Exoproduction and Biochemical Characterization of a Novel Thermophilic Serine Protease From Ornithinibacillus Caprae L9T With Hide-dehairing Activity

Exoproduction and Biochemical Characterization of a Novel Thermophilic Serine Protease From Ornithinibacillus Caprae L9T With Hide-dehairing Activity | 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 Exoproduction and Biochemical Characterization of a Novel Thermophilic Serine Protease From Ornithinibacillus Caprae L9 T With Hide-dehairing Activity Xiaoguang Li, Qian Zhang, Longzhan Gan, Guangyang Jiang, Yongqiang Tian, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-573853/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Nov, 2021 Read the published version in Journal of Microbiology and Biotechnology → Version 1 posted You are reading this latest preprint version Abstract BLAST, basic local alignment search tool; BBD, Box-Behnken design; BSA, bovine serum albumin; CGMCC, China General Microbiological Culture Collection Center; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; EDTA, ethylene-diamine-tetraacetic acid; EGTA, ethylene glycol-bis (β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid; GBDP, genome blast distance phylogeny; HE, hematoxylin and eosin; KCTC, Korean Collection for Type Cultures; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LB, Luria-Bertani; β-ME, β-mercaptoethanol; NR, non-redundant protein database; PMSF, phenylmethylsulfonyl fluoride; RSM, response surface methodology; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SD, standard deviation; TCA, trichloroacetic acid; TSB, tryptic soytone broth; TYGS, type strain genome server; UV-Vis, ultraviolet-visible. General Microbiology Ornithinibacillus caprae Response surface methodology Serine protease Dehairing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction China, one of the major leather-producing countries, is well known for its most active and potential leather trading markets worldwide. According to the China Leather Industry Association data, around 529 million square meters of genuine leather was produced in China in 2019. The leather industry mainly uses hides and skins, the livestock by-products, as the raw materials for processing. Leather processing involves many physical and chemical treatments such as soaking, dehairing, bating and tanning (Ward 2011). The dehairing of skin/hide is considered as the most polluting process (Kandasamy et al. 2012). The traditional hair-removal methods require the use of several chemical depilatory agents, such as sodium sulfide, sodium bisulfide and calcium chloride (Ockerman and Basu 2014), which contribute to environmental pollutions, including the generation of noxious gases and solid wastes (Kanagaraj et al. 2015; Thanikaivelan et al. 2004). Therefore, the introduction of more eco-friendly methods is highly desirable. In this regard, microbial proteases could fill this niche (Paul et al. 2016). Enzymatic dehairing, which has been proved to be the most cost-effective and eco-friendly process, exerts tremendous potential in obtaining high-quality leather (Wang et al. 2009). This phenomenon has increased the attention toward the exploitation of enzyme-producing microorganisms (Barzkar 2020). So far, a variety of extracellular proteases have been reported and characterized from different microorganisms, including fungi like Paecilomyces marquandii (Gradisar et al. 2005), and bacteria, such as Bacillus subtilis (Dettmer et al. 2013), Lactobacillus curvatus (Sun et al. 2019), Nocardiopsis dassonvillei (Sharma et al. 2020) and Streptomyces koyangensis (Ben Elhoul et al. 2015). Interestingly, most of the reported extracellular proteases are serine proteases (Brandelli 2007; Jaouadi et al. 2013), while a few belong to metalloproteases (Wang et al. 2008; Chandrasekaran et al. 2016). As a member of the proteolytic enzyme family, serine proteases exert a wide range of biotechnological applications, including skin dehairing (Jaouadi et al. 2013), feather degradation (Jagadeesan et al. 2020), stain washing (Haddar et al. 2009), food production (Kostyleva et al. 2016), silk degumming and sericin degrading (Suwannaphan et al. 2017). Of the reported proteases to date, the extracellular serine proteases were preponderantly secreted by Bacillus strains. However, little attention has been paid to the rare strains, especially the species of Ornithinibacillus . Ornithinibacillus species are generally Gram-stain-positive, aerobic, rod-shaped and moderately halophilic bacteria from the family Bacillaceae (Li et al. 2020). Over the past decades, a total of 12 strains have been isolated and placed in the genus Ornithinibacillus (Parte et al. 2020) . Despite these studies, no protease from Ornithinibacillus strains has been reported so far. Therefore, further research on the proteases secreted by Ornithinibacillus species is highly necessitated to expand the existing industrial enzyme toolbox and achieve potential applications. In the previous study, an extracellular protease-producing bacterium, Ornithinibacillus sp. L9 T , was isolated from a goatskin and scientifically classified as Ornithinibacillus caprae (Li et al. 2020). In addition, strain L9 T could produce a transparent hydrolysis ring on milk plates supplemented with 100 g/L NaCl. Herein, the extended research was set out to enhance the extracellular protease production from O. caprae L9 T by optimizing the medium components and fermentation conditions. Simultaneously, the crude enzyme was investigated for the biochemical and molecular characteristics, and used as a biocatalyst for dehairing hides to provide a theoretical foundation for further application in the leather industry. Materials And Methods Microorganism and reagents The moderate halobacterium O. caprae L9 T was isolated from a goat hide obtained from a local farm in Chengdu, south-west China (Li et al. 2020). The bacterium was deposited in the laboratory of industrial biotechnology, the Korean Collection for Type Cultures (KCTC, Korea) and China General Microbiological Culture Collection Center (CGMCC, China) with accession number L9, KCTC 43176 and CGMCC 1.17659, respectively. Unless specified, all the chemical reagents used in this experiment were of analytical. Casein, gelatin and collagen were purchased from Sangon Biotech (China). Keratin from wool, azocasein, bovine serum albumin (BSA), Folin & Ciocalteu's phenol reagent and protein marker were obtained from Tokyo Chemical Industry Co., Ltd. (Japan), Sigma-Aldrich (American), BioFroxx (Germany), Solarbio (China) and Takara (Japan), respectively. All other tested chemicals, surfactants, organic solvents and media components were obtained from several commercial companies. Genomic analysis The genome sequence data has been uploaded to the type strain genome server (TYGS) (Meier-Kolthoff and Goker 2019) to determine the genome-based phylogeny of strain L9 T . Meanwhile, the DNA sequences were performed BLASTX search against the non-redundant protein database (NR) (Li et al. 2002) in the National Center for Biotechnology Information to assess the variety of genes in the L9 T strain. Inoculum preparation and protease production The pure culture of bacterium L9 T was precultured in LB broth medium supplemented with 100 g/L NaCl [used to inoculate (2%, v/v) all fermentation medium for protease production]. After the fermentation was completed, the crude enzyme extract was obtained by removing the cell biomass through high-speed centrifugation for 5 min at 4 o C, and stored at 0 o C for further analyses. Determination of protease activity Protease activity was measured using casein as a substrate according to the national standardization administration commission GB/T 23527-2009 with minor modifications. The reaction was performed in an Eppendorf tube with a total volume of 2 mL, containing 0.2 mL of appropriately diluted enzyme solution and 0.2 mL casein (20 g/L) dissolved in Tris-HCl (pH 7). The mixed components were incubated at 70 o C for 10 min, and the reaction was stopped using 0.4 mL of 65.4 g/L trichloroacetic acid (TCA). After shaking evenly, the unreacted casein precipitate was removed by centrifugation at 12000 rpm for 5 min. Later, 0.4 mL of clear supernatant was mixed with 2 mL of 42.4 g/L Na 2 CO 3 and 0.4 mL of 1 M Folin & Ciocalteu's phenol reagent. The mixture was incubated for 20 min at 40 o C and cooled under running water. Then the absorbance was monitored at 680 nm against the control using a UV-Vis spectrophotometer (UV-1100, Mapada, China). The assay was conducted in triplicate, and a control was run in parallel in which the substrate was added after the addition of TCA. One unit of protease activity (U/mL) was designated as the amount of enzyme required to release 1 μg of tyrosine per minute under specified assay conditions. Optimization of protease production by strain L9 T The effect of fermentation process variables on the extracellular protease derived from O. caprae L9 T was investigated using the one-variable-at-a-time approach and response surface methodology (RSM). Three independent variables (C source, N source and initial pH), with the most significant effect on protease production, were selected. Then, the optimum levels and interaction of these three factors for enhancing the protease production were evaluated by RSM using Box-Behnken design (BBD). Each factor was investigated at three different levels: low (-1), central (0) and high (1) (Table S1). The experimental matrix was designed using DesignExpert 8.0.6 (Stat-Ease Inc., Minneapolis, USA), consisting of 17 experimental runs in which 5 runs were repeated at the central level (Table S2). The proteolytic activity (U/mL) was recorded as a response, and the response surface model was constructed and analyzed. SDS-PAGE, zymography and peptide identification Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the crude enzyme was performed using a PAGE gel preparation kit (BaiHe, China) according to the method described by Laemmli (Laemmli 1970) under denaturing conditions. The caseinolytic activity was confirmed by zymography analysis according to the method described by Garciacarreno et al. (1993) with minor modifications. The sample was mixed with an equal volume of 2×SDS non-reducing loading buffer, and then electrophoresed. The gel was soaked twice in 50 mM Tris-HCl pH 7 buffer containing 2.5% (v/v) Triton X-100 with agitation to remove SDS. Subsequently, the gel was submerged in Tris-HCl buffer for 40 min to remove Triton X-100. Later, the hydrolysis reaction occurred inside the gel during incubation at 40 o C for 12 h in Tris-HCl containing 10 g/L casein. At last, the gel was stained with the dye solution composed of 25% (v/v) ethanol, 8% (v/v) acetic acid and 1 g/L Coomassie Brilliant Blue R-250 for 12 h. The appearance of a white band on the dark blue background indicated the presence of protease activity. Later, the white band was extracted from the gel, and subjected to reduction, alkylation, trypsin digestion and desalination. The obtained peptide mixture was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with a TripleTOF 5600 system at Sangon Biotech. All mass spectra were acquired in the m/z 350–1350 mass range, and the obtained peptide sequences were searched against the GenBank database using ProteinPilot software (version 4.5) with the Paragon algorithm. Protease biochemical characterization Temperature optima and thermal stability The optimum temperature of protease activity was evaluated at 30–80 o C for 10 min using Tris-HCl buffer (pH 7), and the maximum value of enzyme activity was set as 100%. Subsequently, the enzyme solution was pre-incubated at 30–80 o C for 1 h, and the residual enzymatic activities were measured at the optimum temperature to evaluate the thermal stability of the protease. All the analyses were conducted in triplicate. pH optima and stability For optimum pH assay, the crude enzyme solution was diluted in the following buffer system 50 mM of glycine-HCl (pH 2–4), sodium acetate-acetic acid (pH 5–6), Tris-HCl (pH 7–9), glycine-NaOH (pH 10–11) and KCl-NaOH (pH 12–13). The proteolytic activity was determined at each pH under the optimum temperature. Later, the enzyme solution was mixed with different pH buffers (pH 2–13) at 25 o C for 1 h to explore the enzyme stability and residual activity at different pH conditions. The unincubated enzyme activity measured by Tris-HCl buffer (pH 7) was considered as 100%. Effect of NaCl on enzyme activity and stability The fermentation supernatant was dialyzed using a dialysis bag (molecular weight cut-off = 8000 Da) against 50 mM Tris-HCl pH 7 buffer for 4 h at 25 o C, and the dialysis buffer was updated hourly to ensure the reliability of the experimental results. Later, the enzyme solution was amended with NaCl at final concentrations of 0–220 g/L, and the effect of NaCl on hydrolytic activity against casein was measured. The salt-containing enzyme solution was preincubated at 25 o C for 1 h prior to determination of residual activities to