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

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 uoride; 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.


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 sul de, sodium bisul de 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 ll 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 (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. 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 modi cations. 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 speci ed 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 signi cant 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 con rmed by zymography analysis according to the method described by Garciacarreno et al. (1993) with minor modi cations. 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 nal 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 nal concentration of 5 mM; protease inhibitors: phenylmethylsulfonyl uoride (PMSF), ethylene-diaminetetraacetic 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 speci city Substrate speci city 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 nal 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 ask 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 asks 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 e cacy and histological examination. About 1 cm 2 pelts from the hides treated with Tris-HCl buffer, Na 2 S and enzyme, were incised and xed with 40 g/L paraformaldehyde (Servicebio, China) for 24 h. The immobilized samples were dehydrated by ethanol, embedded with para n 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 rst 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  . 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.  Table S2. Later, the DesignExport software was employed for regression analysis and signi cance 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 signi cant 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 signi cant (p-value less than 0.05), whereas the interaction between urea and yeast extract was not signi cant (p-value greater than 0.1). This result indicated that the combination of urea and yeast extract had no signi cant effect on protease production by O. caprae L9 T . The 'lack of t F-value' of 3.56 implied that the lack of t was not signi cant relative to the pure error. Moreover, the good ts were realized, and the variability of most of the test data was explained by the model, with the correlation coe cient (R 2 ) of 99.28% and adjusted R 2 of 98.36%. The adequate precision was 28.942 (> 4), which was desirable, con rming 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 rst and then decreased ( Fig. 2a and 2b). Fig. 2b depicts the shape of the contour to be ellipse, indicating a signi cant interaction between yeast extract and pH.

Validation of the optimized process conditions
Based on the regression equation, three repeated veri cation 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), con rming 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 con rmed 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 puri ed from Bacillus mojavensis A21 (Haddar et al. 2009).
The unique protein band was extracted and detected by mass spectrometry. The o ine 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 pro le (Fig. 4b) showed that the extracellular protease was highly stable in the pH range of 3-13. The enzyme 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 ndings 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 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+ , signi cantly 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 re ect 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 con rmed 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, con rming the enzyme to be a serine protease (Jagadeesan et al. 2020).

Substrate speci city pro le 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 e cacy 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 sul de 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 sul de dissolved in water could produce large amounts of hydroxide, and the disul de 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 ber 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)  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 modi ed 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 con rmed 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. Compliance with ethical standards Con icts of interest The authors declare that there are no con icts of interest.

Declarations
Ethical statement This article does not contain any studies with human participants and/or animals performed by any of the authors.  Figure 2 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.    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.