombinant E. coli cells (5 g/L, wet weight) we re added, and the reaction was carried out at 50 ℃ for 24 h with constant temperature shaking. The resulting β-alanine analyzed using high-performance liquid chromatography (HPLC). Preparation of immobilized whole-cell To prepare a ready-to-use THP aqueous solution (30 %), 12 g of THPC was added to 90 mL of ultrapure water, followed by the addition of a solution containing 3.4 g of KOH. The two solutions were mixed at room temperature (crosslinking agents is GLU, THP, PVA and TPAL with the same volume fraction). In a separate step, a mixture of recombinant E. coli (cell loading is 4 g, 5 g, 6 g, 7 g and 8 g) and 0.3 g of diatomite (particle size of DE is 9.6 μm, 19.6 μm, 25.4 μm, 29.3 μm, 36.8 μm and 40.0 μm) was combined with 50 mL of buffer. While stirring, 1 mL of PEI (volume fraction of PEI is 4 %, 6 %, 8 %, 10 %, 12 %, and 14 %, molecular weight of PEI is 600 Da, 1800 Da, 10 000 Da and 70000 Da) with a volume fraction of 8 % was added, and the mixture was allowed to undergo flocculation (flocculation time is 0 h, 0.5 h, 1 h, 1.5 h, 2 h, and 2.5 h). Subsequently, a GLU aqueous solution (concentrations is 2.5 %, 5 %, 7.5 %, 10 %, 12.5 %, 15 % and 20 %) was added, and the cross-linking reaction was allowed to proceed for an additional (crosslinking times is 1 h, 1.5 h, 2 h, 2.5 h, 3 h and 3.5 h). The resulting mixture was then filtered, and the collected precipitate was washed three times with Tris-HCl buffer. Following this, the precipitate was shaped into long strips, air-dried at room temperature, and finally pulverized into granules. Analysis methods Cell density was determined by measuring the optical density of culture samples at 600 nm. The concentration of β-alanine in the supernatant was analyzed using HPLC equipped with an amino column (4.6 mm × 250 mm, 5 μm) at 205 nm. The mobile phase consisted of 50 mmol/L potassium dihydrogen phosphate (pH 4.5)-acetonitrile 30:70 (v/v), with a flow rate of 1.0 mL/min. The column temperature was maintained at a 30 ℃. Error bars were calculated using the standard deviation (STDEV) from triplicate experiments. Declarations Supporting Information Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 22308083 and 22178083), the National Key Research and Development Program of China (2023YFA0914500), the Natural Science Foundation of Hebei Province (B2022202014), Baoding science and technology plan project (2494F016) References M. Könst M C R F, Elinor L. Scotta,Johan P. M. Sandersa (2009) A study on the applicability of L-aspartame α-ddeclaratoryin the bboastedproduction of nitrogen containing chemicals. Green Chem. 11:1646-1652. https://doi.org/10.1039/b902731a Coxon K M, Chakauya E, Ottenhof H H et al (2005) Pantothenate biosynthetic routes in higher plants. Biochem. Soc. Trans. 33:743-746. https://doi.org/10.1042/Bst0330743. Jang Y-S, Kim B, Shin J H et al (2012) Bio-based production of C2–C6 platform chemicals. Biotechnol. Bioeng. 109:2437-2459. https://doi.org/10.1002/bit.24599. Ford J H, Buc S R, Greiner J W (1947) An Improved Synthesis of β-Alanine. III. The Addition of Ammonia to Acrylonitrile at 50-150°. J. Am. Chem. Soc. 69:844-846. https://doi.org/10.1021/ja01196a029. Ji X, Luo (2005) Synthesis and Application of β-Alanine. Amino Acids & Biotic Resources. Liang L-Y, Zheng Y-G, Shen Y-C (2008) Optimization of β-alanine production from β-aminopropionitrile by resting cells of Rhodococcus sp. G20 in a bubble column reactor using response surface methodology. Process Biochemistry.43:758-764. https://doi.org/10.1016/j.procbio.2008.03.002 Zhang T, Zhang R, Xu M et al. (2018) Glu56Ser mutation improves the enzymatic activity and catalytic stability of Bacillus subtilis L-aspartate α-decarboxylase for an efficient β-alanine production. Process Biochemistry. 70:117-123. https://doi.org/10.1016/j.procbio.2018.04.004. Li S, Fu W, Su R et al (2022) Producing malonate inSaccharomyces cerevisiaevia the β-alanine pathway. Syst Microbiol and Biomanufacturing. 3:328-338. https://doi.org/10.1007/s43393-022-00113-8 Li R, Wijma H J, Song L et al (2018) Computational redesign of enzymes for regio- and enantioselective hydroamination. Nat. Chem. Biol. 14:664-670. https://doi.org/10.1038/s41589-018-0053-0. Norman S J, Reeves D J, Saum L M (2021) Use of Pamidronate for Hypercalcemia of Malignancy in Renal Dysfunction. J. Pharm. Pract. 34:553-557. https://doi.org/10.1177/0897190019883162. Cerón-Carrasco J P, Jacquemin D (2021) Using Theory To Extend the Scope of Azobenzene Drugs in Chemotherapy: Novel Combinations for a Specific Delivery. ChemMedChem. 16:1764-1774. https://doi.org/10.1002/cmdc.202100046. Liu CJ, Tsai YS, Huang HS (2022) Atorvastatin Decreases Renal Calcium Oxalate Stone Deposits by Enhancing Renal Osteopontin Expression in Hyperoxaluric Stone-Forming Rats Fed a High-Fat Diet. Int. J. Mol. Sci. 23 :3048. https://doi.org/10.3390/ijms23063048. Kumar A, Shariff M, Doshi R (2020) Impact of rosuvastatin versus atorvastatin on coronary atherosclerotic plaque volume-a systematic review and meta-analysis with trial sequential analysis of randomized control trials. Eur. J. Prev. Cardiol. 27:2138-2141. https://doi.org/10.1177/2047487319868035. Jayasena A, Atapattu N, Lekamwasam S (2015) Treatment of glucocorticoid-induced low bone mineral density in children: a systematic review. Int. J. Rheum. Dis. 18:287-93. https://doi.org/10.1111/1756-185x.12560. Milioni F, de Poli R A B, Saunders B et al (2019) Effect of β-alanine supplementation during high-intensity interval training on repeated sprint ability performance and neuromuscular fatigue. J. Appl. Physiol. 27:1599-1610. https://doi.org/10.1152/japplphysiol.00321.2019. Zhang Y, Zhang X, Wu M et al (2018) Application of Diatomite in New Energy Field. Materials China 37:331-340. https://doi.org/10.7502/j.issn.1674-3962.2018.05.02. Marques T M F, Sales D A, Silva L S et al (2020) Amino-functionalized titanate nanotubes for highly efficient removal of anionic dye from aqueous solution. Appl. Surf. Sci. 512:145659. https://doi.org/10.1016/j.apsusc.2020.145659 Jin L-Q, Chen X-X, Jin Y-T et al (2021) Immobilization of recombinant Escherichia coli cells expressing glucose isomerase using modified diatomite as a carrier for effective production of high fructose corn syrup in packed bed reactor. Bioprocess. Biosyst. Eng. 44:1781-1792. https://doi.org/10.1007/s00449-021-02560-4. Zhang X-H, Liu Z-Q, Xue Y-P et al (2018) Production of R-Mandelic Acid Using Nitrilase from Recombinant E. coli Cells Immobilized with Tris(Hydroxymethyl)Phosphine. Appl. Biochem. Biotechnol. 184:1024-1035. https://doi.org/10.1007/s12010-017-2604-3. Hu Z, Zheng S, Jia M et al (2017) Preparation and characterization of novel diatomite/ground calcium carbonate composite humidity control material. Adv. Powder Technol. 28:1372-1381. https://doi.org/https://doi.org/10.1016/j.apt.2017.03.005. Benayache S, Alleg S, Mebrek A et al (2018) Thermal and microstructural properties of paraffin/diatomite composite. Vacuum. 157:136-144. https://doi.org/10.1016/j.vacuum.2018.08.044 Zhao X, Che Y, Mo Y et al (2021) Fabrication of PEI modified GO/MXene composite membrane and its application in removing metal cations from water. J. Membr. Sci. 640:119847. https://doi.org/https://doi.org/10.1016/j.memsci.2021.119847. Chen J, Zheng Y-G, Shen Y-C (2008) Biosynthetic routes of p-methoxyphenylacetic acid from p-methoxyphenylacetonitrile by immobilized Bacillus subtilis ZJB-063. Process Biochem. 43:978-983. https://doi.org/https://doi.org/10.1016/j.procbio.2008.05.002. H Y-F, W J-F, Liu M et al (2016) Preparation and performance optimization of PVA/PEI composite nanofiltration membrane. Membr. Sci. Tech Supplementary Files 8.18SupportingInformation.docx Cite Share Download PDF Status: Published Journal Publication published 06 Mar, 2026 Read the published version in Biotechnology Letters → Version 1 posted Reviewers agreed at journal 16 Sep, 2025 Reviewers invited by journal 11 Sep, 2025 Editor assigned by journal 08 Sep, 2025 First submitted to journal 06 Sep, 2025 Editorial decision: Major revisions 28 Aug, 2025 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. 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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-7412633","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":513575922,"identity":"6e352647-1377-4abc-b555-aa56a4d0f866","order_by":0,"name":"Li 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09:40:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":251890,"visible":true,"origin":"","legend":"\u003cp\u003e(a) SEM image of free cells ; (b) SEM image of immobilized cells.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7412633/v1/c55d2a29e001ba6a04d9085c.png"},{"id":91837637,"identity":"1c58092a-07ec-4d7f-860a-21e0375ff638","added_by":"auto","created_at":"2025-09-22 09:40:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":18556,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR spectra of \u003cem\u003eE. coli\u003c/em\u003e, DE, \u003cem\u003eE. coli\u003c/em\u003e@ DE/PEI/GLU.