assess its NaCl stability. The enzyme solution without NaCl served as the control and was considered as 100%. Effect of chemical agents on protease activity The effect of chemical agents on protease catalysis was investigated by preincubating the crude enzyme at 25 o C for 1 h with each reagent. Organic solvents, such as glycerol, methanol, benzene, acetone, ethanol, n-hexane, isopropanol and ethanediol were provided at the working concentrations of 5 and 10% (v/v), respectively. Similarly, the surfactants, such as Tween 20, Tween 80, Triton X-100, dimethyl sulfoxide (DMSO) and H 2 O 2 , were evaluated at 1% (v/v). Other classes of chemical agents were used at a final concentration of 5 mM; protease inhibitors: phenylmethylsulfonyl fluoride (PMSF), ethylene-diamine-tetraacetic acid (EDTA) and ethylene glycol-bis (β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA); reducing agents: dithiothreitol (DTT) and β-mercaptoethanol (β-ME). The crude enzyme solution incubated in the ultrapure water was used as a control and considered as 100% enzyme activity. Effect of different metal ions on protease activity The effect of different metal ions were determined at 5 mM concentration; monovalent cations: Li + , K + and Ag + ; divalent cations: Sr 2+ , Mn 2+ , Fe 2+ , Co 2+ , Cu 2+ , Zn 2+ , Ca 2+ , Ba 2+ and Mg 2+ ; trivalent cations: Fe 3+ and Cr 3+ . The enzyme solution was incubated with the respective metal ion solution at 25 o C for 1 h, and the residual activity was measured following the optimal conditions. The enzyme solution treated with ultrapure water served as the control. Substrate specificity Substrate specificity was determined under standard assay conditions using different protein substrates, including casein, gelatin, collagen, keratin, azocasein and BSA. All the substrates were added to a final concentration of 10 g/L. Dehairing performance of O. caprae L9 T protease The fresh goatskins, cowhides and rabbit skins were procured from a local farm, and required consent was obtained from the farmer to use these animal parts for experiments. Some pieces of skin with hair were cut with a sharp knife from the same area of the obtained hides, then rinsed with tap water several times and drained at 25 o C. Most hides were independently placed into a flask containing 100 mL diluted crude enzyme (600 U) and 30 mg/g Na 2 S, respectively, while the rest of the hides were submerged in 100 mL of Tris-HCl buffer (pH 7) containing 20 g/L NaCl that served as a control. All the flasks were incubated in a shaker incubator for 24 h at 150 rpm and 38 o C. Afterward, the application prospect of crude enzyme in the leather industry was evaluated through dehairing efficacy and histological examination. About 1 cm 2 pelts from the hides treated with Tris-HCl buffer, Na 2 S and enzyme, were incised and fixed with 40 g/L paraformaldehyde (Servicebio, China) for 24 h. The immobilized samples were dehydrated by ethanol, embedded with paraffin wax and cut by microtome. Then the slices were stained by hematoxylin and eosin (HE) and Masson’s trichrome staining, and observed using a microscope (Olympus, Japan). Statistical analysis All experiments were performed in triplicate, and the control experiments were proceeded under the identical conditions. The statistical data analysis was done using the software OriginPro version 8.5, and the results were presented as mean ± standard deviation (SD). Nucleotide sequences accession numbers The 16S rRNA gene sequences and the whole genome of strain L9 T have been submitted to the GenBank database under accession MN577409 and WOCA00000000, respectively. Results And Discussion Genomic characteristics The family Bacillaceae was first proposed by Fischer, and it now includes more than 100 recognized genera (Parte et al. 2020), including Virgibacillus , Oceanobacillus and Ornithinibacillus , the neighbors of the phylogenetic evolution. Additionally, in the phylogenomic tree (Fig. 1), the species of the genera Oceanobacillus and Ornithinibacillus are intertwined with each other, specifying that their genomes have some identical sequences or are highly similar. To date, a few Oceanobacillus species have been isolated and reported to secrete protease (Li et al. 2017; Pandey et al. 2012; Thanapun 2013), lipase, amylase and carboxymethyl cellulase (Rohban et al. 2009; Seghal Kiran et al. 2014). Thus, as the twin brother of Oceanobacillus , the Ornithinibacillus species might also have the ability to produce enzymes. A total of 3944 coding DNA sequences of strain L9 T obtained from homology analyses were annotated by the NR database, including at least 25 enzyme-encoding genes. The most abundant was serine protease, followed by metalloprotease, lipase, amylase and glycosidase. The diversity of enzyme genes provides theoretical support for further research on strain L9 T . Furthermore, a zone of clearing formed by strain L9 T was overtly observed on the milk agar plates, and the protease activity was detected at 60.97 U/mL in the TSB medium supplemented with 100 g/L NaCl. Statistical optimization of protease production Response surface methodology for optimizing experimental design The significant independent variables [yeast extract (A), urea (B) and initial pH (C)] were used to determine the optimum levels of these parameters based on the single-factor experimental results (depicted in Supplementary Material). The response values of 17 experiments are listed in Table S2. Later, the DesignExport software was employed for regression analysis and significance tests. A quadratic model was generated and applied to predict the maximum protease production (Y). The model was as follows: Y (U/mL) = 246.15–12.97A–32.89B–7.51C+0.35AB–37.30AC–8.68BC–42.35A 2 –68.29B 2 –77.57C 2 As summarized in Table 1, the variance analysis results of the regression model indicated that the model was significant as it was obvious from the high model F -value (= 107.4) and very low probability value (< 0.0001). Additionally, the model terms of A, B, C, AC, A 2 , B 2 and C 2 were significant ( p -value less than 0.05), whereas the interaction between urea and yeast extract was not significant ( p -value greater than 0.1). This result indicated that the combination of urea and yeast extract had no significant effect on protease production by O. caprae L9 T . The ‘lack of fit F -value’ of 3.56 implied that the lack of fit was not significant relative to the pure error. Moreover, the good fits were realized, and the variability of most of the test data was explained by the model, with the correlation coefficient (R 2 ) of 99.28% and adjusted R 2 of 98.36%. The adequate precision was 28.942 (> 4), which was desirable, confirming the feasibility of this model to predict protease production by strain L9 T . The interaction effects and optimum values of a combination of the three independent factors for maximum protease production by O. caprae L9 T were represented by three-dimensional response surface graphs and contour plots (Fig. 2). With the increase of yeast extract in the medium, the caseinolytic activity increased first and then decreased (Fig. 2a and 2b). Fig. 2b depicts the shape of the contour to be ellipse, indicating a significant interaction between yeast extract and pH. Validation of the optimized process conditions Based on the regression equation, three repeated verification tests were performed by setting yeast extract, urea and initial pH at the predicted levels of 14.3 g/L, 3.8 g/L and 9.0, respectively. The maximum protease activity of 251.12 U/mL was predicted by the model, which was comparable to the experimental results (255.86 ± 0.71 U/mL), confirming the model’s authenticity. After RSM optimization, the protease yield of O. caprae L9 T increased by 319.65% than the unoptimized medium and original fermentation conditions. Gel electrophoresis and molecular analysis The crude extracellular enzyme produced by strain L9 T was analyzed through SDS-PAGE and zymogram. As depicted in Fig. 3, a single protein band was obtained with the cell-free supernatant, and its proteolytic activity was confirmed by zymogram using casein as a substrate. The molecular weight of L9 T protease was estimated as 26 kDa, which was consistent with some literature reports. Earlier, Deng et al. (2010) reported the characterization of a 28 kDa high-alkaline serine protease from Bacillus sp. B001. Two detergent stable serine proteases named protease BM1 and BM2 with molecular masses of 29 and 15.5 kDa, respectively, were purified from Bacillus mojavensis A21 (Haddar et al. 2009). The unique protein band was extracted and detected by mass spectrometry. The offline data was analyzed by ProteinPilot, and a total of 27 peptide sequences were obtained. Afterward, the quality control of peptide fragment was performed based on the unused ProtScore ≥ 1.3, and 9 peptides (Table S3) with the reliability of no less than 95% were screened out. These peptides exhibited high homology with S8 family serine peptidases, suggesting that the extracellular enzyme of strain L9 T might belong to serine protease family and provides a direction for further research. Biochemical characterization of the protease Effect of pH, temperature and NaCl on enzymatic activity and stability As depicted in Fig. 4a, the protease was active over a wide range of pH (2–13) with the nadir activity at pH 2. More than 80% of the protease activity was retained at pH 3–13. Besides, the pH stability profile (Fig. 4b) showed that the extracellular protease was highly stable in the pH range of 3–13. The enzyme remained 57.54 and 88.64% of its activity at pH 2 and 13, respectively, after 1 h incubation at 25 o C. The protease secreted by O. caprae L9 T has broader pH activity and stability than the previously reported proteases (Ibrahim et al. 2015; Deng et al. 2010). Herein, the protease exhibited a typical characteristic of the serine protease (wider pH action), suggesting it to be a good candidate for industrial applications such as use in the detergents (Ibrahim et al. 2015), tanning processes (Kanagaraj et al. 2015) or biocontrol agent (Darwesh et al. 2020). The proteolytic activity was detected at all test temperatures (30 to 80 o C), and the maximum activity was observed at 70 o C (Fig. 4c), revealing the thermophilic nature of the protease. The crude protease showed only 5.4% of the maximal activity at 30 o C and exhibited a few activities till the enzyme assay at 80 o C. These results were consistent with several studies, including an alkaline serine protease produced by N. dassonvillei OK-18 (Sharma et al. 2020), showing the maximal activity at 70 o C, and protease BM2 secreted by B. mojavensis A21 (Haddar et al. 2009), showing the maximal activity at 60 o C. Thermostability studies have revealed that L9 T protease is quite stable between 30 to 45 o C, retaining 95.75 and 9.02% of the initial activity after 1 h of incubation at 50 and 65 o C (Fig. 4d), respectively. Nevertheless, at 70–80 o C, the enzyme fully denatured and lost activity within 60 min. The effects of NaCl on enzyme activities towards casein were determined in the salinity ranging from 0–220 g/L at 70 o C. The protease exhibited the optimum activity at 20 g/L NaCl (Fig. 5). The protease retained 80.31, 55.23 and 32.49% of its activity at NaCl concentrations of 80, 140 and 220 g/L, respectively, suggesting that high salt concentration suppresses the enzyme activity. Notably, the proteolytic activity measured at different salt concentrations remained unchanged before and after heat preservation, indicating the stability of this protease at all tested NaCl concentrations for 1 h. These results were in accordance with the recent report of alkaline serine protease from Bacillus sp. NPST-AK15 (Ibrahim et al. 2015), showing the maximum activity at 15 g/L NaCl and highly stability in NaCl up to 200 g/L. These findings demonstrate that L9 T protease is a slightly halophilic enzyme (different from extreme halophilic protease), which could be useful for certain biotechnological processes depending on salinity. Effect of various metal ions on protease activity Table 2 summarizes the proteolytic activity of the crude enzyme under incubation with different metal ions. Ag + , Ca 2+ and Sr 2+ significantly enhanced the enzyme activities at a rate of 104.46, 104.40 and 120.37% than the control at a final concentration of 5 mM. In consistent with the present study, many investigators have reported the stimulating effect of Ca 2+ on serine proteases (Ben Elhoul et al. 2015; Jagadeesan et al. 2020; Jaouadi et al. 2013). Gong et al. (2015) has reported that Sr 2+ increased the keratinase activity by 10%. The above-mentioned results suggest that Ag + , Ca 2+ and Sr 2+ could maintain the stability of the active site of L9 T protease, which has a selective preference for Sr 2+ ions at a certain concentration as an inducer. The addition of Li + , K + , Ba 2+ , Mg 2+ , Mn 2+ and Co 2+ had a little effect on the enzyme activities. Other ions, including Fe 2+ , Fe 3+ , Cu 2+ and Zn 2+ , significantly