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 L9T 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 L9T. Furthermore, a zone of clearing formed by strain L9T 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–42.35A2–68.29B2–77.57C2
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, A2, B2 and C2 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 L9T. 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 (R2) of 99.28% and adjusted R2 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 L9T.
The interaction effects and optimum values of a combination of the three independent factors for maximum protease production by O. caprae L9T 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 L9T increased by 319.65% than the unoptimized medium and original fermentation conditions.
Gel electrophoresis and molecular analysis
The crude extracellular enzyme produced by strain L9T 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 L9T 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 L9T 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 oC. The protease secreted by O. caprae L9T 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 oC), and the maximum activity was observed at 70 oC (Fig. 4c), revealing the thermophilic nature of the protease. The crude protease showed only 5.4% of the maximal activity at 30 oC and exhibited a few activities till the enzyme assay at 80 oC. 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 oC, and protease BM2 secreted by B. mojavensis A21 (Haddar et al. 2009), showing the maximal activity at 60 oC. Thermostability studies have revealed that L9T protease is quite stable between 30 to 45 oC, retaining 95.75 and 9.02% of the initial activity after 1 h of incubation at 50 and 65 oC (Fig. 4d), respectively. Nevertheless, at 70–80 oC, 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 oC. 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 L9T 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+, Ca2+ and Sr2+ 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 Ca2+ on serine proteases (Ben Elhoul et al. 2015; Jagadeesan et al. 2020; Jaouadi et al. 2013). Gong et al. (2015) has reported that Sr2+ increased the keratinase activity by 10%. The above-mentioned results suggest that Ag+, Ca2+ and Sr2+ could maintain the stability of the active site of L9T protease, which has a selective preference for Sr2+ ions at a certain concentration as an inducer. The addition of Li+, K+, Ba2+, Mg2+, Mn2+ and Co2+ had a little effect on the enzyme activities. Other ions, including Fe2+, Fe3+, Cu2+ and Zn2+, significantly inhibited the enzyme activities, and a loss of 70% in the activity was observed with Fe3+. Moreover, under the same conditions, Fe3+ ions are more likely to deactivate enzyme than Fe2+, which is presumably attributed to the fact that Fe3+ easily absorbs electrons from the enzyme surface through strong oxidation, thereby destroying the enzyme stability. Herein, the enzyme was moderately affected by Cr3+ 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 L9T 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 L9T protease was moderately stable against H2O2, 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 L9T 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 L9T 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 L9T protease is not a member of the metalloprotease family but uses certain cations as stabilizers (Deng et al. 2010; Jellouli et al. 2009). Notably, L9T 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 L9T protease
The effect of various substrates on L9T 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 L9T 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 L9T 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 H2S. 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, Na2S 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 Na2S 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 Na2S. 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, L9T protease meets these criteria and hence, is considered as a potential dehairing candidate in improving leather quality.