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7412633/v1/85c619f12f2fae3924a2bc2b.png"},{"id":91837628,"identity":"ec1c4bed-b0ef-42ee-a1ec-913d97fc9b5c","added_by":"auto","created_at":"2025-09-22 09:40:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":187546,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Effect of crosslinking agents on the operational stability; (b) Effects of different molecular weights of PEI; (c) effects of different molecular weights of different particle sizes; (d) effects of different molecular weights of different cell loading; (e) effects of different molecular weights of \u0026nbsp;different PEI volume fractions; (f) effects of different molecular weights of different PEI flocculation time; (g) effects of different molecular weights of different GLU concentrations; (h) effects of different molecular weights of and different GLU crosslinking times on the activity of immobilized cells.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7412633/v1/65b10cc8ded3b1bce9d2a7ba.png"},{"id":91837638,"identity":"bd4f418d-6099-4df6-8b0e-9f5d68f2a595","added_by":"auto","created_at":"2025-09-22 09:40:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":93268,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Optimum temperature of free cells and immobilized cells; (b) thermal stability of free cells and immobilized cells; (c) optimum pH of free cells and immobilized cells; (d) pH stability of free cells and immobilized cells.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7412633/v1/dc0ea0cf65ce4c4a615fd478.png"},{"id":91837636,"identity":"20666635-d3b7-425c-9766-2225f7a03cba","added_by":"auto","created_at":"2025-09-22 09:40:47","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":22227,"visible":true,"origin":"","legend":"\u003cp\u003eKinetic curves of free cells and immobilized cells.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7412633/v1/63d5ee2bc4d2b9e0b02a9ce4.jpeg"},{"id":91837629,"identity":"c2973cd6-e395-47f6-95c0-e54824bf4d5f","added_by":"auto","created_at":"2025-09-22 09:40:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":44563,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Storage stability of free cells and immobilized cells; (b) reusability of immobilized cells.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7412633/v1/145a8fa54e9dab732a600057.png"},{"id":91837640,"identity":"fc91b225-2f65-4a35-9c2b-9fdcfd87145f","added_by":"auto","created_at":"2025-09-22 09:40:51","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":41764,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Effects of fow rate and substrate concentration on continuous synthesis of β-alanine in fixed bed bioreactor; (b) continuous synthesis of β-alanine in fixed bed bioreactor.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7412633/v1/589b02c3a8871d2001b291f7.png"},{"id":104250649,"identity":"548a550a-1e78-42e0-a65b-f902506312e5","added_by":"auto","created_at":"2026-03-09 16:04:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1408906,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7412633/v1/20cfc58f-d127-4299-aa24-2b004bee91e0.pdf"},{"id":91837635,"identity":"1e986f97-5c0d-4e25-a350-2e156edd7391","added_by":"auto","created_at":"2025-09-22 09:40:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1016821,"visible":true,"origin":"","legend":"","description":"","filename":"8.18SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7412633/v1/020268b9e02df072dbe78bb8.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eImmobilization of hydroaminase-expressing recombinant \u003cem\u003eEscherichia coli\u003c/em\u003e whole-cell biocatalysts for the production of β-alanine\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eβ-Alanine, the only naturally occurring β-amino acid, plays pivotal physiological roles in living organisms and serves as a key precursor for synthesizing nitrogen-containing bioactive compounds. β-Alanine serves as a crucial precursor for the production of diverse nitrogen-containing chemicals and plays an essential role as an intermediate in synthesizing compounds such as acrylamide, acrylonitrile, pantothenic acid, myostatin, pamidronate, and balsalazide. These compounds have extensive applications in various fields including drug synthesis, fine chemicals, environmental applications, food, and feed additives (M. Könst 2009, Coxon et al. 2005) . Due to its versatility, β-alanine is considered one of the 12 most promising three-carbon compounds for future development (Jang et al. 2012) .\u003c/p\u003e\n\u003cp\u003eThe current industrial production of β-alanine primarily involves the ammonolytic hydrolysis of acrylonitrile and the β-aminopropionitrile hydrolysis. However, the production method faces several limitations: the use of strong acids and bases necessitates corrosion-resistant equipment. High temperature and pressure conditions lead to increased energy consumption and production costs, the production of nitrile, among other by-products, poses potential toxic effects on organisms and the environment (Ford et al. 1947, Ji and Luo 2005)\u0026nbsp;.The biosynthetic routes of β-alanine has\u0026nbsp;attracting growing interest in recent years due to its environmental friendliness, mild reaction conditions, and minimal by-product formation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNitrile hydrolases, primarily sourced from (Liang et al. 2008)\u0026nbsp;\u0026nbsp;Compared with chemical synthesis, the conversion method of nitrile hydrolase is milder, but Substrate inhibition at elevated concentrations of β-aminopropionitrile can inhibit nitrile hydrolase, thereby affecting the synthesis efficiency of the product. The production of by-products also brings inconvenience to separation and purification, which cannot meet the requirements of industrial production. The aspartic acid decarboxylase conversion method utilizes aspartic acid-α-decarboxylase, or aspartate lyase combined with PanD, to catalyze the production of β-alanine from aspartic acid. PanD is a key enzyme in the synthesis of β-alanine in organisms, which can efficiently catalyze the cleavage of the C α carboxyl group in L-aspartic acid (L-Asp) to generate β-alanine.At present, researchers mostly screen for PanD in prokaryotes such as \u003cem\u003eEscherichia coli\u003c/em\u003e (Zhang et al. 2018)\u0026nbsp;, \u003cem\u003eBacillus subtilis\u003c/em\u003e, \u003cem\u003eCorynebacterium glutamicum\u003c/em\u003e, as well as some pathogenic bacteria such as \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e and \u003cem\u003eHelicobacter pylori\u003c/em\u003e. The synthesis of β-alanine by aspartate decarboxylase conversion method has a simple reaction mechanism and high yield, but the purification of AspA and PanD requires expensive costs and is not suitable for large-scale production. Therefore, the synthesis of β-alanine by biotransformation mainly relies on whole cell transformation. In addition to using L-aspartic acid as a substrate, Lou Jian\u0026nbsp;(Li et al. 2022)\u0026nbsp;used acrylic acid as a substrate and utilized an acrylic acid aminase to catalyze the synthesis of β-alanine from acrylic acid. Acrylic acid is relatively inexpensive and structurally similar to β-alanine. Using acrylic acid as a substrate to produce β-alanine can complete the reaction in a short period of time, reducing the loss of raw materials. Theoretically, it does not produce harmful substances, is environmentally friendly, and has significant economic benefits. It is worth further exploration. The one-step generation of β-alanine from acrylic acid and ammonia belongs to the hydroamination reaction, which is the β-enantioselective addition of ammonia to unsaturated acids.\u0026nbsp;performed molecular redesign of aspartate decarboxylase from Bacillus subtilis and successfully incorporated it into \u003cem\u003eEscherichia coli\u003c/em\u003e, resulting in the development of an engineered strain capable of synthesizing β-amino acids efficiently. The biocatalytic system facilitates the synthesis of β-amino acids under mild conditions, utilizing cost-effective substrates and aqueous ammonia in a single-step process (Li et al. 2018) \u0026nbsp;catalyst preparation process\u0026nbsp;(Norman et al. 2021, Cerón-Carrasco and Jacquemin 2021, Liu et al. 2022, Kumar et al. 2020, Jayasena et al. 2015)\u0026nbsp;. The selection of the carrier significantly impacts catalytic performance. Consequently, a wide range of polymeric, organic, and inorganic materials has been investigated as carriers. In particular, inorganic materials have garnered attention due to their mechanical stability, resistance to microbial corrosion, modifiability and reusability\u0026nbsp;(Milioni et al. 2019)\u0026nbsp;. Diatomite (DE), composed of amorphous silica with abundant siloxanes, features a highly porous surface and exhibits high mechanical strength, chemical stability, and biocompatibility. Additionally, it is cost-effective and readily available\u0026nbsp;(Zhang et al. 2018)\u0026nbsp;. However, direct cell adsorption onto diatomite is susceptible to shedding. Polyethyleneimine is synthesized through the polymerization of cyclic ethylamine, resulting in a polymer with repeating -CH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eNH\u003csub\u003e2\u003c/sub\u003e units\u0026nbsp;(Marques et al. 2020)\u0026nbsp;. Cross-linking agents react with the primary amine groups present in polyethyleneimine (PEI) to enhance the stability of immobilized cells. Jin et al. prepared immobilized \u003cem\u003eE. coli\u003c/em\u003e by modifying diatomite with PEI and utilizing glutaraldehyde\u0026nbsp;\u0026nbsp;(GLU)\u0026nbsp;as a cross-linking agent\u0026nbsp;(Jin et al. 2021)\u0026nbsp;. The reusability of the immobilized cells showed significant improvement. Hydroxymethylphosphine\u0026nbsp;(THP) employs a nonessential NH\u003csub\u003e2\u003c/sub\u003e group to mitigate enzyme inactivation during immobilization while also forming a stable P-CH\u003csub\u003e2\u003c/sub\u003e-N linkage.