inhibited the enzyme activities, and a loss of 70% in the activity was observed with Fe 3+ . Moreover, under the same conditions, Fe 3+ ions are more likely to deactivate enzyme than Fe 2+ , which is presumably attributed to the fact that Fe 3+ easily absorbs electrons from the enzyme surface through strong oxidation, thereby destroying the enzyme stability. Herein, the enzyme was moderately affected by Cr 3+ at 5 mM, revealing that the protease might act as an adjuvant to the chrome tannage and bating processes in leather industry. Effect of chemical substances on protease activity The effects of various organic solvents on protease were appraised to examine the potential application. Results showed that the crude enzyme was considerably stable in the presence of the tested solvents (Table 3), particularly at a concentration of 5% (v/v). Upon incubation with 10% (v/v) of glycerol, benzene and n-hexane for 1 h, the enzyme also displayed high stability, with respective residual activity of 99.50, 100.76 and 95.70%. This remarkable stability exhibited by L9 T protease in common organic solvents supported the potential candidacy of it as a biocatalyst for the synthesis of peptide and ester. Later, the effects of surfactants on the extracellular protease were evaluated, and the corresponding results are presented in Table 4. The stimulatory effects were observed in the presence of some nonionic surfactants, such as Tween 20 (163.41%) and Tween 80 (115.01%), which were higher than previous reports (Jagadeesan et al. 2020; Ibrahim et al. 2015). On the contrary, the inhibitory effect of Triton X-100 was observed at a concentration of 1% (v/v). The enhanced activity of the enzyme in presence of DMSO reflect the fact that DMSO is an amphipathic molecule that easily penetrates the enzyme with low toxicity, and 99.94% of the activity was maintained (data not shown) at 10% concentration. The strong anionic surfactant sodium dodecyl sulfate (SDS) at 10 g/L inhibited the protease activity up to 76.98%, which was consistent with most of the previous conclusions reporting that SDS denatures and inactivates the enzyme (Ibrahim et al. 2015; Suwannaphan et al. 2017). Furthermore, the L9 T protease was moderately stable against H 2 O 2 , retaining 88.18% of its initial activity at a concentration of 1%. All these prominent features ensure that the enzyme is compatible with certain detergent formulations. Moreover, the effect of various inhibitors and chelators on L9 T protease activity was examined (Table 4). The protease remained unstimulated against thiol reagents, such as DTT and β-ME at a concentration of 5 mM, suggesting that L9 T protease is a thiol-independent enzyme, and the thiol group does not directly participate in the catalytic reaction (Ben Elhoul et al. 2016). On the contrary, the protease activity was partially inhibited by the chelators EDTA and EGTA, which confirmed that L9 T protease is not a member of the metalloprotease family but uses certain cations as stabilizers (Deng et al. 2010; Jellouli et al. 2009). Notably, L9 T protease showed no activity in the presence of 5 mM PMSF, confirming the enzyme to be a serine protease (Jagadeesan et al. 2020). Substrate specificity profile of the L9 T protease The effect of various substrates on L9 T protease activity is summarized in Fig. S4. The highest activity was observed against casein, but a relatively high activity was observed against azocasein. However, no activity was observed toward gelatin, collagen and keratin. Therefore, the absence of collagenase activity indicates the potential application of L9 T protease in hair removal in the leather industry without damaging the skin collagen. Hide dehairing ability of the crude enzyme The dehairing efficacy of the crude enzyme from O. caprae L9 T on hides was evaluated by touch-visual tests and histological analyses. The pelt with hair, traditional dehaired pelt, and enzymatic dehaired hide are depicted in Fig. 6. The results elucidated that both the traditional sodium sulfide depilation and enzymatic dehairing could effectively remove the hair from goatskins, cowhides and rabbit skins; however, the control hides, incubated under the same conditions, showed no sign of hair removal. Notably, the enzymatic dehaired pelts were white in color and displayed smooth grain surface, soft touch and clean hair pores; while, organoleptic tests showed that the skins obtained after chemical dehairing were dark brown or yellow in color, wrinkled and hard in touch. Sodium sulfide dissolved in water could produce large amounts of hydroxide, and the disulfide bond of the hair breaks under the action of strong alkali to complete the hair removal process (Sujitha et al. 2018), which leads to the production of toxic gas H 2 S. In contrast, as a biologically active catalyst, the crude enzyme cannot directly act on hair shafts; instead, it may hydrolyze the mucinoid, mucin, albumin and globulin in raw skin tissue, thereby destroying the bond between the hair follicle and the hair root, the hair bulb and the hair papilla, to achieve the purpose of hair removal (Sujitha et al. 2018; Yates 1972). The histological images of control, Na 2 S and crude enzyme treated goatskins stained with HE and Masson's trichrome staining are depicted in Fig. S5. The results demonstrated that compared with the blank control, the epidermis was removed from the goatskins via two different treatments; and the collagen fiber structure of the enzymatically dehaired pelt was more regular and intact compared with the Na 2 S treatment. It could be observed from Masson’s trichrome staining images that the collagen components appeared in blue color, and the non-collagen substances appeared in deep red color. This result suggested that the enzyme treatment could well maintain the inherent collagen component in the dermal structure, while part of the damaged collagen existed in the fur treated with Na 2 S. Overall, the crude enzyme exhibited no collagenase activity, and was advantageous over chemical depilatory agents. In this study, the crude protease completely dehaired the skins from goat and rabbit (Fig. 6a and 6c) in 24 h without any chemicals. This result strengthened the hypothesis that this protease could be beneficial to protect the environment and reduce energy waste (Jaouadi et al. 2013; Kandasamy et al. 2012). Moreover, the reasons for incomplete enzymatic depilation of cowhide (Fig. 6b) might be attributed to the fact that the thick cowhide leads to slower enzyme penetration and lack of mechanical pulling force. It is noteworthy to mention that the microbial enzymes with excellent dehairing ability are stable in an alkaline environment, especially between pH 8 and 10 (Jaouadi et al. 2013; Bouacem et al. 2016). Fortunately, L9 T protease meets these criteria and hence, is considered as a potential dehairing candidate in improving leather quality. Conclusions In the present study, a novel thermophilic and slightly halophilic serine protease from O. caprae L9 T was reported. The halophile exhibited a higher protease production capacity (255.86 U/mL) under the following conditions: 72 h of fermentation time, initial pH 9, culture temperature 37 o C, and the modified medium containing 14.3 g yeast extract, 3.8 g urea, 130 g NaCl and 1 L distilled water. The crude enzyme appeared as a single band on SDS-PAGE with a molecular mass of 26 kDa. The optimum temperature for caseinolytic activity was 70 o C. Furthermore, the protease activity was enhanced by metal ions Ca 2+ , Sr 2+ , Ag + and 20 g/L NaCl. The enzyme exhibited excellent stability toward surfactants, organic solvents and a wide range of pH from 3 to 13. Further studies confirmed that the crude protease has an excellent ability to remove hair in a short time without damaging the collagen. In summary, the protease could be a propitious candidate for various industrial applications. Declarations Acknowledgement This work was supported by the National Key Research and Development Program of China (2017YFB0308401). Author contributions XL performed the experiment and wrote the manuscript. QZ revised the grammar and typography of the manuscript. LG and GJ provided guidance on response surface analyses. YT supervised and guided the experiments. BS critically reviewed the structure of manuscript. Compliance with ethical standards Conflicts of interest The authors declare that there are no conflicts of interest. Ethical statement This article does not contain any studies with human participants and/or animals performed by any of the authors. References Barzkar N (2020) Marine microbial alkaline protease: An efficient and essential tool for various industrial applications. Int J Biol Macromol 161:1216–1229 Ben Elhoul M, Zarai Jaouadi N, Rekik H, Bejar W, Boulkour Touioui S, Hmidi M, Badis A, Bejar S, Jaouadi B (2015) A novel detergent-stable solvent-tolerant serine thiol alkaline protease from Streptomyces koyangensis TN650. Int J Biol Macromol 79:871–882 Ben Elhoul M, Zarai Jaouadi N, Rekik H, Omrane Benmrad M, Mechri S, Moujehed E, Kourdali S, El Hattab M, Badis A, Bejar S, Jaouadi B (2016) Biochemical and molecular characterization of new keratinoytic protease from Actinomadura viridilutea DZ50. 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Int J Biol Macromol 133:987–997 Suwannaphan S, Fufeungsombut E, Promboon A, Chim-anage P (2017) A serine protease from newly isolated Bacillus sp for efficient silk degumming, sericin degrading and colour bleaching activities. Int Biodeter Biodegr 117:141–149 Thanapun T (2013) Screening and characterization of protease-producing Virgibacillus , Halobacillus and Oceanobacillus strains from Thai fermented fish. J App Pharm Sci 3:025–030 Thanikaivelan P, Rao JR, Nair BU, Ramasami T (2004) Progress and recent trends in biotechnological methods for leather processing. Trends Biotechnol 22:181–188 Wang R, Min C, Haiming C, Li ZQ (2009) Enzyme unhairing- an eco-friendly process. J Soc Leath Tech Ch 93:51–55 Wang SL, Hsu WT, Liang TW, Yen YH, Wang CL (2008) Purification and characterization of three novel keratinolytic metalloproteases produced by Chryseobacterium indologenes TKU014 in a shrimp shell powder medium. Bioresour Technol 99:5679–5686 Ward OP (2011) Proteases, in: M. Moo-Young (Ed.), Comprehensive Biotechnology (Second Edition), Academic Press, Burlington, pp 571–582 Yates JR (1972) Studies in depilation. Part X. The mechanism of the enzyme depilation process. J Soc Leather Trades Chem 56:158–177 Tables Table 1. Analysis of variance for the regression equation Source Sum of squares df Mean squares F -value p -value Model 74551.59 9 8283.51 107.40 < 0.0001** A-Yeast extract 1346.02 1 1346.02 17.45 0.0042** B-Urea 8652.39 1 8652.39 112.18 < 0.0001** C-pH 451.79 1 451.79 5.86 0.0461* AB 0.48 1 0.48 0.01 0.9392† AC 5565.63 1 5565.63 72.16 < 0.0001** BC 301.41 1 301.41 3.91 0.0886 A 2 7552.10 1 7552.10 97.91 < 0.0001** B 2 19637.99 1 19637.99 254.61 < 0.0001** C 2 25334.82 1 25334.82 328.47 < 0.0001** Residual 539.90 7 77.13 Lack of fit 392.94 3 130.98 3.56 0.1256† Pure error 146.97 4 36.74 Cor total 75091.49 16 F , F ratio; p-value , probability value; df, degree of freedom. *, Significant ( p < 0.05); **, very significant ( p 0.1). R 2 = 0.9928; adjusted R 2 = 0.9836; adequate precision = 28.942. Table 2. Effect of different metal ions on the protease activity Metal ions Final Concentration (mM) Relative activity (± SD) (%) Control - 100.00 ± 0.79 Li + 5 90.19 ± 0.97 K + 5 97.71 ± 2.49 Ag + 5 104.46 ± 1.27 Fe 2+ 5 84.85 ± 1.25 Cu 2+ 5 85.32 ± 1.40 Zn 2+ 5 78.39 ± 2.47 Ca 2+ 5 104.40 ± 2.12 Mg 2+ 5 97.77 ± 2.00 Ba 2+ 5 93.31 ± 2.92 Mn 2+ 5 96.36 ± 2.13 Co 2+ 5 95.54 ± 0.35 Sr 2+ 5 120.37 ± 3.10 Fe 3+ 5 28.55 ± 3.16 Cr 3+ 5 84.15 ± 1.64 Table 3. Effect of organic solvents on L9 T protease stability Solvents Relative activity (± SD) (%) 5% (v/v) 10% (v/v) Control 100.00 ± 0.1 - Acetone 88.44 ± 5.31 85.09 ± 3.72 Benzene 100.76 ± 2.24 95.83 ± 6.36 Ethanediol 99.12 ± 2.28 93.37 ± 2.10 Ethanol 90.90 ± 0.77 74.92 ± 4.05 Glycerol 88.76 ± 3.03 99.50 ± 1.22 n-Hexane 91.16 ± 1.91 95.70 ± 3.94 Isopropanol 91.98 ± 1.37 71.64 ± 4.18 Methanol 97.22 ± 3.90 89.77 ± 2.22 Table 4. Effect of various surfactants, oxidizing agent, alkahest and inhibitors on crude serine protease from O. caprae L9 T Chemical substances Working concentration Relative activity (± SD) (%) Control - 100.00 ± 3.06 Surfactants Tween 20 1% (v/v) 163.41 ± 2.23 Tween 80 1% (v/v) 115.01 ± 1.13 Triton X-100 1% (v/v) 90.34 ± 3.32 SDS 10 g/L 23.02 ± 1.18 Oxidizing agent H 2 O 2 1% (v/v) 88.18 ± 1.18 Alkahest DMSO 1% (v/v) 110.22 ± 1.70 Inhibitors DTT 5 mM 96.96 ± 0.73 β-Me 5 mM 95.05 ± 2.23 EDTA 5 mM 30.44 ± 2.77 EGTA 5 mM 49.85 ± 0.60 PMSF 5 mM 0 Supplementary Files CertificateofEnglishEditing.pdf SupplementaryMaterial.pdf Cite Share Download PDF Status: Published Journal Publication published 19 Nov, 2021 Read the published version in Journal of Microbiology and Biotechnology → Version 1 posted 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. 