\u0026nbsp;(Zhang et al. 2018)\u0026nbsp;\u0026nbsp;immobilized recombinant \u003cem\u003eE. coli\u003c/em\u003e cells using DE/PEI/THP. The immobilized cells exhibited superior thermal stability and reusability compared to free cells, making them suitable for the industrial production of R-mandelic acid.\u003c/p\u003e\n\u003cp\u003eIn this study, We demonstrate that hydroaminase (HAMase), a recombinant enzyme, can effectively catalyze the production of β-alanine. Diatomite was employed as a carrier to immobilize recombinant \u003cem\u003eEscherichia coli\u003c/em\u003e whole cells, resulting in immobilized cells that Enhanced operational stability, mechanical robustness, and reusability. Fig. 1 illustrates preparation process of immobilized \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) /pET-30a (+) -AspB whole-cells.This method facilitated the immobilization of whole cells through a streamlined synthetic procedure while preserving high enzymatic activity. Finally, we assessed the production capacity for the continuous synthesis of β-alanine using immobilized cells in a fixed-bed bioreactor. This approach presents a promising candidate for whole-cell immobilization in various applications.\u0026nbsp;\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003eCell immobilization and characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe scanning electron microscopy (SEM) micrographs in Fig. 2a demonstrate that \u003cem\u003eE. coli\u003c/em\u003e particles exhibit a rod-like morphology with smooth surfaces, measuring between 0.5 \u0026mu;m and 2 \u0026mu;m in length. Fig. 2b present SEM images of DE/PEI/GLU-immobilized \u003cem\u003eE. coli\u003c/em\u003e, where the immobilized \u003cem\u003eE. coli\u003c/em\u003e cells exhibit uniform particle sizes, characterized by membrane-coated surfaces and tightly interconnected cellular junctions, with no noticeable structural damage. This observation suggests that following diatomite adsorption, glutaraldehyde crosslinked with PEI, leading to the changes in cell structure.\u003c/p\u003e\n\u003cp\u003eFT-IR analysis was performed to confirm the successful immobilization of diatomite and cells using GLU as a crosslinking agent. Fig. 3 illustrates the spectra of free cells, diatomite, and diatomite-immobilized cells. Upon cell immobilization, several distinct peaks emerged in the spectrum, differentiating it from those of \u003cem\u003eE. coli\u003c/em\u003e and diatomite alone. Specifically, peaks at 792, 617, 471, and 1090 cm\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003ewere attributed to Si-O stretching vibrations, Si-O-Si symmetric stretching vibrations, Si-O bending vibrations, and the binding vibration of calcite to CO₃\u003csup\u003e2-\u003c/sup\u003e in diatomite, respectively. Furthermore the peak at 3305 cm\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003ecorresponded to the stretching vibrations of -OH and -NH, indicating the presence of these functional groups\u0026nbsp;(Hu et al. 2017, Benayache et al. 2018)\u0026nbsp;. Additionally, the peaks at 1656 cm\u003csup\u003e-1\u003c/sup\u003e and 1537 cm\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003erepresented the bending vibrations within the N-H plane of primary and secondary amines.\u003c/p\u003e\n\u003cp\u003eUpon treatment with GLU and PEI as cross-linking agent, the stretching vibrations of the C-H bond at 2927 cm\u003csup\u003e-1\u003c/sup\u003e, bending vibrational peak of CH\u003csub\u003e2\u003c/sub\u003e at 1393 cm\u003csup\u003e-1\u003c/sup\u003e, in-plane rocking absorption peak for CH\u003csub\u003e2\u003c/sub\u003e in diatomite immobilized cells at 1230 cm\u003csup\u003e-1\u003c/sup\u003e, which can be attributed to PEI and GLU (Hu et al. 2017)\u0026nbsp;. Additionally, a new stretch at 1453 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e can be assigned to stretching vibration absorption peaks of -C=N-. These peaks in the spectra (1230, 1393, 1453 and 2937 cm\u003csup\u003e-1\u003c/sup\u003e) indicate the successful immobilization of diatomite and cells using GLU as a cross-linking agent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOptimization of parameters for cell immobilization by diatomite\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe use of diatomite as an immobilization matrix presents several advantages, including its high resistance to dissolution and exceptional adsorption capacity. However, relying solely on its adsorption properties for cell immobilization can result in challenges such as cell dislodgement. To improve the stability of immobilized cells, we investigated the efficacy of various cross-linking agents in forming covalent bonds with the cells. The effects of different types of cross-linker on enzyme activity recovery is presented in Table 1, while their impact on reusability is illustrated in Fig. 4a.\u003c/p\u003e\n\u003cp\u003eThe enzyme activity recoveries of the immobilized cells were found to be similar across all four cross-linkers. However, when polyvinyl alcohol and p-phenylenedial were employed as cross-linking agents, the reusability of the immobilized cells was considerably poor, with yields of \u0026beta;-alanine amounting to only 25.15 % and 17.55 % after three recycling cycles, respectively. In contrast, the use of GLU as a cross-linking agent resulted in a yield of 75.88 % after six recycling cycles. This enhanced reusability can be attributed to the utilization of free amino groups by GLU for cross-linking, which minimizes the impact on the activity. Consequently, GLU was selected as the preferred cross-linking agent for subsequent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003eEffect of crosslinking agents on enzyme activity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg 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Ha5ECXjbt29PZs2alU7pwZvcCDS8dchHfnZ1XYQFUfKjEZH/BrZG1HO1yg8V3ibHPwrx8T+jfTS0okFTFl2ltlBlQ4uIFGKNg7Iaxdg0Gs4wnft73Hu0e5Hc92OYNNwbZDrz2L1NY2msIRDzM86/d+qrdk/UXyZ90lA2f5nck7QyVbtPynykC+8nN4q8iqBctg19jCMPys/navdUiy5LmqctLSKFcHLn5GxdGAzBSd1aoxLEwla8BC0LvuErQg3DjGc66cgvL1gYlpUXEG2Zlh8Njywt+TLO7/KwLpQlpmrLA9vBWkZnYZptb9JmqbUMiUuvKRQRKYCGQjyb6zeEiqHMqllV+7aegqqISA3cG+Y/R+U1empWGcFPAbV/qKGSiEgGGlARmHhN4hNPPJFs2LAhnRJfGcFPAbV/KKiKiGSYMGGC+5/H2LlzZ/RqXxmcVP0rIiISia5URUREIlFQFRERiURBVUREJBIFVRERkUgUVEVERCJRUBUREYlEQVVERCQSBVUREZFIFFRFREQiUVAVERGJpK7XFPJyaRERkcEoxlt7daUqIiISiV6oLyIiEomuVEVERCJRUBUREYlEQVVERCQSBVUREZFIFFRFREQiUVAVERGJREFVREQkEgVVERGRSBRURUREIlFQFRERiURBVUREJBIFVRERkUgUVEVERCJRUBUREYmkLYPq9evXk61btybjx49Pzp49m45tf8eOHUsWLVqUzJ07Nx2T7datW279Ro0alY4ppmj+zWi0bEXs2bMnuXz5cjrU3jjuKK+ISD1aFlQtIHCy7ujocB3DBw8edEHUD573799PLl68mFy7di0d0/4IRjh06JDrV7Nv377ks88+S+7cuZOOqa2e/JvRSNlqoezs62nTpiUTJ05Mxz5Qz7Fh08MuCz8OstKGP9TC6fxomT59uivvypUr01TiY9+E243tbfjxFE73u1o4JvhRTVr6RX7gUCb/OBLpF/yT8jLdvHmze+HChfwj9O4tW7Z0X7p0KZ3S7T7703wMM/7MmTPpmPocPXq04XmbQZnnzJmTDuWz9atX0fyb0WjZ8rCP2R+hRo+N3bt3u/Hjxo3rrvzwSsdmYxlsL9KTXx7KRxq/DGDZK1asSIfEdHZ2uu3qd2xrwzZje7P9/I501fYD2Bek4fvLZ5bFvsk6huDvY5bbH997EVN6ULWD/cCBA+mYh/FF4AvnY5j5Gv2CsFwF1cY0WrYs7HdOilkaPTbYr/VsB1ufMJ9QXn4E77wT+lDE/uKHTR5+6OTtU46FavsbYd7kx/7LCsYEVPIcOXLkQz+IRPpDqdW/VNmcOHEiqZwYXbVMnvfffz957LHH0qHm2XKl/1F9umDBgnTogf46NhpROZknb775Zjo0tFGVv27duuS9995z+y2rzcPYsWMz9ylV+RcuXEhmzZqVjsm2fPny9FMP8kPlx43r+1555RWX5+nTpzNvLYi0WqlB9cMPP3T98EsSGj16dNUTK/z7MfZF9u+Z2f2ctWvXJq+//rr7PGPGjD7p/fs0dCzTbzhj92S4p8Z40nJ/hpMBJxMChM3LNP8eUi2k9e8Rcc84xDIov90Tmjx5cuZJC355yM/Ww98mdH6jpqzthdhlM2xP7s0+99xz6ZgHYh4bZXv66afd/f1a6zsUnDt3LqlcFbr9yv19vmMchxwftZw6dcr9iGKf1oPtzjLfeuutdEwPji/7YaaAKm0jvWKNjqoYsm90EVZl51fhUv0TjqMqiXF+1V7WvFZlaFVLNmxVSlQj2Tiqk8iDzu7bUTXIZ9LRWVnCKifGhdWIVGFSPWXlsXuCdD6Wa+VjmQyThs+GYcZ3dXW5Kkk68qZsxvInXYiy+dVrMcsWIm/SsL18zR4btp/yqmtDdjz4x0iWvPxseWxzeYDvHscd24Z9XQvHTD3V6Bw37DP/+PSxv2y/2mfSaj9JfyotqNqJiC6PTfc7+/LYidD/MmWNs+X4J8ysdHyZGecHQb6I4YmUNH6AMpwQ/Hs6tlx/GWCcn6elC08mFpQNJ6gwCFqZ/RMWw2E6C14+ysAJxg9ofPbXLXbZQnaiC9lys6YZm+53tq1t/nDf5bHjwT9GsuTlx3arZ3lDCduGY4PtU+2eJj++OB7rQZ7WMW94L9YfTzno7LugwCr9pSWP1ORVDVWWn1Suftznysk+qXwp3KMMZZg3b55bHtVEVBtRLZp335Uq0ND58+fdfFQFUw3629/+Np1S3f79+12f5fsmTZqUfurxzTffuHtDVj1L98ILL7hpLNMXVp89/vjj6acHKgHEVdF99NFH6Zie6rdly5alQ+WUzVfkvnY7HBu11FtdOZSwbfbu3es+V9vfR44cqbsan2OgEox7j2W/mtlud0yZMsXlSznodu7c6Y6XTZs2FaqSFomttKA6YcKE9FPPfZg8Tz75pOsTyMo+edk91e+++859UStXHumU2viC8qWePXu2u8f2l7/8JZ1SXbWg4/vll19ceTiRhN3x48fTVMXx46Hyq92dXKwMNDCZP3+++4z+Kls7HhvSOI61ytVqOpSNZ59ffPHFdKg4GimtWbOmN7D+9NNPbvyYMWNcP8vChQtd39KKtFJpQZWTICd17Nixw/X7E1eZXF19/fXX7tdsPQ0bCKhc2d6+fTu5evWq+2U8fPjwdGoxtX41P/LIIy7vLJS9EatXr3b9rq4ulwc/CKwlpa+ssuX9aGnFsUHjFvvRYK2H79696/pZ1AipeXn7m6tKvjthjUg9/B+D4BgaOXJk5nE5YsQI1x82bJjri7RSqdW/PA5BVQzVQlSZluHevXvpp+o+//xz9yVspJUgv3ip/pw5c2Y6prhnn33W9an+qoa8qeriatjHCalaMKiGAErwopUm+YYtbcsumwXwrCviso8NqrZt+VQRotrbqD755JPkpZdeSof6soBr20v64jggyOV9t7766qveH1FZitSY/N///Z/7/vq1HO+++25mq2xar3Pl3Mh3XaRp3SWjgYI1ZKj8ku3TKIaGDTQosGk+ayzjt1T1G6jwmYYnNj/prSGL34iGRgx0lh/zkI75Kid119CB9JSTjjSM47Ox1qqsB2n9/MjH1snSkS+NJkCf/Oj81rOsg5WH+Uhn24m+rZu/Xax8fv6wstj6+5iHZYfbFzHLloXtlFcusCzLl7yKHhuU1coSIk+2B/P6LC+mkbdhmYwjL3+b+kjDvH75hio7ltjOYN8ynLftwPHqb3Of7Uv/+GMZfHfDZYTb345LOktLPsyftzyRspUeVI0FIg54vkR09gXli2BfCjDO0tAxbOzkyLzkyReOLy0nesuDL5vlYSdXvmSks/xIyzQ/H1uedX4w8Jdr5bXys7xq85PWAh/zUFY65mXZhnw4mZCGtMxjJ6u8/MNx5BsiH385vhhlq8byrIblkJ/lbWWhDOGxYdNrdVkBkHHk6afjhFyrfOx7jh158D3wt12148C+d3myfrDY8UjHvH6ADdlxaemZVwFV+lMHfyoHowxS3C+dOnVq7j3RsnHPdfv27a719EBFQynu/TZzT1BEhoaWPFIj/Yf/OvP222+nQ61Ho67K1YZreT0QWQMvBVQRKUJBdRDiyopnSXmV4OHDhx9qOdlqn376afLFF1/0Pls4UFDeH3/80bUWFxEpQkF1EOLKClwh8hxpfz/jyfK54vvf//3fARNYKSetvjdu3JiOERGpTfdURUREItGVqoiISCQKqiIiIpEoqIqIiESioCoiIhKJgqqIiEgkCqoiIiKRKKiKiIhEoqAqIiISiYKqiIhIJAqqIiIikSioioiIRKKgKiIiEomCqoiISCQKqiIiIpEoqIqIiESioCoiIhKJgqqIiEgkCqoiIiKRKKiKiIhEoqAqIiISiYKqiIhIJAqqIiIikSioioiIRDLoguqtW7eSrVu3JqNGjUrHiLl+/brbNuPHj0/Onj2bjm3OsWPHkkWLFiVz585NxxR3+fLlZOXKlW23r+pZp4MHDyYdHR3RtqeIDGylBVVOSJxsws7Umt6offv2JZ999lly586ddIyY+/fvJxcvXkyuXbuWjmkOP2Bw6NAh168X5Tl58mRb7at612n48OHpJxGREoPq8ePH3cl73Lhxbnj37t1Jd3e3+4xa0xu1Zs2aZNmyZemQrF27Nv2UJBMnTkwmTZqUDjVv9OjRybx589Kh+k2fPt1dNbeTeteJtBy3rIuISKnVv2PHju09ac6fP9/1ff70J5980vUlHqok//73v6dDIiJStpbdU+UKQFqHaswlS5akQyIi0gpt11CJhjT+PVa/sYg/jc/GGt8wnj73DX00JrGGJzSOIQ2NY2i4A8Yx3fLms9/wJLz/y/KYbsNWRj8NnZ+HPz5sMERn5ScvAiKdlcnShfbs2dM7H32GQf5PPPGEq14/ceJEb74hGuTY/H41McN+l7ceeciXBkiUn/XgM+lZFvsiD9uE/UKXlY58J0+e7PIiDfnaPjT+OpGWjvJTDjsOWA773PYraRjOEpbfT8ey2W6Mt20UjsvbxoY0/jqRhmWKyADVXbI5c+ZwozQdephNP3PmTDqmu3v37t1uXGdnZzrmAdIz3axYsaJ75MiRvfPbvLbMmzdvummW35YtW1w3bty47krQ6b506ZKbZnkyjnSMO3r0qBsHy5f5zMKFC93yWQby5gVlYF7SskzmI11XV5crD9P9ceRNHnSsH52PNLZsOpv3wIEDaQp3g9ptLx/LyluuzVt0PYy/HMZbGsaxHiyLjnRhnrb/KQvLZ15bNuUwbH9/P9O37WLp6DNfmIY+01gu0ymLHQuUj3F+PmAcaUjr7wd///v70ZZZa9/6+4c8WYYt144x0orIwNSyoFqrs5OSYT5OYv7Jm8/+SY15mNc/ScNOlD6G/XkN40jvs5NzmN4PSJwcs05+VibS+hi2wI28sjOOE63PApKx8oXbhnF+mRm2YGdsHfzlWn5+mYuuB7KWwzBlIcgYlklaf/3s+PDXhW3LODsmrHzhci2d7Qcrc5iX5QOms0w/jW1f+obhcD9YYPTZ9vSXUXQbs33C/c8ySUd6ERl4Wlb9W1lWZlc5waUp+qqcfNyjFh999FE6JklOnTrVp2Xv/v37XT9srZnXwpVqOB9VeZWT10PpaUBVCbRuml/1Sctixm/atCn55ptvkp07d6ZTHqAVKGk2b97cp2ry8OHDmY21sh7JCO8/jxgxwvWtLGwHPProo67akI7PoMxF+MtlfUP1rkcWtjctjg37qRJIkgsXLqRjHvDXecyYMemnHra+YWM2qnIrP7ySXbt2uWHKXAlKrlqXaleQJmyZ+/zzz/dZ3tKlS10/bNQV7ofHH388/VRMtW1sx94LL7zQuw/pbNv8+9//dn0RGVja7p6q4WRcuTJwAcxO6uvWretzQvdP9o3gOck8eYGZ8oDnK/OWTxp+EGzbts0Ncy+P+2axGmvdvXvX9bN+pNDFUsZ6hD9sirD1zTJlypT0Uw8e1VqwYEHy6quvumVVu4drsn5UlM2OvcoVbuY+1CM6IgNT2wZVrF692vW7urrcyXH27NmZJ8BmG3aEDZt8w4YNSz89aLRy9OhRN8yVXNayKSM/CLiC4oqEHwO2LjHYlWtWUC8SRIoqaz24umzElStX0k99+fkR8KlR+Oc//+lqNRYvXuwaJhXxyCOPpJ/KZ8fVzz//7Po+9qtfQyIiA0fLgmojgc9O6rzdhmC2fPnydEqPZ5991vWPHDni+vXiaoDqSPIPAxSBlqppv/py1apVrlqaasy//OUvrqpu/fr16dS+LPjMnDnTVUnGvBqaNm2a64dBnc8//vhjOhRHM+vxww8/pJ96UD7GUSVbD6ud+PDDD13fR37vvvuu+2ytbWHBlePn22+/deNM+CPKAth//ud/un4rcFzxY4Dj2m9RDGoGdKUqMkB1l4jGFjQ2YjF+q0fDdBprMD1shGIsDxqXhGhswjQ6m5/01viFRiE0lGEcw6Tjs89vPGPTyIu01siG5VROzn0asqAS1Ny8TMtiDWD8RizGGrP4623lZJv4DWlsOf42ZJmWluWQn78OYJj1YB2tjDafv1zWk3FZ2xjV1sPmDcts+8CWY9uQ8lg6+pSRdH6DJuZhHOtkrAzkwXyWH/NbfpTP1hdsC8pl6w7yII2tC2nIw1/3WvvB3w5Z29PKWmsbW0MrykM+rC/Tw8ZLIjJwlBZU7aQadqbWdB8ns6ygDE6AdrLj5MSJyU5O1vIzXEYYHBi2kzsd+fkneb+sNi/LsHF0pAmRByfmULjuDOeVMxznBxo+kz/jKb9fZrD+bBPSMC1rueF60IXy1iOvzCBvymRBh47t6gd9fz4622/+OIYNQcrW1wKRH/RYNstkmqUhwPlpGE85bDlhmnr2Q1jWvPJX28YEUDv2WLe847zd2A8Cv2M9fbY/bDqf7fiohf3BfrF9Ge5rX5GyiLRKB38qB2Hbospw6tSpydWrV9MxAwcP8j/99NN1V3e2m0bWw142QcOhdkIL28oJ11UNS+OyGqx9+eWXveOo0n7mmWfcO73ttg0vJ3n99deTyo+0PrdVsthLULg1QxsCO57Onz/v+r5aZRFpKRda2xi/OP1qtIGCX9VZV3cDTaPrwRUaXbvhkNdVTHO4Mqz1neQqkyvTEMdSre1vV57+Va1VyYfzFimLSCu1ZVC1Kj6+lHR51T7txk4GlJ9qK4YHombXwwIxebTTvqMa244raYztW44LqtHzqnOt6jbc/0WOJ/YP6UIsl84ULYtIK7VlULX7cHxR2umkXAsnbb7gfNEHakBFM+vBiY1953ftcGVIGcJy6SRcP/8esHVZ9zv9HzB8BoGWtNWQD/Nl1XLY/Wq7L1+0LCKt1PbVvyLSnvjBxQ8vC2YhpjPNriSLVNPaj7JqQTXrx1Ctsoi0Slu//EFE2hcN186dO+eeX7YXhPiYviV93SgNjpjuP1cdU62yiLSKgqqINIwWtnv37nWf+TeDPt7udePGjeTmzZvuRSUEO2vVW4ZqZRFpFQVVEWkKj8dwhejjzVa81pJ/OkGwI8CuWLHCvYVs3759aaqH/epXv0o/5ZswYUL66WFZZRFpJQVVEYlijvcfp3bs2PHQP08gwJImfG2kj9dgjhs37qFXXIJxTCvy/KlfFpFWUlAVkaZw/5JA57/QgX9OkPXCFntfty987/aGDRvcfVj/nwqwDMYxrZqssoi0VNpgqW3QHJ5Wgv7r7NoNjwa0c/lEykJLXlrh2vFPS1yGw8dYrBWv/4gLj8Awv/9uY77rpAtbBvOoDB3zsiwb9hUti0grtVVQ5Xk2/9nUrGcL8zr7QmVNM3nTqy2HLzLTwy8qJwt7/k5kqOAHZa3vhuE7SRo/PeN8BFim+YEW5Ml3jGkET5YbLqeesoi0StsEVb4MfDH8K0C+JOEvWL5ABEdjXz7SgvntebWs5+LyppOPBV37gpPWvtiUzf/C8pl8FFhFRMS0zT1V/i9p+E/IH3vssYf+h2qI+yc0gDDMbw0k7P9w+vzpTz75pOuDfJ5//nn3efjw4a5PWvKuBFTXatH/v62k5/7Oa6+9lo4REZGhri2CKo0LeIbtN7/5TTqmRz3/FSXrv44Q+GJYsGCB6//1r391fTNr1iwXbHlcQEREpC2CKv8SCtOnT3f9dmOPAIQtFwnaNPH//PPP0zEiIjKUtUVQPXnyZFs9sP3zzz+7Pm9+2bp1q3s7y8iRI5OlS5e68T6qkple1ltiRERk4GiLoHrt2rVoVbU+/iF1VlfrFWbbt29PRo0alTz66KPJ5s2b3SvWTp8+3ed+r7Gr159++sn1RURk6Or3oOo/4B1bd0/r5oe6OTXetvLxxx8nt2/fdmnpc88072HyESNGpJ9ERGSoa4srVRERkcGg34NquzZOEhERqVdbXKnSgjbrPaHNarTx0L1799JPtVnL4Gr/OUNERIaGtgiqvPSBxkq12POgNDQKX8JtGG//4eLUqVOu72O6BfArV664PgjAFiC/+OIL1y+C+Wi5XEZDKxERGWC62wCv+qMo4XtBfUwPO/91hch7t6/Jm84rDrPGVysPeFUh6Q4cOJCOERGRoayDP5XA0O9WrlzpngXduHFjOqb9ceXM4zfnz59Px4iIyFDWNkGVatS5c+cmhw4dynwetB1Nnjw52bt3r/53o4iIOG3zSA33JAlQXV1dA+LtRGvXrlVAFRGRPtrmStUQUPlvMLysvl2vWHlXcTuXT0RE+kfbBVUREZGBSm9UEhERiURBVUREJBIFVRERkUgUVEVERCJRUBUREYlEQVVERCQSBVUREZFIFFRFREQiUVAVERGJREFVREQkEgVVERGRSBRURUREIlFQFRERiURBVUREJBIFVRERkUgUVEVERCJRUBUREYlEQVVERCQSBVUREZFIFFRFREQiUVAVERGJREFVREQkEgVVERGRSAZcUD127FiyaNGiZO7cuemYcly+fDkZNWpUsnXr1nRMfCxj5cqVbjnNunXrVnLw4MFk8uTJpZT5+vXrLt/x48cnZ8+eTcc2p9F9yXbbs2dPOtS+bJuxb0RkaCgtqHKi7OjoyOyYRgCol52cDh065PplGjZsWPqpPPfv309OnjyZ3LlzJx3TuLt37yb/+te/kgsXLqRj4qKsFy9eTK5du5aOaU6j+5Lj5quvvkqWL1/uAlbW8UVnPy5sOfwQyEoXCvOsFvDJc+3atenQw8aOHZssXbo0WbVq1aALrOwHfzvRse1CRdNVQx78+OLHJ/OHYixDJJruElVOwN0jR47sZjE3b9504y5dutTd2dnpxq1YscKNqxfzzpkzJx0a2FiPWLvhzJkzLq8tW7akY+IiX/JnObHUsy/t2PFxXNk2PHr0qBvHccexxTjS27HHeDv2/PEh247kkZcGLJfju1oakF+15Q1ErA/r73dZ61c0XRbSkd72Rd5x18wyRGIrNajCvhQ+DnjG0XGirBfzke9gkLV9GjXYg+q4ceN6A6cvr1ycbBm/e/fudMyDbcS0PARfllUNaciHrsj2Jih0dXWlQwPbgQMH+mzTPEXTZeEcwT7iR0u1c0QzyxApQ7/cUx09enRSOZG6zydOnHB9kWq4h1oJZMm8efPSMbUtWLDA9f/617+6PqZPn+6OParJ8+4N79u3L9mwYUM6lG3btm1JJZi6z5s3b3b9al588cVk06ZN7j7rQEY19rp165L33nvPVcnmbcOi6fK88sorbh+dPn06mThxYjq2r2aXIVKGfgmqfBl++OEH9/mpp55yfdAAhS+H3Rcp8kVhun8vhc7407Lu8Vrefjq7D8dJ3G/0E46jrJYnfaYjXI5/b8efVmu9Qv7y6CgHDX2K4ERuDaKYl3zIz1fvtmfZNFoibdZ9xUb2ZTUffvhh7w+xor799lvXf/bZZ13fLFmyxPWzAif7kfvclDcP25M0a9asSSpXoO6eOPf1qvmP//gP1z9y5IjrD1Tnzp1LKlePbp25Hz5jxgx3bNnxb4qmy8K25Mc22zYvoKKZZYiUJr1iLY1Vb1JdBvoLFy58qAqOKh7GWVUO6az6LqzyY5xfZUiVHtVEjA/vpVA1x/JAnlTrZaWjGokqOpCOcpDOqvb8ceRHVR7LtXE2L/nymXFZ1X2M96uzbPtUQ56sX7gejPNZ1aZfHUla0vnb1ea1bVB025Mv41gvPrM8W1e2n2lmX2ZhfltuFiuXlYH1snGsJ/OH7DhgHXzMR1cN62xpbJvXqi4G6fxjfqBje9t2tOM/S9F0xr4TbGP7zH6sVn1e7zJEytKyoGp9Og5+Dnw/sDHOgoaxkynTfJafz06i4UmSdH4Q48tHOjsBG9L55ckKUDYu/HJTPr88FgTDMjJ/uI6kIc9q2A5+YIStry+rzGzn8ERO+f1gU3Tb2zL9wGjp/GU2uy9DWevls3KxnqwXn+lTBn/f++w4CMtJHv5xELJ966exfZh1v9dnPywGE7aDrVfetkbRdCAN25h9xHx0HMeMrxZY61mGSFlaVv17/Phxziauu3r1arJz5053bxVUFVZOusmkSZPcsOGRhMpJz02rVXXIowuVE3byhz/8IR3TU00HvwqJaj3ScS/GkI5lWXlqGTFiRPqpB9WgPvL54IMPXBWWX0W7f//+5I033kiHiqNst2/fdo+RsB2o4nrnnXfSqdXt2rWr996i2bhxo8uPfBvZ9sOHD08/9aTzxdiXoe+//z79VN3HH3/s1otjjD7ViHnVh3YcUG1oxwnpqVavdhxwv7Vygu+TxqqTd+zY4fp5ih5fAwnrtHfvXve5WvuIouk4fjBlyhS3j5iPjvMF+4v70nnVu0WXIVKmtnj5A89A5glPznn4QnGPjMYNdn+LxiR2wvO9/fbb7uTup1u9erX7HAsBkJPAm2++6YbtPhwNZRrByYZ7oawjjV4qV2fplObE2Pa+2PmVye6psv/BD61qxwEncxolcWK3e8V0ixcvdtM5kTdz33ig4odL5QoxHcpXJN2YMWPSTw/jRxl++ukn189StCwiZWmLoGp4uUCeIi9j8K9CCUJ5DU7mz5/vGjhs37699yolvOKKgZM2wZsGTpy4sxrGFMG6zJw5M3nppZfcFX89LWBx+PDh9NMDBAj/KrrZbR+Kmd9zzz2XforLjheu5vmBNXv27KrHAVepXEFZjYvfdXV1uTSffPKJ6w9Fcwo2JKuWjh/HfDepzQpZDVGR46doWURia1lQrdYij6u3sCrOcHLmC5JXjReyQEYQ4oo0C1/cd999113V8uv35ZdfTqfEZSdtmvxz0s4K8EVwBUQLx2nTpqVjimP9/Kt3s379+mTq1KlRtz1i54df/epXrn/jxg3Xz3Pv3r30U3HLli1zfa42qx0HdpVKNWSWt956ywUD1tuqMEPsx8F4FcX68p2qtV/z0oXHCd/NrNsEHD9sv2rLKVoWkdJUfmWXpvLF6G044jeyyUIjD9JVvjS9DWiYh/n9Rgd8Jl3lxO0aJmQhD+bLmw6mkaZykk/H9JXVkMXK6I8jH8qStzybJ6uRjc3L9GoNK9gOtlwa7ZAX5WYc5WRcmM6Qr+0DGnvYvH55im57ayzi70vbH/52jLkvDWmy9hXz2bbw17so5q92HIA0rHutcrK+4Xob5mNatYY2A4FtK1s/jj2Gw+1SNJ0ds/4xRRq2Ya3jp+gyRFqptKDKwc2XJeyq4UthJyY6TpL+l4jpfl50jAvxBeQkWAsnOAJAiIDjL4N1CcfRFS0PX/7wJItwXj/Q+ThJ2PbkxM4yLHDZSTqrfIZt6M/vn8AMeVbb9uH+zNsmplZ+RbedsZOvL2v5dNXyyUI+YWtwX7juWfn707PS2f7KOg4GEo43Wz/2L9suK4gVTWfbJfwektZ+xNGFxw+KLkOklTr4UzkoBxVacFINV8Z90npx3/K///u/c6sNpThaWdPCtt57yu2AFtuVH1eu5bWIDF5t1VApBu7DVK7G2iKggiBAS2Bp3tdff5386U9/SocGDu4Znj9/3t13FZHBbVAEVa4CeLSBK9Tf/va3rmFQf+EESllGjRrlrqwI7mo0EQfb8Y9//GPVf7fWbmjgRMtvWm3TgEZEBrdBEVR//etfp5+Sqi/gbgWa/XemLTwXLlyoat/IaEH9+9//fsD8k/JTp04l77//vgKqyBAxKO+pioiI9IdBd09VRESkvyioioiIRKKgKiIiEomCqoiISCQKqiIiIpEoqIqIiESioCoiIhKJgqqIiEgkCqoiIiKRKKiKiIhEoqAqIiISiYKqiIhIJAqqIiIikSioioiIRKKgKiIiEomCqoiISCQKqiIiIpEoqIqIiESioCoiIhKJgqqIiEgkCqoiIiKRKKiKiIhEoqAqIiISiYJqhsuXLycrV65MRo0alY4p7tatW8nBgweTyZMnJ1u3bk3HNu7YsWPJokWLkrlz56Zjirl+/bpb/vjx45OzZ8+mY7MxnfzrKW89+TejkbKJiPSX0oIqJ8OOjo5CXbudMO/fv5+cPHkyuXPnTjqmuLt37yb/+te/kgsXLqRjGkeAxqFDh1y/HqzDxYsXk2vXrqVjsrGf9u/fn5w4cSIdU0zR/JvRaNlERPpLqVeqK1asSG7evJl0d3e7bs6cOW68DdPt3r07+fbbb934djF9+nR3BVbU2rVr009JMnbs2OS5555Lh5ozevToZN68eelQfSZOnJhMmjQpHcrHuu7cuTMdKq5o/s1otGwiIv2ltKB679695P3333eBoZrly5cnzz//fDo08HA1uWvXrnRIRESGstKCKldYtQKqWbNmTfpp4Fm1alVD1cQiIjL4tF1DJRrA0DDH7rfymXE2japWaxzDZ9L41a979uxx0xlPn2FDox8aIJEnV5h8tnQ0LsrDPV8aLdH56WiMZPc7rbwhGj3R0IZp9O0+qbG8mU6fMoVpsrAutp50rBPLKoJ0/jb2t5+Pbcw6WtlIl1c2vzx+frYM68jT+ONNGWUTEWmVtgqqBM3Zs2cnb7zxhrvfeubMGddgiHGgcQxXhTSOoQHL008/3XufFpxYOSmfO3fO3ctlvtdff90FQk64w4cPd/n98ssv7gpz5MiRSVdXl8tv8eLFLjCECHqPPfZY8re//S0ZN26cS2dB/vz58w/dJ/bRkOerr75K1q1b5+4d0+Bm/fr16dSevN95553k9OnTbt53333XVSUfOXIkTZGNgPLCCy8kb7/9du92Irh/8MEHaYp8bJ+ZM2e6QMQ2Yt3ZJiG2xR/+8AeXL8sg702bNvUpv/mf//mf5B//+Efy+eefu/vopLMfH+Tf2dnpPh89etTdJzWUm21KOVBG2UREWqpyUmqZSgAi6qRDD6uckLsrQS4d6sEw8xw4cMANb9mypc+wqZyA3fjKyTgd0+0+M65y4k7H9JSB4UuXLqVjursrJ3uXrnLyT8c8KKufH8tkXCUYpGOy14npjAvXheWS3jC9EtjToR7Mxzr6GOfPZ+X114HpfhrYtgrL668nyCdcLmVlOT7mIx3bGpa/n872g5+XbY9wvRiu/NhIh+KWTUSkP7TVlSpXN1xxWNUfHcPgMRXfmDFj0k89Tp065fqPPvpo77x8Blc8Pqopab1quP9bOVFnPgbj3xcOl1nLiBEj0k89WK5v48aNye3bt91VtFVbF0F5K/vOrQPbjGrlIo+dcIVNugULFqRjevjbAlwxss24Gvb3hW2ff//7365vqAEwtH4OcXW6cOHCZPPmzb1X+Th8+HAyf/5897mssomItFJbBVWqditXJC5ghF2txkw8H4qseelqKRrQYqMKeOrUqa78WVWdeewe5nfffee2WeUqL52Sr2jAoZodVM9mbUu/Crcofhyxf7dt2+aG+THAPVH70dKfZRMRiaWtgir3OG/cuJEO9WX36PLYVaF/JWRqzWtYfivRKOmzzz5z94D50ZB1lZeF9eFK7euvv3bPcYZXc7XYD5A8w4YNc/2ff/7Z9X1sX7+xUVGsG/dbuWfM1Sb3mVevXp1OfaA/yiYiEktbBVUaFnHSDYMgVaMTJkxIh7JNmzbN9alm9FuB8vnHH39Mh3r88MMP6acepGEcrU5biXXt7Ows/OiRoUEQPwDqDaa2DWng42+jEPmSP0GfAOjjSrPRq0ELojRGYr39HxH9XTYRkRhaFlTtnhnyrhzfe+89d8KkhS0BjqpRTp7cT7UAYleytDj1MZ0rIe6tUZ1KS2Dm537j0qVL01Q9qIa0R204gVurUV5WAcbZid0/cV+5csX1v//+e9fHs88+6/rkxzJZT7uKovWvIb+rV6+64G15s65U+bI9qM61x0d4w5RtI1s+89p8jzzyiFsH1o8rM+azvMnHrtZt+VZugjdVxdyTfOWVV3rT2bK4amZ+cAXMMgiA7APbli+++KKbDtsXlj+svFlvySKI0tqafGnh7YtdNhGRftHdArTqZFFhl4XWnpWrTTe9EnT6tKAN82E4RCtRWocynRahfgtZWAtTWhpbPiyvcjJPU3T3jreOPPOWTf4sj7LSOpi0fjo6a/0ajiM98zHMetLSmLKRH/nmzWfLZJhyUHZrSWytovPKC1rc+vOTn21rfzvQwpbykI70fovrrPyz1j1kZc8To2xSPra3v5/p2P+G/RZO97ta2L92HLD/ydtviY9mlyFShg7+VA7AIYMrGhw/ftz1pbW4qub54lZXtUtcfiMz8+WXX/aOowaBlu3h+6GpwaCmJa+2CtRIvPrqq67mibYS1FJQg1H5kdXne9vMMkTKoqAqLUP1NVXzVFXLwEWwokEZ7+3OQtV9XhsFgvEf//jHqj+q+I76AdqOGwJr5erU3eppdhkipSGoDhVUH1mVUliVJOWwakK2u189LQOTfYfYl9w24XZEUVTf1/rukSbrGLHjqNbyiixDpExt1fq3TDTo4WUQ/NoFn2ngIuWiVW/lBOw+08BIVw8DG49/sT9pKEZL7RkzZrhqWGtEVw0vaKFKN6w29tGYLesYsRev2CNVeYosQ6RMQ676V0TioBqY5435oUogq/W/b6mW/fOf/9zQ/wjmXvzf//73mrdtmlmGSAwKqiLSMK5QuQfKo2x2vzML90A7Oztdw6J62T1VXnaSlz+aWYZILEOm+ldE4qOade/eve5ztfdP85+XGq36/+ijj5INGzZUDahoZhkisSioikhTCHZcIVbDYzGNvJyDKmb+9WKRYNnoMkRiUlAVkSh4jjQLb9miSrbe+5zMxz+MyHt0x9foMkRiU1AVkaYQ0KgGzque/eqrr1xDpjz2SkofeTJfrcZPptYyRFqlZUGVxga8HzfrCzRQsT76rygylIwaNco1TLLvMcf/O++8417WkIdHb37/+9+nQ33xHRo3blzvu7hBle/vfvc79zYlHnvzO3t5S6jaMkRaita/ZeMdnTwobg9kZ70jNq/jYe/wPbN+x7QiLxQgH/89wlmy8qerhnfV6oUGMlTwHbLvBe9eznonr4/vPi+LyMM7fsmLvj+c1/nvFza1liHSSqUHVb5wfPl404nhi0Ew8vGFIUAa5luxYkXvl4j5eYsL6exLzJeJvBlH2mrIm/mrnQBAuciPL6lf5mqKBnYRERncSq/+5d+q8X9S/f+dSWu+Wo0PuEfj309h/ilTprjP9rYU7uHYw+D2z6+zUFVFc3/eArNv3750bLYnn3zS9cePH9+nzNVQTt4qM5iqtkVEpH6lBlWCHMHuN7/5TTqmRz3Pkq1Zsyb9lI0AW7lSdJ/znpPjn1dXrnjd582bN7t+TARf7guxHBERGbpKDarW+GD69OmuXwYaQPHfKvDUU0+5vo+rR/4ROMGZ1oFcrZbxL6F4PRr5FnkHqoiIDE6lBlWCWa2Hwhth1az0V61a5QIly8l6Ro2rx2XLlrnPL7/8suvzvtLYHn/8cVcOXjguIiJDU6lBlRdt2/3PmLh/2dHR4apceecoV6BZL9rmqpGrx6VLl7phrpipKqZc/CPkmOwq+R//+Ifri4jI0FNaUC3z+U0CaHdPy2X3D69pKJQVvGmURMD1py1ZssT1d+zY4fqxDB8+PP0kIiJDValXqv2Jq1QaJW3atMld1Vq3ePFiN51GTXpxg4iIxFRaUC2zcVIRXKXyCI5d0fpdV1eXS/PJJ5+4fr0Ixnp8RkREQqVeqXLPk+rZ2Gq1sLWr1Lz3hr711lvJyJEj3avN8p5trWb//v0PPcP6/fffu35WC2QRERkaSg2qvPSBRkG12CMuVMnmXQEy3h6d4f8m5iGg8sIJ3lHKu0OzcI+VgI/XXnutzzKvXLni+lmBm3Q8Y0tADt29e9f1+WfKIiIyNHV0Ux9aEq4Cn3nmmeTMmTO51cHc5wzRQtdvzctLtLNe7JBV9DBt1rKzlkm6GTNmpEPVHT169KHHd3hOla7of9UQEZHBp9SgCh5/4cpu48aN6ZjBhytYnpPl8Z6irzYUEZHBp/SgSjUqV4/cvxysAYcfDr/+9a/rev2iiIgMPqUHVVAN/MEHHySffvppKS+D6E92P1gBVUREWhJUwRUrDYxmzZo1aK5YeSsTL33o78eHRESkPbQsqIqIiAx2g/aNSiIiIq2moCoiIhKJgqqIiEgkCqoiIiKRKKiKiIhEoqAqIiISiYKqiIhIJAqqIiIikdT18oes/+4iIiIyGMR4F5KuVEVERCJRUBUREYlE7/4VERGJRFeqIiIikSioioiIRKKgKiIiEomCqoiISCQKqiIiIpEoqIqIiESioCoiIhKJgqqIiEgkCqoiIiKRKKiKiIhEoqAqIiISiYKqiIhIFEny/wGckIldZ7eXjAAAAABJRU5ErkJggg==\"\u003e\u003c/p\u003e\n\u003cp\u003ePEI serves as a flocculant for the immobilization of cells onto diatomite. The primary amine groups in PEI can cross-link with glutaraldehyde, resulting in the formation of a stable mesh structure. Variation in the relative molecular mass of PEI lead to changes in its viscosity and the proportion of secondary to tertiary amines, thereby influencing the immobilization effectiveness. Consequently, we conducted an investigation on the impact of PEI molecular weight on the immobilization effect, as illustrated in Fig. 4b.The viability of immobilized cells exhibited a gradual increase with the molecular weight of PEI up to 10 kDa, followed by a decline upon reaching 70 kDa. This phenomenon can be attributed to the increased viscosity and greater branching of the polymer when the molecular weight of PEI reaches 10 kDa. Consequently, a denser polymer film \u0026nbsp;forms on the cell surface, leading to an enhanced effective specific surface area during interactions with the cross-linking agent, thereby improving the efficiency of immobilization (Zhao et al. 2021) . An increase in the molecular weight of PEI to 70 kDa may result in a reduction in the proportion of secondary amine groups within the amine group, potentially decreasing the efficiency of immobilization. Ultimately, PEI with a molecular mass of 10 kDa was chosen as the optimal flocculant.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eDiatomite serves as a carrier for immobilized cells, and its particle size significantly influences the effectiveness of immobilization. Fig. 4c demonstrates that the viability of immobilized cells initially increases and then decreases as the diatomite particle size increases. This phenomenon can be attributed to the enhancement of the pore structure, total pore volume, and micropore volume of diatomite with increasing particle size. As a result, both the loading capacity and loading efficiency of cells are improved. Consequently, diatomite with a particle size of 36.8 \u0026mu;m was chosen as the optimal immobilization carrier.\u003c/p\u003e\n\u003cp\u003eA quantity of 0.3 g of diatomite was measured and added to varying masses of recombinant \u003cem\u003eE. coli\u003c/em\u003e cells, aiming to investigate the impact of cell loading on the viability of immobilized cells, as depicted in Fig. 4d. The viability of immobilized cells exhibited an increase as the cell loading was raised to 5 g, but declined beyond this threshold. This phenomenon can be attributed to the accumulation of a high concentration of bacteria within the immobilized cells, which results in the blockage of channels and a reduced probability of substrate-cell contact. Consequently, the viability of the immobilized cells decreases. Therefore, a cell loading of 5 g was selected.\u003c/p\u003e\n\u003cp\u003eThe optimal volume fraction of PEI for immobilization was determined, as shown in Fig. 4e. The viability of immobilized cells exhibited a gradual increase with an increase as the PEI volume fraction continues to increase, the viability of immobilized cells began to decline. At low concentrations of PEI, the structure formed by the cross-linking of GLU and cells is loose, making the cells prone to detachment (Chen et al. 2008)\u0026nbsp;. Conversely,\u0026nbsp;as the\u0026nbsp;concentration of PEI in the system increases, the molecules of PEI cross-link, resulting in heightened mass transfer resistance and a subsequent decrease in the viability of\u0026nbsp;the\u0026nbsp;immobilized cells\u0026nbsp;(HOU et al. 2016)\u0026nbsp;. Consequently, a volume fraction of 8 % PEI was chosen as the optimal flocculant.\u003c/p\u003e\n\u003cp\u003eThe selection of flocculation time for PEI is illustrated in Fig. 4f. Initially, the viability of immobilized cells increased with longer PEI flocculation time increased, peaking at 2.5 h. However, further increase in flocculation time resulted in a decrease in cell viability. This phenomenon may be attributed to the tightening of the crosslinked structure of the immobilized cells as the flocculation time is extended, which leads to excessive mass transfer resistance and ultimately reducing cell viability. Therefore, a flocculation time of 2.5 h was determined to be optimal.\u003c/p\u003e\n\u003cp\u003eWhen immobilizing cells using cross-linking, the choice of crosslinker volume fraction significantly and irreversibly influences cell structure. Therefore, optimal immobilization is often achieved through precise control of the concentration and duration. The efffect of GLU on the viability of immobilized cells is depicted in Fig. 4g, revealing a positive correlation between GLU concentrations and cell viability. Optimal viability was observed at a GLU volume fraction of 5 %. This maximum viability is attributed to the toxicity of GLU to recombinant \u003cem\u003eE. coli\u003c/em\u003e; excessive GLU concentration can reduce the viability of immobilized cells. Consequently, a GLU the concentration of 5 % was selected.\u003c/p\u003e\n\u003cp\u003eThe impact of GLU crosslinking duration on the activity of immobilized is illustrated in Fig. 4h. The viability of the immobilized cells increased as the crosslinking time extended, peaking at 1.5 h (The specific activity was 310.73 U/g, with an activity recovery yield of 70.71%.). This enhancement is attributed to the low molecular weight of GLU and the limited availability of aldehyde groups for crosslinking with PEI, a process that is completed within 1.5 h. Beyond this duration, structural changes in the immobilized cells remained minimal, but any residual GLU in the system began to affect viability, resulting in a decline in cell activity. Therefore, a final crosslinking time of 1.5 h for GLU was established.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProperties of immobilized cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn biocatalysis, the rate of catalytic reactions typically increases with increasing temperatures. However, excessively high temperatures can lead to a decline in cellular activity. The impact of temperature on the specific activity of both immobilized and free cells is illustrated in Fig. 5a. Viability for both types of cells showed an upward trend with increasing temperature, peaking at 50 ℃. Beyond this point, viability for both immobilized and free cells began to decrease. The optimal temperature for cell activity, whether immobilized or free, was determined to be 50 ℃.\u003c/p\u003e\n\u003cp\u003eThe effect of temperature on the stability of free and immobilized cells is depicted in Fig. 5b. Following a 12-hour incubation at 45 ℃ , the activities of immobilized cells and free cells were 90.15 % and 82.50 % of their initial activities, respectively, indicating minimal activity loss and good stability. After the same duration at 50 ℃ , the activities decreased to 81.29 % and 65.59 % for immobilized and free cells, respectively. Subsequent incubation at 55 ℃ resulted in further activity decline, with immobilized and free cells reaching 26.60 % and 12.56 % of their initial activities. Immobilization enhanced thermal stability, with diatomite providing additional cellular protection. Optimal stability for both cell types was observed at 45 ℃ , which was consequently chosen as the preferred reaction temperature.\u003c/p\u003e\n\u003cp\u003eThe effect of temperature on the stability of free and immobilized cells is depicted in Fig. 5b. After a 12 h incubation at 45 ℃ , the activities of immobilized cells and free cells were 90.15 % cells achieving 26.60 % and 12.56 % of their initial activities. Immobilization enhanced thermal stability, with diatomite providing and 82.50 % of their initial activities, respectively, indicating minimal activity loss and good stability. Following the same duration at 50 ℃ , the activities decreased to 81.29 % and 65.59 % for immobilized and free cells, respectively. Subsequent incubation at 55℃ resulted in a further decline in activity, with immobilized and free cells additional cellular protection. Optimal stability for both cell types was observed at 45 ℃ , which was subsequently chosen as the preferred reaction temperature.\u003c/p\u003e\n\u003cp\u003eIn biocatalytic reactions, variations in the pH of the reaction solution lead to the dissociation of specific groups in aspartate lyase to varying degrees, which affects their substrate binding capacities and results in differing activities at different pH levels. The impact of pH on the specific activity of both immobilized and free cells is illustrated in Fig. 5c. Viability for both types of cells exhibited a gradual increase with rising pH. The peak viability for immobilized cells was observed at pH 9.5, followed by a decline with further increases in pH. Conversely, free cell activity reached its maximum at pH 10.0, decreasing beyond this point. The optimal pH was determined to be 9.5 for immobilized cells and 10.0 for free cells.\u003c/p\u003e\n\u003cp\u003eThe effect of pH on cell stability before and after immobilization is shown in Fig. 5d. Following a 12 h storage in Tris-HCl buffer at pH 8.0, both immobilized and free cells exhibited higher viability compared to those stored in other buffers, with free cells demonstrating superior pH stability. Immobilized cells displayed robust stability within the pH range of 8.0 to 9.5, retaining over 85 % of their activity. Overall, pH stability increased following immobilization likely due to the protective cross-linking network formed by PEI and GLU, which shielded the cells and minimized environmental influences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKinetic Properties\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInvestigating kinetic parameters is essential for assessing the efficiency of an immobilization process. The kinetics of the catalytic reaction were analyzed, and the Michaelis-Menten parameters were determined using a Lineweaver-Burk plot. Fig. 6 depicts the resultant fitted curves, revealing a \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e of 636.93 mmol/L and\u003cem\u003e\u0026nbsp;V\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e of 99.66 U/g for free cells, and a \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e of 748.13 mmol/L and \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e of 43.55 U/g for immobilized cells. The \u003cem\u003eK\u003csub\u003em\u003c/sub\u003e\u003c/em\u003e value for immobilized cells increased by a factor of 1.17, primarily due to the heightened mass transfer resistance induced by the mesh structure within the immobilized cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStorage stability and reusability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmobilized and free cells were stored at 4 \u0026deg;C for 60 days to evaluate changes in activity (Fig. 7a). Immobilized cells remained above 93 % for the first 30 days, demonstrating a 64 % higher residual viability compared to free cells. By day 60, immobilized cells retained a viability of 72.29 %, while free cells only reached 14.26 %. Immobilized cells exhibit significantly enhanced storage stability, effectively preserving cell activity during storage.\u003c/p\u003e\n\u003cp\u003eSystematic evaluation of biocatalyst recyclability revealed that the immobilized cell system maintained excellent operational stability during repeated-batch \u0026beta;-alanine synthesis in Fig. 7b. The initial 6-hour conversion cycle achieved near-quantitative yield (96.6 \u0026plusmn; 1.2%), with subsequent cycles showing minimal biocatalyst deactivation (0.89 \u0026plusmn; 0.15% per cycle), ultimately retaining 80.06% production yield and 86.26% initial activity after 10 consecutive batches. These results demonstrate exceptional sustainability for potential industrial implementation. In contrast, batches using free cells achieved only around a 10 % yield after four consecutive reactions. Diatomite-immobilized cells demonstrate \u0026nbsp;superior reusability, offering promising prospects for industrial applications.