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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-573853","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":30360783,"identity":"5c5a7673-f5cf-40c7-ab6b-1c85d30f5a81","order_by":0,"name":"Xiaoguang Li","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoguang","middleName":"","lastName":"Li","suffix":""},{"id":30360784,"identity":"d75d7469-0161-46b4-b953-e185f955c98f","order_by":1,"name":"Qian Zhang","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Zhang","suffix":""},{"id":30360785,"identity":"344ac162-df64-43f9-a47b-995d9fcaf20c","order_by":2,"name":"Longzhan Gan","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Longzhan","middleName":"","lastName":"Gan","suffix":""},{"id":30360786,"identity":"e1a43aa0-dec7-41fd-be5f-af8c1e489886","order_by":3,"name":"Guangyang Jiang","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Guangyang","middleName":"","lastName":"Jiang","suffix":""},{"id":30360787,"identity":"b1d70206-a31c-41f5-867a-e4221245dd57","order_by":4,"name":"Yongqiang Tian","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYJCCAwwMNhAWD3EamEFa0kjUAgSHSdBicDz/4IGfO87b889IYHzwto1B3pygljOPGQ72nrmdOONGArPh3DYGw50NhLTcSGY4wNt2O8FAIoFNmreNIcHgABFaDv5tO2cP1ML+m2gth3nbDjBuANrCTJQWyTOPDQ7LtiUnzjjzsFlyzjkJww2EtPAdT3z88W2bnT1/e/LBD2/KbOQJ2qJwIAHGZGwAEhIE1AOBfEMCQTWjYBSMglEw0gEADmFDAF2cN8IAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-5729-209X","institution":"Sichuan University","correspondingAuthor":true,"prefix":"","firstName":"Yongqiang","middleName":"","lastName":"Tian","suffix":""},{"id":30360788,"identity":"4a1c23d0-ecaa-4167-ad4c-c0eef4ad506f","order_by":5,"name":"Bi Shi","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Bi","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2021-05-29 21:21:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-573853/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-573853/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.4014/jmb.2108.08037","type":"published","date":"2021-11-20T01:17:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":10090970,"identity":"27031fa4-e9fa-4def-8b63-55e219a9e884","added_by":"auto","created_at":"2021-06-07 20:00:10","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":85192,"visible":true,"origin":"","legend":"The phylogenomic tree based on genome sequences of O. caprae L9T and the related type species using the genome blast distance phylogeny (GBDP) method. The numbers above branches are GBDP pseudo-bootstrap support values \u003e 70% from 100 replications.","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-573853/v1/8c255e55200c79c5e6607f1c.jpg"},{"id":10091296,"identity":"e28435c1-2fa3-4620-80c1-2679501da099","added_by":"auto","created_at":"2021-06-07 20:06:10","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":214486,"visible":true,"origin":"","legend":"Three-dimensional response surface graphs and contour plots of extracellular protease production by O. caprae L9T elucidating the interaction between: a, yeast extract and urea; b, yeast extract and pH; c, urea and pH.","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-573853/v1/7c8bc194300fd54b019bc140.jpg"},{"id":10091244,"identity":"5ef1e35e-4c46-498c-8f33-d4fc827d706c","added_by":"auto","created_at":"2021-06-07 20:03:10","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":20035,"visible":true,"origin":"","legend":"SDS-PAGE (12.5%) of extracellular protease from O. caprae L9T. Lane 3, cell-free supernatant; lane M, molecular weight marker; lane 6, crude enzyme zymogram.\n\n","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-573853/v1/13a4afc59b8e35c8cad1e2fd.jpg"},{"id":10091245,"identity":"fc265c46-f733-46fd-b1bf-6984858f09c7","added_by":"auto","created_at":"2021-06-07 20:03:10","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":198221,"visible":true,"origin":"","legend":"Effect of pH and temperature on the protease activities from O. caprae L9T. a, Relative activities at different pH values; b, pH stability of the protease; c, relative activities at different temperatures; d, thermal stability of the protease. Results represent means of three experiments, and vertical bars indicate standard error of the mean. \n\n","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-573853/v1/ff23b89cfeaec19b2962de11.jpg"},{"id":10091310,"identity":"47e28b05-8bea-4da7-b9de-cbcef02f3e96","added_by":"auto","created_at":"2021-06-07 20:09:10","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":42283,"visible":true,"origin":"","legend":"Effect of various concentrations of NaCl (0–220 g/L) on activity and stability of the dialysed L9T protease. Each value represents mean ± SD, n = 3.","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-573853/v1/d5a776d3d4b72a70849e8531.jpg"},{"id":10091247,"identity":"bcda2a54-2b06-4489-93d4-24019ca44737","added_by":"auto","created_at":"2021-06-07 20:03:10","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":75637,"visible":true,"origin":"","legend":"Dehairing performance on goatskin (a), cowhide (b) and rabbit skin (c) of crude enzyme from O. caprae L9T.","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-573853/v1/1efc3cb368a280ea0680120d.jpg"},{"id":15879325,"identity":"5b1e4377-1656-4fb2-a2e5-c4a22dfa6baf","added_by":"auto","created_at":"2021-11-25 01:17:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":773552,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-573853/v1/e127ccf3-66c2-4fd9-9617-5cd4a2c3671b.pdf"},{"id":10091250,"identity":"972bed59-7462-4675-9930-091af3e92c2d","added_by":"auto","created_at":"2021-06-07 20:03:10","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":307205,"visible":true,"origin":"","legend":"","description":"","filename":"CertificateofEnglishEditing.pdf","url":"https://assets-eu.researchsquare.com/files/rs-573853/v1/a1148244ce715a70a3360b41.pdf"},{"id":10091249,"identity":"b1b5d000-fab9-433b-a7f3-5b45ca374815","added_by":"auto","created_at":"2021-06-07 20:03:10","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1211769,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-573853/v1/f62f115686ebef9e64897434.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eExoproduction and Biochemical Characterization of a Novel Thermophilic Serine Protease From Ornithinibacillus Caprae L9\u003csup\u003eT \u003c/sup\u003eWith Hide-dehairing Activity\u003c/p\u003e","fulltext":[{"header":"Introduction ","content":"\u003cp\u003eChina, one of the major leather-producing countries, is well known for its most active and potential leather trading markets worldwide. According to the China Leather Industry Association data, around 529 million square meters of genuine leather was produced in China in 2019. The leather industry mainly uses hides and skins, the livestock by-products, as the raw materials for processing. Leather processing involves many physical and chemical treatments such as soaking, dehairing, bating and tanning (Ward 2011). The dehairing of skin/hide is considered as the most polluting process (Kandasamy et al. 2012). The traditional hair-removal methods require the use of several chemical depilatory agents, such as sodium sulfide, sodium bisulfide and calcium chloride (Ockerman and Basu 2014), which contribute to environmental pollutions, including the generation of noxious gases and solid wastes (Kanagaraj et al. 2015; Thanikaivelan et al. 2004). Therefore, the introduction of more eco-friendly methods is highly desirable. In this regard, microbial proteases could fill this niche (Paul et al. 2016).\u003c/p\u003e\n\u003cp\u003eEnzymatic dehairing, which has been proved to be the most cost-effective and eco-friendly process, exerts tremendous potential in obtaining high-quality leather (Wang et al. 2009). This phenomenon has increased the attention toward the exploitation of enzyme-producing microorganisms (Barzkar 2020). So far, a variety of extracellular proteases have been reported and characterized from different microorganisms, including fungi like \u003cem\u003ePaecilomyces marquandii\u003c/em\u003e (Gradisar et al. 2005), and bacteria, such as \u003cem\u003eBacillus subtilis\u003c/em\u003e (Dettmer et al. 2013), \u003cem\u003eLactobacillus curvatus\u003c/em\u003e (Sun et al. 2019), \u003cem\u003eNocardiopsis dassonvillei\u003c/em\u003e (Sharma et al. 2020) and \u003cem\u003eStreptomyces koyangensis\u003c/em\u003e (Ben Elhoul et al. 2015). Interestingly, most of the reported extracellular proteases are serine proteases (Brandelli 2007; Jaouadi et al. 2013), while a few belong to metalloproteases (Wang et al. 2008; Chandrasekaran et al. 2016). As a member of the proteolytic enzyme family, serine proteases exert a wide range of biotechnological applications, including skin dehairing (Jaouadi et al. 2013), feather degradation (Jagadeesan et al. 2020), stain washing (Haddar et al. 2009), food production (Kostyleva et al. 2016), silk degumming and sericin degrading (Suwannaphan et al. 2017). Of the reported proteases to date, the extracellular serine proteases were preponderantly secreted by \u003cem\u003eBacillus\u003c/em\u003e strains. However, little attention has been paid to the rare strains, especially the species of \u003cem\u003eOrnithinibacillus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eOrnithinibacillus\u003c/em\u003e species are generally Gram-stain-positive, aerobic, rod-shaped and moderately halophilic bacteria from the family \u003cem\u003eBacillaceae\u003c/em\u003e (Li et al. 2020). Over the past decades, a total of 12 strains have been isolated and placed in the genus \u003cem\u003eOrnithinibacillus \u003c/em\u003e(Parte et al. 2020)\u003cem\u003e.\u003c/em\u003e Despite these studies, no protease from \u003cem\u003eOrnithinibacillus\u003c/em\u003e strains has been reported so far. Therefore, further research on the proteases secreted by \u003cem\u003eOrnithinibacillus \u003c/em\u003especies is highly necessitated to expand the existing industrial enzyme toolbox and achieve potential applications. In the previous study, an extracellular protease-producing bacterium, \u003cem\u003eOrnithinibacillus \u003c/em\u003esp. L9\u003csup\u003eT\u003c/sup\u003e, was isolated from a goatskin and scientifically classified as \u003cem\u003eOrnithinibacillus caprae\u003c/em\u003e (Li et al. 2020). In addition, strain L9\u003csup\u003eT\u003c/sup\u003e could produce a transparent hydrolysis ring on milk plates supplemented with 100 g/L NaCl. Herein, the extended research was set out to enhance the extracellular protease production from \u003cem\u003eO. caprae \u003c/em\u003eL9\u003csup\u003eT\u003c/sup\u003e by optimizing the medium components and fermentation conditions. Simultaneously, the crude enzyme was investigated for the biochemical and molecular characteristics, and used as a biocatalyst for dehairing hides to provide a theoretical foundation for further application in the leather industry.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eMicroorganism and reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe moderate halobacterium \u003cem\u003eO. caprae \u003c/em\u003eL9\u003csup\u003eT\u003c/sup\u003e was isolated from a goat hide obtained from a local farm in Chengdu, south-west China (Li et al. 2020). The bacterium was deposited in the laboratory of industrial biotechnology, the Korean Collection for Type Cultures (KCTC, Korea) and China General Microbiological Culture Collection Center (CGMCC, China) with accession number L9, KCTC 43176 and CGMCC 1.17659, respectively.\u003c/p\u003e\n\u003cp\u003eUnless specified, all the chemical reagents used in this experiment were of analytical. Casein, gelatin and collagen were purchased from Sangon Biotech (China). Keratin from wool, azocasein, bovine serum albumin (BSA), Folin \u0026amp; Ciocalteu's phenol reagent and protein marker were obtained from Tokyo Chemical Industry Co., Ltd. (Japan), Sigma-Aldrich (American), BioFroxx (Germany), Solarbio (China) and Takara (Japan), respectively. All other tested chemicals, surfactants, organic solvents and media components were obtained from several commercial companies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenomic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genome sequence data has been uploaded to the type strain genome server (TYGS) (Meier-Kolthoff and Goker 2019) to determine the genome-based phylogeny of strain L9\u003csup\u003eT\u003c/sup\u003e. Meanwhile, the DNA sequences were performed BLASTX search against the non-redundant protein database (NR) (Li et al. 2002) in the National Center for Biotechnology Information to assess the variety of genes in the L9\u003csup\u003eT\u003c/sup\u003e strain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInoculum preparation and protease production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe pure culture of bacterium L9\u003csup\u003eT\u003c/sup\u003e was precultured in LB broth medium supplemented with 100 g/L NaCl [used to inoculate (2%, v/v) all fermentation medium for protease production]. After the fermentation was completed, the crude enzyme extract was obtained by removing the cell biomass through high-speed centrifugation for 5 min at 4 \u003csup\u003eo\u003c/sup\u003eC, and stored at 0 \u003csup\u003eo\u003c/sup\u003eC for further analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of protease activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtease activity was measured using casein as a substrate according to the national standardization administration commission GB/T 23527-2009 with minor modifications. The reaction was performed in an Eppendorf tube with a total volume of 2 mL, containing 0.2 mL of appropriately diluted enzyme solution and 0.2 mL casein (20 g/L) dissolved in Tris-HCl (pH 7). The mixed components were incubated at 70 \u003csup\u003eo\u003c/sup\u003eC for 10 min, and the reaction was stopped using 0.4 mL of 65.4 g/L trichloroacetic acid (TCA). After shaking evenly, the unreacted casein precipitate was removed by centrifugation at 12000 rpm for 5 min. Later, 0.4 mL of clear supernatant was mixed with 2 mL of 42.4 g/L Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e and 0.4 mL of 1 M Folin \u0026amp; Ciocalteu's phenol reagent. The mixture was incubated for 20 min at 40 \u003csup\u003eo\u003c/sup\u003eC and cooled under running water. Then the absorbance was monitored at 680 nm against the control using a UV-Vis spectrophotometer (UV-1100, Mapada, China). The assay was conducted in triplicate, and a control was run in parallel in which the substrate was added after the addition of TCA. One unit of protease activity (U/mL) was designated as the amount of enzyme required to release 1 \u0026mu;g of tyrosine per minute under specified assay conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOptimization of protease production by strain L9\u003csup\u003eT\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effect of fermentation process variables on the extracellular protease derived from \u003cem\u003eO. caprae \u003c/em\u003eL9\u003csup\u003eT\u003c/sup\u003e was investigated using the one-variable-at-a-time approach and response surface methodology (RSM). Three independent variables (C source, N source and initial pH), with the most significant effect on protease production, were selected. Then, the optimum levels and interaction of these three factors for enhancing the protease production were evaluated by RSM using Box-Behnken design (BBD). Each factor was investigated at three different levels: low (-1), central (0) and high (1) (Table S1). The experimental matrix was designed using DesignExpert 8.0.6 (Stat-Ease Inc., Minneapolis, USA), consisting of 17 experimental runs in which 5 runs were repeated at the central level (Table S2). The proteolytic activity (U/mL) was recorded as a response, and the response surface model was constructed and analyzed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSDS-PAGE, zymography and peptide identification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the crude enzyme was performed using a PAGE gel preparation kit (BaiHe, China) according to the method described by Laemmli (Laemmli 1970) under denaturing conditions.\u003c/p\u003e\n\u003cp\u003eThe caseinolytic activity was confirmed by zymography analysis according to the method described by Garciacarreno et al. (1993) with minor modifications. The sample was mixed with an equal volume of 2\u0026times;SDS non-reducing loading buffer, and then electrophoresed. The gel was soaked twice in 50 mM Tris-HCl pH 7 buffer containing 2.5% (v/v) Triton X-100 with agitation to remove SDS. Subsequently, the gel was submerged in Tris-HCl buffer for 40 min to remove Triton X-100. Later, the hydrolysis reaction occurred inside the gel during incubation at 40 \u003csup\u003eo\u003c/sup\u003eC for 12 h in Tris-HCl containing 10 g/L casein. At last, the gel was stained with the dye solution composed of 25% (v/v) ethanol, 8% (v/v) acetic acid and 1 g/L Coomassie Brilliant Blue R-250 for 12 h. The appearance of a white band on the dark blue background indicated the presence of protease activity.\u003c/p\u003e\n\u003cp\u003eLater, the white band was extracted from the gel, and subjected to reduction, alkylation, trypsin digestion and desalination. The obtained peptide mixture was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with a TripleTOF 5600 system at Sangon Biotech. All mass spectra were acquired in the m/z 350\u0026ndash;1350 mass range, and the obtained peptide sequences were searched against the GenBank database using ProteinPilot software (version 4.5) with the Paragon algorithm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtease biochemical characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTemperature optima and thermal stability\u003c/p\u003e\n\u003cp\u003eThe optimum temperature of protease activity was evaluated at 30\u0026ndash;80 \u003csup\u003eo\u003c/sup\u003eC for 10 min using Tris-HCl buffer (pH 7), and the maximum value of enzyme activity was set as 100%. Subsequently, the enzyme solution was pre-incubated at 30\u0026ndash;80 \u003csup\u003eo\u003c/sup\u003eC for 1 h, and the residual enzymatic activities were measured at the optimum temperature to evaluate the thermal stability of the protease. All the analyses were conducted in triplicate.\u003c/p\u003e\n\u003cp\u003epH optima and stability\u003c/p\u003e\n\u003cp\u003eFor optimum pH assay, the crude enzyme solution was diluted in the following buffer system 50 mM of glycine-HCl (pH 2\u0026ndash;4), sodium acetate-acetic acid (pH 5\u0026ndash;6), Tris-HCl (pH 7\u0026ndash;9), glycine-NaOH (pH 10\u0026ndash;11) and KCl-NaOH (pH 12\u0026ndash;13). The proteolytic activity was determined at each pH under the optimum temperature. Later, the enzyme solution was mixed with different pH buffers (pH 2\u0026ndash;13) at 25 \u003csup\u003eo\u003c/sup\u003eC for 1 h to explore the enzyme stability and residual activity at different pH conditions. The unincubated enzyme activity measured by Tris-HCl buffer (pH 7) was considered as 100%.\u003c/p\u003e\n\u003cp\u003eEffect of NaCl on enzyme activity and stability\u003c/p\u003e\n\u003cp\u003eThe fermentation supernatant was dialyzed using a dialysis bag (molecular weight cut-off = 8000 Da) against 50 mM Tris-HCl pH 7 buffer for 4 h at 25 \u003csup\u003eo\u003c/sup\u003eC, and the dialysis buffer was updated hourly to ensure the reliability of the experimental results. Later, the enzyme solution was amended with NaCl at final concentrations of 0\u0026ndash;220 g/L, and the effect of NaCl on hydrolytic activity against casein was measured. The salt-containing enzyme solution was preincubated at 25 \u003csup\u003eo\u003c/sup\u003eC for 1 h prior to determination of residual activities to assess its NaCl stability. The enzyme solution without NaCl served as the control and was considered as 100%.\u003c/p\u003e\n\u003cp\u003eEffect of chemical agents on protease activity\u003c/p\u003e\n\u003cp\u003eThe effect of chemical agents on protease catalysis was investigated by preincubating the crude enzyme at 25 \u003csup\u003eo\u003c/sup\u003eC for 1 h with each reagent. Organic solvents, such as glycerol, methanol, benzene, acetone, ethanol, n-hexane, isopropanol and ethanediol were provided at the working concentrations of 5 and 10% (v/v), respectively. Similarly, the surfactants, such as Tween 20, Tween 80, Triton X-100, dimethyl sulfoxide (DMSO) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, were evaluated at 1% (v/v). Other classes of chemical agents were used at a final concentration of 5 mM; protease inhibitors: phenylmethylsulfonyl fluoride (PMSF), ethylene-diamine-tetraacetic acid (EDTA) and ethylene glycol-bis (\u0026beta;-aminoethyl ether)-N,N,N\u0026rsquo;,N\u0026rsquo;-tetraacetic acid (EGTA); reducing agents: dithiothreitol (DTT) and \u0026beta;-mercaptoethanol (\u0026beta;-ME). The crude enzyme solution incubated in the ultrapure water was used as a control and considered as 100% enzyme activity.\u003c/p\u003e\n\u003cp\u003eEffect of different metal ions on protease activity\u003c/p\u003e\n\u003cp\u003eThe effect of different metal ions were determined at 5 mM concentration; monovalent cations: Li\u003csup\u003e+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e and Ag\u003csup\u003e+\u003c/sup\u003e; divalent cations: Sr\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e, Co\u003csup\u003e2+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, Ba\u003csup\u003e2+\u003c/sup\u003e and Mg\u003csup\u003e2+\u003c/sup\u003e; trivalent cations: Fe\u003csup\u003e3+\u003c/sup\u003e and Cr\u003csup\u003e3+\u003c/sup\u003e. The enzyme solution was incubated with the respective metal ion solution at 25 \u003csup\u003eo\u003c/sup\u003eC for 1 h, and the residual activity was measured following the optimal conditions. The enzyme solution treated with ultrapure water served as the control.\u003c/p\u003e\n\u003cp\u003eSubstrate specificity\u003c/p\u003e\n\u003cp\u003eSubstrate specificity was determined under standard assay conditions using different protein substrates, including casein, gelatin, collagen, keratin, azocasein and BSA. All the substrates were added to a final concentration of 10 g/L.\u003c/p\u003e\n\u003cp\u003eDehairing performance of \u003cem\u003eO. caprae\u003c/em\u003e L9\u003csup\u003eT\u003c/sup\u003e protease\u003c/p\u003e\n\u003cp\u003eThe fresh goatskins, cowhides and rabbit skins were procured from a local farm, and required consent was obtained from the farmer to use these animal parts for experiments. Some pieces of skin with hair were cut with a sharp knife from the same area of the obtained hides, then rinsed with tap water several times and drained at 25 \u003csup\u003eo\u003c/sup\u003eC. Most hides were independently placed into a flask containing 100 mL diluted crude enzyme (600 U) and 30 mg/g Na\u003csub\u003e2\u003c/sub\u003eS, respectively, while the rest of the hides were submerged in 100 mL of Tris-HCl buffer (pH 7) containing 20 g/L NaCl that served as a control. All the flasks were incubated in a shaker incubator for 24 h at 150 rpm and 38 \u003csup\u003eo\u003c/sup\u003eC. Afterward, the application prospect of crude enzyme in the leather industry was evaluated through dehairing efficacy and histological examination.\u003c/p\u003e\n\u003cp\u003eAbout 1 cm\u003csup\u003e2\u003c/sup\u003e pelts from the hides treated with Tris-HCl buffer, Na\u003csub\u003e2\u003c/sub\u003eS and enzyme, were incised and fixed with 40 g/L paraformaldehyde (Servicebio, China) for 24 h. The immobilized samples were dehydrated by ethanol, embedded with paraffin wax and cut by microtome. Then the slices were stained by hematoxylin and eosin (HE) and Masson\u0026rsquo;s trichrome staining, and observed using a microscope (Olympus, Japan).\u003c/p\u003e\n\u003cp\u003eStatistical analysis\u003c/p\u003e\n\u003cp\u003eAll experiments were performed in triplicate, and the control experiments were proceeded under the identical conditions. The statistical data analysis was done using the software OriginPro version 8.5, and the results were presented as mean \u0026plusmn; standard deviation (SD).\u003c/p\u003e\n\u003cp\u003eNucleotide sequences accession numbers\u003c/p\u003e\n\u003cp\u003eThe 16S rRNA gene sequences and the whole genome of strain L9\u003csup\u003eT\u003c/sup\u003e have been submitted to the GenBank database under accession MN577409 and WOCA00000000, respectively.\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cp\u003e\u003cstrong\u003eGenomic characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe family \u003cem\u003eBacillaceae\u003c/em\u003e was first proposed by Fischer, and it now includes more than 100 recognized genera (Parte et al. 2020), including \u003cem\u003eVirgibacillus\u003c/em\u003e, \u003cem\u003eOceanobacillus\u003c/em\u003e and \u003cem\u003eOrnithinibacillus\u003c/em\u003e, the neighbors of the phylogenetic evolution. Additionally, in the phylogenomic tree (Fig. 1), the species of the genera \u003cem\u003eOceanobacillus \u003c/em\u003eand \u003cem\u003eOrnithinibacillus\u003c/em\u003e are intertwined with each other, specifying that their genomes have some identical sequences or are highly similar. To date, a few \u003cem\u003eOceanobacillus \u003c/em\u003especies have been isolated and reported to secrete protease (Li et al. 2017; Pandey et al. 2012; Thanapun 2013), lipase, amylase and carboxymethyl cellulase (Rohban et al. 2009; Seghal Kiran et al. 2014). Thus, as the twin brother of \u003cem\u003eOceanobacillus\u003c/em\u003e, the \u003cem\u003eOrnithinibacillus\u003c/em\u003e species might also have the ability to produce enzymes. A total of 3944 coding DNA sequences of strain L9\u003csup\u003eT\u003c/sup\u003e obtained from homology analyses were annotated by the NR database, including at least 25 enzyme-encoding genes. The most abundant was serine protease, followed by metalloprotease, lipase, amylase and glycosidase. The diversity of enzyme genes provides theoretical support for further research on strain L9\u003csup\u003eT\u003c/sup\u003e. Furthermore, a zone of clearing formed by strain L9\u003csup\u003eT\u003c/sup\u003e was overtly observed on the milk agar plates, and the protease activity was detected at 60.97 U/mL in the TSB medium supplemented with 100 g/L NaCl.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical optimization of protease production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResponse surface methodology for optimizing experimental design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe significant independent variables [yeast extract (A), urea (B) and initial pH (C)] were used to determine the optimum levels of these parameters based on the single-factor experimental results (depicted in Supplementary Material). The response values of 17 experiments are listed in Table S2. Later, the DesignExport software was employed for regression analysis and significance tests. A quadratic model was generated and applied to predict the maximum protease production (Y). The model was as follows:\u003c/p\u003e\n\u003cp\u003eY (U/mL) = 246.15\u0026ndash;12.97A\u0026ndash;32.89B\u0026ndash;7.51C+0.35AB\u0026ndash;37.30AC\u0026ndash;8.68BC\u0026ndash;42.35A\u003csup\u003e2\u003c/sup\u003e\u0026ndash;68.29B\u003csup\u003e2\u003c/sup\u003e\u0026ndash;77.57C\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eAs summarized in Table 1, the variance analysis results of the regression model indicated that the model was significant as it was obvious from the high model \u003cem\u003eF\u003c/em\u003e-value (= 107.4) and very low probability value (\u0026lt; 0.0001). Additionally, the model terms of A, B, C, AC, A\u003csup\u003e2\u003c/sup\u003e, B\u003csup\u003e2\u003c/sup\u003e and C\u003csup\u003e2\u003c/sup\u003e were significant (\u003cem\u003ep\u003c/em\u003e-value less than 0.05), whereas the interaction between urea and yeast extract was not significant (\u003cem\u003ep\u003c/em\u003e-value greater than 0.1). This result indicated that the combination of urea and yeast extract had no significant effect on protease production by \u003cem\u003eO. caprae \u003c/em\u003eL9\u003csup\u003eT\u003c/sup\u003e. The \u0026lsquo;lack of fit \u003cem\u003eF\u003c/em\u003e-value\u0026rsquo; of 3.56 implied that the lack of fit was not significant relative to the pure error. Moreover, the good fits were realized, and the variability of most of the test data was explained by the model, with the correlation coefficient (R\u003csup\u003e2\u003c/sup\u003e) of 99.28% and adjusted R\u003csup\u003e2\u003c/sup\u003e of 98.36%. The adequate precision was 28.942 (\u0026gt; 4), which was desirable, confirming the feasibility of this model to predict protease production by strain L9\u003csup\u003eT\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe interaction effects and optimum values of a combination of the three independent factors for maximum protease production by \u003cem\u003eO. caprae \u003c/em\u003eL9\u003csup\u003eT\u003c/sup\u003e were represented by three-dimensional response surface graphs and contour plots (Fig. 2). With the increase of yeast extract in the medium, the caseinolytic activity increased first and then decreased (Fig. 2a and 2b). Fig. 2b depicts the shape of the contour to be ellipse, indicating a significant interaction between yeast extract and pH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation of the optimized process conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the regression equation, three repeated verification tests were performed by setting yeast extract, urea and initial pH at the predicted levels of 14.3 g/L, 3.8 g/L and 9.0, respectively. The maximum protease activity of 251.12 U/mL was predicted by the model, which was comparable to the experimental results (255.86 \u0026plusmn; 0.71 U/mL), confirming the model\u0026rsquo;s authenticity. After RSM optimization, the protease yield of \u003cem\u003eO. caprae \u003c/em\u003eL9\u003csup\u003eT\u003c/sup\u003e increased by 319.65% than the unoptimized medium and original fermentation conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGel electrophoresis\u003c/strong\u003e\u003cstrong\u003e and molecular analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe crude extracellular enzyme produced by strain L9\u003csup\u003eT\u003c/sup\u003e was analyzed through SDS-PAGE and zymogram. As depicted in Fig. 3, a single protein band was obtained with the cell-free supernatant, and its proteolytic activity was confirmed by zymogram using casein as a substrate. The molecular weight of L9\u003csup\u003eT\u003c/sup\u003e protease was estimated as 26 kDa, which was consistent with some literature reports. Earlier, Deng et al. (2010) reported the characterization of a 28 kDa high-alkaline serine protease from \u003cem\u003eBacillus\u003c/em\u003e sp. B001. Two detergent stable serine proteases named protease BM1 and BM2 with molecular masses of 29 and 15.5 kDa, respectively, were purified from \u003cem\u003eBacillus mojavensis\u003c/em\u003e A21 (Haddar et al. 2009).\u003c/p\u003e\n\u003cp\u003eThe unique protein band was extracted and detected by mass spectrometry. The offline data was analyzed by ProteinPilot, and a total of 27 peptide sequences were obtained. Afterward, the quality control of peptide fragment was performed based on the unused ProtScore \u0026ge; 1.3, and 9 peptides (Table S3) with the reliability of no less than 95% were screened out. These peptides exhibited high homology with S8 family serine peptidases, suggesting that the extracellular enzyme of strain L9\u003csup\u003eT\u003c/sup\u003e might belong to serine protease family and provides a direction for further research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical characterization of the protease\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEffect of pH, temperature and NaCl on enzymatic activity and stability\u003c/p\u003e\n\u003cp\u003eAs depicted in Fig. 4a, the protease was active over a wide range of pH (2\u0026ndash;13) with the nadir activity at pH 2. More than 80% of the protease activity was retained at pH 3\u0026ndash;13. Besides, the pH stability profile (Fig. 4b) showed that the extracellular protease was highly stable in the pH range of 3\u0026ndash;13. The enzyme remained 57.54 and 88.64% of its activity at pH 2 and 13, respectively, after 1 h incubation at 25 \u003csup\u003eo\u003c/sup\u003eC. The protease secreted by \u003cem\u003eO. caprae \u003c/em\u003eL9\u003csup\u003eT\u003c/sup\u003e has broader pH activity and stability than the previously reported proteases (Ibrahim et al. 2015; Deng et al. 2010). Herein, the protease exhibited a typical characteristic of the serine protease (wider pH action), suggesting it to be a good candidate for industrial applications such as use in the detergents (Ibrahim et al. 2015), tanning processes (Kanagaraj et al. 2015) or biocontrol agent (Darwesh et al. 2020).\u003c/p\u003e\n\u003cp\u003eThe proteolytic activity was detected at all test temperatures (30 to 80 \u003csup\u003eo\u003c/sup\u003eC), and the maximum activity was observed at 70 \u003csup\u003eo\u003c/sup\u003eC (Fig. 4c), revealing the thermophilic nature of the protease. The crude protease showed only 5.4% of the maximal activity at 30 \u003csup\u003eo\u003c/sup\u003eC and exhibited a few activities till the enzyme assay at 80 \u003csup\u003eo\u003c/sup\u003eC. These results were consistent with several studies, including an alkaline serine protease produced by \u003cem\u003eN. dassonvillei\u003c/em\u003e OK-18 (Sharma et al. 2020), showing the maximal activity at 70 \u003csup\u003eo\u003c/sup\u003eC, and protease BM2 secreted by \u003cem\u003eB. mojavensis \u003c/em\u003eA21 (Haddar et al. 2009), showing the maximal activity at 60 \u003csup\u003eo\u003c/sup\u003eC. Thermostability studies have revealed that L9\u003csup\u003eT\u003c/sup\u003e protease is quite stable between 30 to 45 \u003csup\u003eo\u003c/sup\u003eC, retaining 95.75 and 9.02% of the initial activity after 1 h of incubation at 50 and 65 \u003csup\u003eo\u003c/sup\u003eC (Fig. 4d), respectively. Nevertheless, at 70\u0026ndash;80 \u003csup\u003eo\u003c/sup\u003eC, the enzyme fully denatured and lost activity within 60 min.\u003c/p\u003e\n\u003cp\u003eThe effects of NaCl on enzyme activities towards casein were determined in the salinity ranging from 0\u0026ndash;220 g/L at 70 \u003csup\u003eo\u003c/sup\u003eC. The protease exhibited the optimum activity at 20 g/L NaCl (Fig. 5). The protease retained 80.31, 55.23 and 32.49% of its activity at NaCl concentrations of 80, 140 and 220 g/L, respectively, suggesting that high salt concentration suppresses the enzyme activity. Notably, the proteolytic activity measured at different salt concentrations remained unchanged before and after heat preservation, indicating the stability of this protease at all tested NaCl concentrations for 1 h. These results were in accordance with the recent report of alkaline serine protease from \u003cem\u003eBacillus\u003c/em\u003e sp. NPST-AK15 (Ibrahim et al. 2015), showing the maximum activity at 15 g/L NaCl and highly stability in NaCl up to 200 g/L. These findings demonstrate that L9\u003csup\u003eT\u003c/sup\u003e protease is a slightly halophilic enzyme (different from extreme halophilic protease), which could be useful for certain biotechnological processes depending on salinity.\u003c/p\u003e\n\u003cp\u003eEffect of various metal ions on protease activity\u003c/p\u003e\n\u003cp\u003eTable 2 summarizes the proteolytic activity of the crude enzyme under incubation with different metal ions. Ag\u003csup\u003e+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e and Sr\u003csup\u003e2+\u003c/sup\u003e significantly enhanced the enzyme activities at a rate of 104.46, 104.40 and 120.37% than the control at a final concentration of 5 mM. In consistent with the present study, many investigators have reported the stimulating effect of Ca\u003csup\u003e2+\u003c/sup\u003e on serine proteases (Ben Elhoul et al. 2015; Jagadeesan et al. 2020; Jaouadi et al. 2013). Gong et al. (2015) has reported that Sr\u003csup\u003e2+\u003c/sup\u003e increased the keratinase activity by 10%. The above-mentioned results suggest that Ag\u003csup\u003e+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e and Sr\u003csup\u003e2+\u003c/sup\u003e could maintain the stability of the active site of L9\u003csup\u003eT\u003c/sup\u003e protease, which has a selective preference for Sr\u003csup\u003e2+\u003c/sup\u003e ions at a certain concentration as an inducer. The addition of Li\u003csup\u003e+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e, Ba\u003csup\u003e2+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e and Co\u003csup\u003e2+\u003c/sup\u003e had a little effect on the enzyme activities. Other ions, including Fe\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e and Zn\u003csup\u003e2+\u003c/sup\u003e, significantly inhibited the enzyme activities, and a loss of 70% in the activity was observed with Fe\u003csup\u003e3+\u003c/sup\u003e. Moreover, under the same conditions, Fe\u003csup\u003e3+\u003c/sup\u003e ions are more likely to deactivate enzyme than Fe\u003csup\u003e2+\u003c/sup\u003e, which is presumably attributed to the fact that Fe\u003csup\u003e3+\u003c/sup\u003e easily absorbs electrons from the enzyme surface through strong oxidation, thereby destroying the enzyme stability. Herein, the enzyme was moderately affected by Cr\u003csup\u003e3+\u003c/sup\u003e at 5 mM, revealing that the protease might act as an adjuvant to the chrome tannage and bating processes in leather industry.\u003c/p\u003e\n\u003cp\u003eEffect of chemical substances on protease activity\u003c/p\u003e\n\u003cp\u003eThe effects of various organic solvents on protease were appraised to examine the potential application. Results showed that the crude enzyme was considerably stable in the presence of the tested solvents (Table 3), particularly at a concentration of 5% (v/v). Upon incubation with 10% (v/v) of glycerol, benzene and n-hexane for 1 h, the enzyme also displayed high stability, with respective residual activity of 99.50, 100.76 and 95.70%. This remarkable stability exhibited by L9\u003csup\u003eT\u003c/sup\u003e protease in common organic solvents supported the potential candidacy of it as a biocatalyst for the synthesis of peptide and ester.\u003c/p\u003e\n\u003cp\u003eLater, the effects of surfactants on the extracellular protease were evaluated, and the corresponding results are presented in Table 4. The stimulatory effects were observed in the presence of some nonionic surfactants, such as Tween 20 (163.41%) and Tween 80 (115.01%), which were higher than previous reports (Jagadeesan et al. 2020; Ibrahim et al. 2015). On the contrary, the inhibitory effect of Triton X-100 was observed at a concentration of 1% (v/v). The enhanced activity of the enzyme in presence of DMSO reflect the fact that DMSO is an amphipathic molecule that easily penetrates the enzyme with low toxicity, and 99.94% of the activity was maintained (data not shown) at 10% concentration. The strong anionic surfactant sodium dodecyl sulfate (SDS) at 10 g/L inhibited the protease activity up to 76.98%, which was consistent with most of the previous conclusions reporting that SDS denatures and inactivates the enzyme (Ibrahim et al. 2015; Suwannaphan et al. 2017). Furthermore, the L9\u003csup\u003eT\u003c/sup\u003e protease was moderately stable against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, retaining 88.18% of its initial activity at a concentration of 1%. All these prominent features ensure that the enzyme is compatible with certain detergent formulations.