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026beta;-alanine synthesis in fixed bed bioreactor\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo achieve continuous production, the immobilized cells were placed in a fixed bed reactor. This method eliminates the need for mechanical stirring and prevents direct contact between the enzyme and acidoid, thereby ensuring the overall stability of the whole cell. The effects of acrylic acid concentration and volumetric flow rate on the continuous production yield of \u0026beta;-alanine were systematically evaluated in a fixed-bed bioreactor packed with immobilized \u003cem\u003eE. coli\u003c/em\u003e cells. The experimental results are shown in Fig. 8a. When the concentration of acrylic acid is 2 mol/L, the optimal flow rate is 0.03 mL/min, resulting in a space-time yield of 193.08 g\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e\u0026middot;h\u003csup\u003e-1\u003c/sup\u003e. At acrylic acid concentrations of 1.5 mol/L and 2 mol/L, the space-time yield remians the same at the optimal flow rate. Higher concentrations of acrylic acid and aqueous ammonia can cause damage to the cells. Therefore, the subsequent synthesis of \u0026beta;-alanine was carried out in a fixed-bed reactor under the conditions of 1.5 mol/L acrylic acid concentration and a flow rate of 0.04 mL/min. The ability of immobilized cells to continuously synthesize \u0026beta;-alanine was investigated in the fixed-bed reactor under optimal conditions for 360 h, as shown in Fig. 8b. In the fixed-bed reactor, the immobilized cells demonstrated stable synthesis of \u0026beta;-alanine, achieving a yield of over 71.30 % after 360 h of continuous reaction.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, a straightforward method for preparing immobilized cells was devised. Diatomite served as the carrier, facilitating the crosslinking of whole-cell catalysts with PEI and glutaraldehyde. The immobilized cells enhanced thermal and pH stability, while retaining retained high enzyme activity. Furthermore, the immobilized cells have shown favourable reusability , which is a prerequisite for applications in industrial continuous production. The performance of the immobilized cells was investigated in both batch reactor and fixed bed bioreactors. The immobilized cells demonstrated stable synthesis of \u0026beta;-alanine, with a yield of over 71.30 % after 360 h of continuous reaction. An assessment of the immobilized whole-cell catalyst highlighted its exceptional reusability and robust storage stability. This method presents a viable and economical approach to preparing whole-cell catalysts for industrial applications of \u0026beta;-alanine.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Experimental Section","content":"\u003cp\u003e\u003cstrong\u003eMaterials and Strain\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental strain \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3)/pET-30a (+)-HAMase was obtained from Anhui Pengpai Biological Co., Ltd. (Anhui, China). Yeast maceration powder and tryptone were purchased from Beijing Auboxing Biotechnology Co. Glycerol and isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from Tianjin Dingguo Biological Co. Dipotassium hydrogen phosphate and potassium dihydrogen phosphate were purchased from Tianjin Komeo Chemical Reagent Co. Ammonia, acrylic acid, acetonitrile, ammonium persulfate, sodium dodecyl sulfate (SDS), tris (hydroxymethyl) aminomethane (Tris), diatomite, polyethyleneimine, glutaraldehyde , potassium hydroxide, Tetrakis(hydroxymethyl) phosphonium chloride (THPC), poly(vinyl alcohol) (PVA), p-phenylene dichalcogenide (p-phenylene dichalcogenide) were purchased from Aladdin Technology (Shanghai) Co. Concentrated hydrochloric acid was purchased from Huanghua Century Kobo Technology Development Co. Among these, acetonitrile was chromatographically pure, diatomite consisted of calcined products, and the others were analytically pure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of whole-cell biocatalysts\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe preserved recombinant strains were cultured in 10 mL of LB medium for 14 h using a volume of 10 microliters. Subsequently, the cultures were transformed by transferring 500 μL into 50 mL of TB and cultivated at 37 ℃. Upon reaching an OD\u003csub\u003e600\u003c/sub\u003e of 0.6-0.8, protein overexpression was induced by adding IPTG to achieve a final concentration of 0.3 mM. Strains were cultured at 25 ℃ for another 20 h. After the cultivation and induction, the cells were collected by centrifugation at 8000 g and 4 ℃ for 6 min. The cells were washed once with 50 mM Tris-HCl buffer (pH 7.5). Whole-cell catalysts were obtained. The cells were homogenized using a high-pressure homogenizer, and the resulting supernatant was centrifuged at 4 ℃ and 12000 r/min for 10 min to obtain the crude enzyme solution. The crude enzyme solution was subsequently analyzed using SDS-PAGE.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole-cell catalyst activity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe reaction system was supplemented with 2 mL of 600 mM acrylic acid and 600 mM ammonia. The pH was then adjusted to 10.0 using HCl. The appropriate whole-cell catalyst was added, and the reaction was conducted at 50 ℃ for 30 min. The reaction was terminated by heating at 95 ℃ \u0026nbsp;for 5 min. β-Alanine was analyzed using HPLC. One unit of activity is defined as the amount of enzyme that produces 1 μmol of β-alanine per minute. The unit of activity per gram of wet cells is defined as the specific activity (U/g\u003csub\u003ecell\u003c/sub\u003e).\u003c/p\u003e\n\u003cp\u003eIn a 20 mL reaction system, 1 mol/L acrylic acid and 1.5 mol/L ammonia were added, and the pH of the reaction system was adjusted to 9.5 with concentrated hydrochloric acid recombinant \u003cem\u003eE. coli\u003c/em\u003e cells (5 g/L, \u003cem\u003ewet weight) we\u003c/em\u003ere added, and the reaction was carried out at 50 ℃ for 24 h with constant temperature shaking. The resulting β-alanine analyzed using high-performance liquid chromatography (HPLC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of immobilized whole-cell\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo prepare a ready-to-use THP aqueous solution (30 %), 12 g of THPC was added to 90 mL of ultrapure water, followed by the addition of a solution containing 3.4 g of KOH. The two solutions were mixed at room temperature (crosslinking agents is GLU, THP, PVA and TPAL with the same volume fraction). In a separate step, a mixture of \u0026nbsp; recombinant \u003cem\u003eE. coli\u003c/em\u003e (cell loading is 4 g, 5 g, 6 g, 7 g and 8 g) and 0.3 g of diatomite (particle size of DE is 9.6 μm, 19.6 μm, 25.4 μm, 29.3 μm, 36.8 μm and 40.0 μm) was combined with 50 mL of buffer. While stirring, 1 mL of PEI (volume fraction of PEI is 4 %, 6 %, 8 %, 10 %, 12 %, and 14 %, molecular weight of PEI is 600 Da, 1800 Da, 10 000 Da and 70000 Da) with a volume fraction of 8 % was added, and the mixture was allowed to undergo flocculation (flocculation time is 0 h, 0.5 h, 1 h, 1.5 h, 2 h, and 2.5 h). Subsequently, a GLU aqueous solution (concentrations is 2.5 %, 5 %, 7.5 %, 10 %, 12.5 %, 15 % and 20 %) was added, and the cross-linking reaction was allowed to proceed for an additional (crosslinking times is 1 h, 1.5 h, 2 h, 2.5 h, 3 h and 3.5 h). The resulting mixture was then filtered, and the collected precipitate was washed three times with Tris-HCl buffer. Following this, the precipitate was shaped into long strips, air-dried at room temperature, and finally pulverized into granules.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell density was determined by measuring the optical density of culture samples at 600 nm. The concentration of β-alanine in the supernatant was analyzed using HPLC equipped with an amino column (4.6 mm × 250 mm, 5 μm) at 205 nm. The mobile phase consisted of 50 mmol/L potassium dihydrogen phosphate (pH 4.5)-acetonitrile 30:70 (v/v), with a flow rate of 1.0 mL/min. The column temperature was maintained at a 30 ℃.\u003c/p\u003e\n\u003cp\u003eError bars were calculated using the standard deviation (STDEV) from triplicate experiments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupporting Information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 22308083 \u0026nbsp;and 22178083), \u0026nbsp;the National Key Research and Development Program of China (2023YFA0914500), the Natural Science Foundation of Hebei Province (B2022202014), Baoding science and technology plan project (2494F016)\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eM. K\u0026ouml;nst M C R F, Elinor L. Scotta,Johan P. M. Sandersa (2009) A study on the applicability of L-aspartame \u0026alpha;-ddeclaratoryin the bboastedproduction of nitrogen containing chemicals. Green Chem. 11:1646-1652. https://doi.org/10.1039/b902731a\u003c/li\u003e\n \u003cli\u003eCoxon K M, Chakauya E, Ottenhof H H et al (2005) Pantothenate biosynthetic routes in higher plants. Biochem. Soc. Trans. 33:743-746. https://doi.org/10.1042/Bst0330743.\u003c/li\u003e\n \u003cli\u003eJang Y-S, Kim B, Shin J H et al (2012) Bio-based production of C2\u0026ndash;C6 platform chemicals. Biotechnol. Bioeng. 109:2437-2459. https://doi.org/10.1002/bit.24599.\u003c/li\u003e\n \u003cli\u003eFord J H, Buc S R, Greiner J W (1947) An Improved Synthesis of \u0026beta;-Alanine. III. The Addition of Ammonia to Acrylonitrile at 50-150\u0026deg;. J. Am. Chem. Soc. 69:844-846. https://doi.org/10.1021/ja01196a029.\u003c/li\u003e\n \u003cli\u003eJi X, Luo (2005) Synthesis and Application of \u0026beta;-Alanine. Amino Acids \u0026amp; Biotic Resources.