\u003c/p\u003e\n\u003cp\u003eMoreover, the effect of various inhibitors and chelators on L9\u003csup\u003eT\u003c/sup\u003e protease activity was examined (Table 4). The protease remained unstimulated against thiol reagents, such as DTT and \u0026beta;-ME at a concentration of 5 mM, suggesting that L9\u003csup\u003eT\u003c/sup\u003e protease is a thiol-independent enzyme, and the thiol group does not directly participate in the catalytic reaction (Ben Elhoul et al. 2016). On the contrary, the protease activity was partially inhibited by the chelators EDTA and EGTA, which confirmed that L9\u003csup\u003eT\u003c/sup\u003e protease is not a member of the metalloprotease family but uses certain cations as stabilizers (Deng et al. 2010; Jellouli et al. 2009). Notably, L9\u003csup\u003eT\u003c/sup\u003e protease showed no activity in the presence of 5 mM PMSF, confirming the enzyme to be a serine protease (Jagadeesan et al. 2020).\u003c/p\u003e\n\u003cp\u003eSubstrate specificity profile of the L9\u003csup\u003eT\u003c/sup\u003e protease\u003c/p\u003e\n\u003cp\u003eThe effect of various substrates on L9\u003csup\u003eT\u003c/sup\u003e protease activity is summarized in Fig. S4. The highest activity was observed against casein, but a relatively high activity was observed against azocasein. However, no activity was observed toward gelatin, collagen and keratin. Therefore, the absence of collagenase activity indicates the potential application of L9\u003csup\u003eT\u003c/sup\u003e protease in hair removal in the leather industry without damaging the skin collagen.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHide dehairing ability of the crude enzyme\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dehairing efficacy of the crude enzyme from \u003cem\u003eO. caprae \u003c/em\u003eL9\u003csup\u003eT\u003c/sup\u003e on hides was evaluated by touch-visual tests and histological analyses. The pelt with hair, traditional dehaired pelt, and enzymatic dehaired hide are depicted in Fig. 6. The results elucidated that both the traditional sodium sulfide depilation and enzymatic dehairing could effectively remove the hair from goatskins, cowhides and rabbit skins; however, the control hides, incubated under the same conditions, showed no sign of hair removal. Notably, the enzymatic dehaired pelts were white in color and displayed smooth grain surface, soft touch and clean hair pores; while, organoleptic tests showed that the skins obtained after chemical dehairing were dark brown or yellow in color, wrinkled and hard in touch. Sodium sulfide dissolved in water could produce large amounts of hydroxide, and the disulfide bond of the hair breaks under the action of strong alkali to complete the hair removal process (Sujitha et al. 2018), which leads to the production of toxic gas H\u003csub\u003e2\u003c/sub\u003eS. In contrast, as a biologically active catalyst, the crude enzyme cannot directly act on hair shafts; instead, it may hydrolyze the mucinoid, mucin, albumin and globulin in raw skin tissue, thereby destroying the bond between the hair follicle and the hair root, the hair bulb and the hair papilla, to achieve the purpose of hair removal (Sujitha et al. 2018; Yates 1972).\u003c/p\u003e\n\u003cp\u003eThe histological images of control, Na\u003csub\u003e2\u003c/sub\u003eS and crude enzyme treated goatskins stained with HE and Masson's trichrome staining are depicted in Fig. S5. The results demonstrated that compared with the blank control, the epidermis was removed from the goatskins via two different treatments; and the collagen fiber structure of the enzymatically dehaired pelt was more regular and intact compared with the Na\u003csub\u003e2\u003c/sub\u003eS treatment. It could be observed from Masson\u0026rsquo;s trichrome staining images that the collagen components appeared in blue color, and the non-collagen substances appeared in deep red color. This result suggested that the enzyme treatment could well maintain the inherent collagen component in the dermal structure, while part of the damaged collagen existed in the fur treated with Na\u003csub\u003e2\u003c/sub\u003eS. Overall, the crude enzyme exhibited no collagenase activity, and was advantageous over chemical depilatory agents.\u003c/p\u003e\n\u003cp\u003eIn this study, the crude protease completely dehaired the skins from goat and rabbit (Fig. 6a and 6c) in 24 h without any chemicals. This result strengthened the hypothesis that this protease could be beneficial to protect the environment and reduce energy waste (Jaouadi et al. 2013; Kandasamy et al. 2012). Moreover, the reasons for incomplete enzymatic depilation of cowhide (Fig. 6b) might be attributed to the fact that the thick cowhide leads to slower enzyme penetration and lack of mechanical pulling force. It is noteworthy to mention that the microbial enzymes with excellent dehairing ability are stable in an alkaline environment, especially between pH 8 and 10 (Jaouadi et al. 2013; Bouacem et al. 2016). Fortunately, L9\u003csup\u003eT\u003c/sup\u003e protease meets these criteria and hence, is considered as a potential dehairing candidate in improving leather quality.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn the present study, a novel thermophilic and slightly halophilic serine protease from \u003cem\u003eO. caprae \u003c/em\u003eL9\u003csup\u003eT\u003c/sup\u003e was reported. The halophile exhibited a higher protease production capacity (255.86 U/mL) under the following conditions: 72 h of fermentation time, initial pH 9, culture temperature 37 \u003csup\u003eo\u003c/sup\u003eC, and the modified medium containing 14.3 g yeast extract, 3.8 g urea, 130 g NaCl and 1 L distilled water. The crude enzyme appeared as a single band on SDS-PAGE with a molecular mass of 26 kDa. The optimum temperature for caseinolytic activity was 70 \u003csup\u003eo\u003c/sup\u003eC. Furthermore, the protease activity was enhanced by metal ions Ca\u003csup\u003e2+\u003c/sup\u003e, Sr\u003csup\u003e2+\u003c/sup\u003e, Ag\u003csup\u003e+\u003c/sup\u003e and 20 g/L NaCl. The enzyme exhibited excellent stability toward surfactants, organic solvents and a wide range of pH from 3 to 13. Further studies confirmed that the crude protease has an excellent ability to remove hair in a short time without damaging the collagen. In summary, the protease could be a propitious candidate for various industrial applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement \u003c/strong\u003eThis work was supported by the National Key Research and Development Program of China (2017YFB0308401).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e XL performed the experiment and wrote the manuscript. QZ revised the grammar and typography of the manuscript. LG and GJ provided guidance on response surface analyses. YT supervised and guided the experiments. BS critically reviewed the structure of manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest \u003c/strong\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statement\u003c/strong\u003e This article does not contain any studies with human participants and/or animals performed by any of the authors.\u003c/p\u003e"},{"header":"References ","content":"\u003col\u003e\n\u003cli\u003eBarzkar N (2020) Marine microbial alkaline protease: An efficient and essential tool for various industrial applications. 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J Ind Microbiol Biotechnol 36:333\u0026ndash;340\u003c/li\u003e\n\u003cli\u003eSeghal Kiran G, Nishanth Lipton A, Kennedy J, Dobson AD, Selvin J (2014) A halotolerant thermostable lipase from the marine bacterium\u003cem\u003e Oceanobacillus\u003c/em\u003e sp PUMB02 with an ability to disrupt bacterial biofilms. Bioengineered 5:305\u0026ndash;318\u003c/li\u003e\n\u003cli\u003eSharma AK, Kikani BA, Singh SP (2020) Biochemical, thermodynamic and structural characteristics of a biotechnologically compatible alkaline protease from a haloalkaliphilic, \u003cem\u003eNocardiopsis dassonvillei\u003c/em\u003e OK-18. Int J Biol Macromol 153:680\u0026ndash;696\u003c/li\u003e\n\u003cli\u003eSujitha P, Kavitha S, Shakilanishi S, Babu NKC, Shanthi C (2018) Enzymatic dehairing: a comprehensive review on the mechanistic aspects with emphasis on enzyme specificity. Int J Biol Macromol 118:168\u0026ndash;179\u003c/li\u003e\n\u003cli\u003eSun F, Sun Q, Zhang H, Kong B, Xia X (2019) Purification and biochemical characteristics of the microbial extracellular protease from \u003cem\u003eLactobacillus curvatus\u003c/em\u003e isolated from Harbin dry sausages. Int J Biol Macromol 133:987\u0026ndash;997\u003c/li\u003e\n\u003cli\u003eSuwannaphan S, Fufeungsombut E, Promboon A, Chim-anage P (2017) A serine protease from newly isolated \u003cem\u003eBacillus\u003c/em\u003e sp for efficient silk degumming, sericin degrading and colour bleaching activities. Int Biodeter Biodegr 117:141\u0026ndash;149\u003c/li\u003e\n\u003cli\u003eThanapun T (2013) Screening and characterization of protease-producing \u003cem\u003eVirgibacillus\u003c/em\u003e, \u003cem\u003eHalobacillus\u003c/em\u003e and \u003cem\u003eOceanobacillus\u003c/em\u003e strains from Thai fermented fish. J App Pharm Sci 3:025\u0026ndash;030\u003c/li\u003e\n\u003cli\u003eThanikaivelan P, Rao JR, Nair BU, Ramasami T (2004) Progress and recent trends in biotechnological methods for leather processing. Trends Biotechnol 22:181\u0026ndash;188\u003c/li\u003e\n\u003cli\u003eWang R, Min C, Haiming C, Li ZQ (2009) Enzyme unhairing- an eco-friendly process. J Soc Leath Tech Ch 93:51\u0026ndash;55\u003c/li\u003e\n\u003cli\u003eWang SL, Hsu WT, Liang TW, Yen YH, Wang CL (2008) Purification and characterization of three novel keratinolytic metalloproteases produced by \u003cem\u003eChryseobacterium indologenes\u003c/em\u003e TKU014 in a shrimp shell powder medium. Bioresour Technol 99:5679\u0026ndash;5686\u003c/li\u003e\n\u003cli\u003eWard OP (2011) Proteases, in: M. Moo-Young (Ed.), Comprehensive Biotechnology (Second Edition), Academic Press, Burlington, pp 571\u0026ndash;582\u003c/li\u003e\n\u003cli\u003eYates JR (1972) Studies in depilation. Part X. The mechanism of the enzyme depilation process. J Soc Leather Trades Chem 56:158\u0026ndash;177\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Analysis of variance for the regression equation\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eSource\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eSum of squares\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003edf\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"105\"\u003e\n\u003cp\u003eMean squares\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e\u003cem\u003eF\u003c/em\u003e-value\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eModel\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e74551.59\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"105\"\u003e\n\u003cp\u003e8283.51\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e107.40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026lt; 0.0001**\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eA-Yeast extract\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e1346.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"105\"\u003e\n\u003cp\u003e1346.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e17.45\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.0042**\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eB-Urea\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e8652.39\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"105\"\u003e\n\u003cp\u003e8652.39\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e112.18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026lt; 0.0001**\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eC-pH\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e451.79\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"105\"\u003e\n\u003cp\u003e451.79\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e5.86\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.0461*\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eAB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e0.48\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"105\"\u003e\n\u003cp\u003e0.48\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e0.01\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.9392\u0026dagger;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eAC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e5565.63\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"105\"\u003e\n\u003cp\u003e5565.63\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e72.16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026lt; 0.0001**\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eBC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e301.41\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"105\"\u003e\n\u003cp\u003e301.41\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e3.91\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.0886\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eA\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e7552.