\u003c/li\u003e\n \u003cli\u003eLiang L-Y, Zheng Y-G, Shen Y-C (2008) Optimization of \u0026beta;-alanine production from \u0026beta;-aminopropionitrile by resting cells of Rhodococcus sp. G20 in a bubble column reactor using response surface methodology. Process Biochemistry.43:758-764.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ehttps://doi.org/10.1016/j.procbio.2008.03.002\u003c/li\u003e\n \u003cli\u003eZhang T, Zhang R, Xu M et al.\u0026nbsp;(2018) Glu56Ser mutation improves the enzymatic activity and catalytic stability of Bacillus subtilis L-aspartate \u0026alpha;-decarboxylase for an efficient \u0026beta;-alanine production. Process Biochemistry.\u0026nbsp;70:117-123. https://doi.org/10.1016/j.procbio.2018.04.004.\u003c/li\u003e\n \u003cli\u003eLi S, Fu W, Su R et al (2022) Producing malonate inSaccharomyces cerevisiaevia the \u0026beta;-alanine pathway.\u003cem\u003e\u0026nbsp;\u003c/em\u003eSyst Microbiol and\u003c/li\u003e\n \u003cli\u003eBiomanufacturing.\u0026nbsp;3:328-338.\u0026nbsp;https://doi.org/10.1007/s43393-022-00113-8\u003c/li\u003e\n \u003cli\u003eLi R, Wijma H J, Song L et al (2018) Computational redesign of enzymes for regio- and enantioselective hydroamination. Nat. Chem. Biol. 14:664-670. https://doi.org/10.1038/s41589-018-0053-0.\u003c/li\u003e\n \u003cli\u003eNorman S J, Reeves D J, Saum L M (2021) Use of Pamidronate for Hypercalcemia of Malignancy in Renal Dysfunction. J. Pharm. Pract. 34:553-557. https://doi.org/10.1177/0897190019883162.\u003c/li\u003e\n \u003cli\u003eCer\u0026oacute;n-Carrasco J P, Jacquemin D (2021) Using Theory To Extend the Scope of Azobenzene Drugs in Chemotherapy: Novel Combinations for a Specific Delivery. ChemMedChem.\u0026nbsp;16:1764-1774. https://doi.org/10.1002/cmdc.202100046.\u003c/li\u003e\n \u003cli\u003eLiu CJ, Tsai YS, Huang HS (2022) Atorvastatin Decreases Renal Calcium Oxalate Stone Deposits by Enhancing Renal Osteopontin Expression in Hyperoxaluric Stone-Forming Rats Fed a High-Fat Diet. Int. J. Mol. Sci.\u0026nbsp;\u003cem\u003e23\u003c/em\u003e:3048. https://doi.org/10.3390/ijms23063048.\u003c/li\u003e\n \u003cli\u003eKumar A, Shariff M, Doshi R (2020) Impact of rosuvastatin versus atorvastatin on coronary atherosclerotic plaque volume-a systematic review and meta-analysis with trial sequential analysis of randomized control trials. Eur.\u0026nbsp;J.\u0026nbsp;Prev.\u0026nbsp;Cardiol.\u0026nbsp;27:2138-2141. https://doi.org/10.1177/2047487319868035.\u003c/li\u003e\n \u003cli\u003eJayasena A, Atapattu N, Lekamwasam S (2015) Treatment of glucocorticoid-induced low bone mineral density in children: a systematic review. Int. J. Rheum. Dis. 18:287-93. https://doi.org/10.1111/1756-185x.12560.\u003c/li\u003e\n \u003cli\u003eMilioni F, de Poli R A B, Saunders B et al (2019) Effect of \u0026beta;-alanine supplementation during high-intensity interval training on repeated sprint ability performance and neuromuscular fatigue. J.\u0026nbsp;Appl.\u0026nbsp;Physiol.\u0026nbsp;27:1599-1610. https://doi.org/10.1152/japplphysiol.00321.2019.\u003c/li\u003e\n \u003cli\u003eZhang Y, Zhang X, Wu M et al (2018) Application of Diatomite in New Energy Field. Materials China 37:331-340. https://doi.org/10.7502/j.issn.1674-3962.2018.05.02.\u003c/li\u003e\n \u003cli\u003eMarques T M F, Sales D A, Silva L S et al (2020) Amino-functionalized titanate nanotubes for highly efficient removal of anionic dye from aqueous solution.\u0026nbsp;Appl. Surf. Sci.\u0026nbsp;512:145659. https://doi.org/10.1016/j.apsusc.2020.145659\u003c/li\u003e\n \u003cli\u003eJin L-Q, Chen X-X, Jin Y-T et al (2021) Immobilization of recombinant \u003cem\u003eEscherichia coli\u003c/em\u003e cells expressing glucose isomerase using modified diatomite as a carrier for effective production of high fructose corn syrup in packed bed reactor.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eBioprocess.\u0026nbsp;Biosyst. Eng. 44:1781-1792. https://doi.org/10.1007/s00449-021-02560-4.\u003c/li\u003e\n \u003cli\u003eZhang X-H, Liu Z-Q, Xue Y-P et al (2018) Production of R-Mandelic Acid Using Nitrilase from Recombinant \u003cem\u003eE. coli\u003c/em\u003e Cells Immobilized with Tris(Hydroxymethyl)Phosphine. Appl. Biochem. Biotechnol. 184:1024-1035. https://doi.org/10.1007/s12010-017-2604-3.\u003c/li\u003e\n \u003cli\u003eHu Z, Zheng S, Jia M et al (2017) Preparation and characterization of novel diatomite/ground calcium carbonate composite humidity control material. Adv. Powder Technol. 28:1372-1381. https://doi.org/https://doi.org/10.1016/j.apt.2017.03.005.\u003c/li\u003e\n \u003cli\u003eBenayache S, Alleg S, Mebrek A et al (2018) Thermal and microstructural properties of paraffin/diatomite composite.\u0026nbsp;\u0026nbsp;Vacuum.\u0026nbsp;157:136-144. https://doi.org/10.1016/j.vacuum.2018.08.044\u003c/li\u003e\n \u003cli\u003eZhao X, Che Y, Mo Y et al (2021) Fabrication of PEI modified GO/MXene composite membrane and its application in removing metal cations from water. J. Membr. Sci. 640:119847. https://doi.org/https://doi.org/10.1016/j.memsci.2021.119847.\u003c/li\u003e\n \u003cli\u003eChen J, Zheng Y-G, Shen Y-C (2008) Biosynthetic routes of p-methoxyphenylacetic acid from p-methoxyphenylacetonitrile by immobilized Bacillus subtilis ZJB-063. Process Biochem. 43:978-983. https://doi.org/https://doi.org/10.1016/j.procbio.2008.05.002.\u003c/li\u003e\n \u003cli\u003eH Y-F, W J-F, Liu M et al (2016) Preparation and performance optimization of PVA/PEI composite nanofiltration membrane. Membr. Sci. Tech\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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biotechnology-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bile","sideBox":"Learn more about [Biotechnology Letters](https://www.springer.com/journal/10529)","snPcode":"10529","submissionUrl":"https://submission.nature.com/new-submission/10529/3","title":"Biotechnology Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7412633/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7412633/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Developing stable and easy-to-operate biocatalysts is crucial for their use as industrial catalysts. Here immobilized whole-cell catalysts were used for β-alanine production by immobilizing recombinant Escherichia coli cells (containing hydroaminase) with diatomite. E. coli BL21 (DE3)/pET-30a (+)-HAMase was genetically engineered for the efficient synthesis of β-alanine from acrylic acid and aqueous ammonia. Using glutaraldehyde as a cross-linking agent, polyethyleneimine (PEI) as a flocculant, and diatomite as the immobilization carrier, optimal immobilization was achieved with 8 % (w/v) PEI solution, 5 % (w/v) glutaraldehyde, and 100 mg wet cell/mL cell suspension, along with a PEI flocculation time of 2.5 h and glutaraldehyde cross-linking time of 1.5 h. The enzyme activity recovery rate reached 70.72 %. Remarkably, the immobilized whole-cell catalysts exhibited excellent stability, retaining over 90 % of initial enzyme activity after 12 h incubation at 45 °C and maintaining over 72 % enzyme activity after storage for 60 days at 4 °C. Additionally, the immobilized cells demonstrated enhanced reusability, maintaining consistent β-alanine yield even after ten consecutive reaction batches with an average yield of approximately 80 %.","manuscriptTitle":"Immobilization of hydroaminase-expressing recombinant Escherichia coli whole-cell biocatalysts for the production of β-alanine","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-22 09:39:19","doi":"10.21203/rs.3.rs-7412633/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-09-16T06:51:22+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-11T10:02:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-08T08:00:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biotechnology Letters","date":"2025-09-06T11:30:03+00:00","index":"","fulltext":""},{"type":"decision","content":"Major revisions","date":"2025-08-28T13:38:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biotechnology-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bile","sideBox":"Learn more about [Biotechnology Letters](https://www.springer.com/journal/10529)","snPcode":"10529","submissionUrl":"https://submission.nature.com/new-submission/10529/3","title":"Biotechnology Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7979745f-96c1-40ee-8a9d-25ace2f2ba02","owner":[],"postedDate":"September 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-09T16:01:31+00:00","versionOfRecord":{"articleIdentity":"rs-7412633","link":"https://doi.org/10.1007/s10529-026-03705-9","journal":{"identity":"biotechnology-letters","isVorOnly":false,"title":"Biotechnology Letters"},"publishedOn":"2026-03-06 15:58:10","publishedOnDateReadable":"March 6th, 2026"},"versionCreatedAt":"2025-09-22 09:39:19","video":"","vorDoi":"10.1007/s10529-026-03705-9","vorDoiUrl":"https://doi.org/10.1007/s10529-026-03705-9","workflowStages":[]},"version":"v1","identity":"rs-7412633","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7412633","identity":"rs-7412633","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}