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"105\"\u003e\n\u003cp\u003e7552.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e97.91\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026lt; 0.0001**\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eB\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e19637.99\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"105\"\u003e\n\u003cp\u003e19637.99\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e254.61\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026lt; 0.0001**\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eC\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e25334.82\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"105\"\u003e\n\u003cp\u003e25334.82\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e328.47\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026lt; 0.0001**\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eResidual\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e539.90\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"105\"\u003e\n\u003cp\u003e77.13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eLack of fit\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e392.94\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"105\"\u003e\n\u003cp\u003e130.98\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e3.56\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e0.1256\u0026dagger;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003ePure error\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e146.97\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"105\"\u003e\n\u003cp\u003e36.74\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003eCor total\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"113\"\u003e\n\u003cp\u003e75091.49\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"105\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003eF\u003c/em\u003e, \u003cem\u003eF \u003c/em\u003eratio; \u003cem\u003ep-value\u003c/em\u003e, probability value; df, degree of freedom.\u003c/p\u003e\n\u003cp\u003e*, Significant (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05); **, very significant (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01); \u0026dagger;, not significant (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.1).\u003c/p\u003e\n\u003cp\u003eR\u003csup\u003e2 \u003c/sup\u003e= 0.9928; adjusted R\u003csup\u003e2\u003c/sup\u003e = 0.9836; adequate precision = 28.942.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. \u003c/strong\u003eEffect of different metal ions on the protease activity\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003eMetal ions\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003eFinal Concentration (mM)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003eRelative activity (\u0026plusmn; SD) (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003eControl\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e100.00 \u0026plusmn; 0.79\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003eLi\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e90.19 \u0026plusmn; 0.97\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003eK\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e97.71 \u0026plusmn; 2.49\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003eAg\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e104.46 \u0026plusmn; 1.27\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003eFe\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e84.85 \u0026plusmn; 1.25\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003eCu\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e85.32 \u0026plusmn; 1.40\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003eZn\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e78.39 \u0026plusmn; 2.47\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003eCa\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e104.40 \u0026plusmn; 2.12\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003eMg\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e97.77 \u0026plusmn; 2.00\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003eBa\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e93.31 \u0026plusmn; 2.92\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003eMn\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e96.36 \u0026plusmn; 2.13\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003eCo\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e95.54 \u0026plusmn; 0.35\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003eSr\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e120.37 \u0026plusmn; 3.10\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003eFe\u003csup\u003e3+\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e28.55 \u0026plusmn; 3.16\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003eCr\u003csup\u003e3+\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"180\"\u003e\n\u003cp\u003e84.15 \u0026plusmn; 1.64\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u003c/strong\u003e Effect of organic solvents on L9\u003csup\u003eT\u003c/sup\u003e protease stability\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"177\"\u003e\n\u003cp\u003eSolvents\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"277\"\u003e\n\u003cp\u003eRelative activity (\u0026plusmn; SD) (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003e5% (v/v)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e10% (v/v)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"177\"\u003e\n\u003cp\u003eControl\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003e100.00 \u0026plusmn; 0.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"177\"\u003e\n\u003cp\u003eAcetone\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003e88.44 \u0026plusmn; 5.31\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e85.09 \u0026plusmn; 3.72\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"177\"\u003e\n\u003cp\u003eBenzene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003e100.76 \u0026plusmn; 2.24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e95.83 \u0026plusmn; 6.36\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"177\"\u003e\n\u003cp\u003eEthanediol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003e99.12 \u0026plusmn; 2.28\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e93.37 \u0026plusmn; 2.10\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"177\"\u003e\n\u003cp\u003eEthanol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003e90.90 \u0026plusmn; 0.77\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e74.92 \u0026plusmn; 4.05\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"177\"\u003e\n\u003cp\u003eGlycerol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003e88.76 \u0026plusmn; 3.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e99.50 \u0026plusmn; 1.22\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"177\"\u003e\n\u003cp\u003en-Hexane\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003e91.16 \u0026plusmn; 1.91\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e95.70 \u0026plusmn; 3.94\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"177\"\u003e\n\u003cp\u003eIsopropanol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003e91.98 \u0026plusmn; 1.37\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e71.64 \u0026plusmn; 4.18\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"177\"\u003e\n\u003cp\u003eMethanol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003e97.22 \u0026plusmn; 3.90\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"133\"\u003e\n\u003cp\u003e89.77 \u0026plusmn; 2.22\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.\u003c/strong\u003e Effect of various surfactants, oxidizing agent, alkahest and inhibitors on crude serine protease from \u003cem\u003eO. caprae \u003c/em\u003eL9\u003csup\u003eT\u003c/sup\u003e\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003eChemical substances\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003eWorking concentration\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"170\"\u003e\n\u003cp\u003eRelative activity (\u0026plusmn; SD) (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003eControl\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"170\"\u003e\n\u003cp\u003e100.00 \u0026plusmn; 3.06\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003eSurfactants\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"170\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003eTween 20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003e1% (v/v)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"170\"\u003e\n\u003cp\u003e163.41 \u0026plusmn; 2.23\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003eTween 80\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003e1% (v/v)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"170\"\u003e\n\u003cp\u003e115.01 \u0026plusmn; 1.13\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003eTriton X-100\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003e1% (v/v)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"170\"\u003e\n\u003cp\u003e90.34 \u0026plusmn; 3.32\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003eSDS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003e10 g/L\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"170\"\u003e\n\u003cp\u003e23.02 \u0026plusmn; 1.18\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003eOxidizing agent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"170\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003e1% (v/v)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"170\"\u003e\n\u003cp\u003e88.18 \u0026plusmn; 1.18\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003eAlkahest\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"170\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003eDMSO\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003e1% (v/v)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"170\"\u003e\n\u003cp\u003e110.22 \u0026plusmn; 1.70\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003eInhibitors\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"170\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003eDTT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"184\"\u003e\n\u003cp\u003e5 mM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"170\"\u003e\n\u003cp\u003e96.96 \u0026plusmn; 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BBD, Box-Behnken design; BSA, bovine serum albumin; CGMCC, China General Microbiological Culture Collection Center; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; EDTA, ethylene-diamine-tetraacetic acid; EGTA, ethylene glycol-bis (β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid; GBDP, genome blast distance phylogeny; HE, hematoxylin and eosin; KCTC, Korean Collection for Type Cultures; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LB, Luria-Bertani; β-ME, β-mercaptoethanol; NR, non-redundant protein database; PMSF, phenylmethylsulfonyl fluoride; RSM, response surface methodology; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SD, standard deviation; TCA, trichloroacetic acid; TSB, tryptic soytone broth; TYGS, type strain genome server; UV-Vis, ultraviolet-visible.\u003c/p\u003e","manuscriptTitle":"Exoproduction and Biochemical Characterization of a Novel Thermophilic Serine Protease From Ornithinibacillus Caprae L9T With Hide-dehairing Activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-06-07 20:00:08","doi":"10.21203/rs.3.rs-573853/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"495efde0-6e6a-4217-a420-9bfd1b19ebcb","owner":[],"postedDate":"June 7th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":4827322,"name":"General Microbiology"}],"tags":[],"updatedAt":"2021-11-25T01:17:19+00:00","versionOfRecord":{"articleIdentity":"rs-573853","link":"https://doi.org/10.4014/jmb.2108.08037","journal":{"identity":"journal-of-microbiology-and-biotechnology","isVorOnly":true,"title":"Journal of Microbiology and Biotechnology"},"publishedOn":"2021-11-20 01:17:19","publishedOnDateReadable":"November 20th, 2021"},"versionCreatedAt":"2021-06-07 20:00:08","video":"","vorDoi":"10.4014/jmb.2108.08037","vorDoiUrl":"https://doi.org/10.4014/jmb.2108.08037","workflowStages":[]},"version":"v1","identity":"rs-573853","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-573853","identity":"rs-573853","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}