Directed evolution of a keratinase BLk from Bacillus licheniformis to enhance the solvent tolerance

Directed evolution of a keratinase BLk from Bacillus licheniformis to enhance the solvent tolerance | 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 Directed evolution of a keratinase BLk from Bacillus licheniformis to enhance the solvent tolerance Fucheng Zhu, Zixu Yan, Jingli Dai, Guosi Li, Qiling Xu, Yunfeng Ma, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3464492/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Keratinase, an important protein used in hair degradation, requires stability enhancement in industrial applications due to the harsh reaction environment for keratin hydrolysis. Previous studies have focused on improving the thermostability of keratinase. In this study, directed evolution was applied to enhance the organic solvent stability of keratinase BLk from Bacillus licheniformis . Three excellent mutants were screened and exhibited significantly improved stability in various solvents, although similar results were not observed in terms of thermostability. The identified mutations were located on the enzyme's surface. The half-life of the D41A, A24E, and A24Q mutants increased by 47-, 63-, and 61-fold, respectively, in the presence of 50% (v/v) acetonitrile compared to the wild-type. Similarly, in the presence of 50% (v/v) acetone, the half-life of these mutants increased by 22-, 27-, and 27-fold compared to the wild-type. Importantly, the proteolytic activity of all selected mutants was similar to that of the parent keratinase BLk. Furthermore, molecular dynamic simulation was employed to analyze the possible reasons for the enhanced solvent stability. The results suggest that increased intramolecular interactions, such as hydrogen bonds and hydrophobic interactions, may contribute to the improved solvent tolerance. The mutants obtained in this study hold significant potential for industrial applications. Keratinase BLk Organic solvent stability Directed evolution MD Simulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Keratin is a fibrous protein that is abundant and insoluble. It is found in mammalian horns, wool, claws, feathers, and beaks. The stability of keratin is attributed to its tight packing, disulfide bonds, and hydrophobic interactions. These properties enable it to resist degradation by proteolytic enzymes such as pepsin and trypsin (Stiborova et al. 2013 ). While chemical or physico-chemical hydrolysis can be used to utilize keratin, these methods consume a significant amount of energy and produce harmful gases, posing a threat to the environment (Dettmer et al. 2013 ).In recent years, keratinase has gained attention in bioremediation as it plays a crucial role in managing keratin resources and degrading keratin waste into amino acids or soluble peptides (Su et al. 2020 ). Various keratinase producing microorganisms have been reported, including fungi such as Aspergillus and Microsporum (Bohacz et al. 2020 ), and bacteria such as Bacillus (Benkiar et al. 2013 ; Jagadeesan et al. 2020 ), Pseudomonas (Moonnee et al. 2021 ), and Streptomyces (Indhuja et al. 2012 ). These keratinases can be divided into 14 different protease families according to MEROPS database ( http://www.ebi.ac.uk/merops/ ). The unique property of keratinase, which sets it apart from other proteases, is its ability to bind to insoluble protein substrates. However, natural insoluble substrates like poultry feathers often create a harsh environment, posing a challenge for the application of keratinase. Therefore, it is necessary to enhance the stability or activity of keratinase to facilitate its industrial application. Multiple strategies have been successfully employed to improve the stability of keratinase. Srivastava et al. significantly enhanced the thermostability and activity of keratinase by immobilizing protease on chitosan (Srivastava et al. 2020 ). The activity and thermostability of keratinase KerSMD were significantly improved through domain replacement (Fang et al. 2016 ). Liu et al. achieved an excellent mutant with increased thermostability and catalytic efficiency through rational design (Liu et al. 2013 ). Enzyme engineering has been recognized as an attractive strategy for enhancing the properties of keratinase (Gupta et al. 2013 ). Currently, the focus of improving the stability of keratinase lies mainly in enhancing its thermostability. However, there have been few reports on improving its solvent tolerance. It is widely acknowledged that the keratin hydrolysis reaction mixture contains complex ingredients, including surfactants or other organic solvents. Therefore, it is also crucial for the keratinase to maintain high activity in organic solvents in order to effectively apply it in the process of keratin hydrolysis. There is a correlation between solvent stability and thermostability, although these two types of stability often exhibit different trends. While it is difficult to predict the solvent stability of a protein and identify the protein's hotspots that affect solvent tolerance using molecular simulation methods, it is relatively easier to enhance the thermostability of a protein (Zhu et al. 2020 ). Additionally, there have been fewer studies on the evolution of protein solvent tolerance, and the mechanisms underlying improved solvent stability are not well understood. Directed evolution, using error-prone polymerase chain reaction (PCR), is a suitable strategy for modifying enzyme tolerance in organic solvents as it does not rely on prior knowledge of the protein's structure and mechanism (McCullum et al. 2010 ). In a previous study, we successfully enhanced the solvent stability of metalloprotease PT121 by using error-prone PCR, resulting in 1.2–3.5-fold increases in half-lives (Zhu et al. 2020 ). Koudelakova et al. also employed the same method to engineer an enzyme with significantly improved stability, increasing its half-life in the presence of 40% (v/v) DMSO by 5040-fold (Koudelakova et al. 2013 ). They found that residues located around the access tunnel, which connects the surrounding solvent with the active pocket of the enzyme, played a crucial role in stabilizing the protein. These studies highlight the effectiveness of directed evolution as a strategy for improving protein solvent tolerance. In a previous study, a keratinase BLk from Bacillus licheniformis was expressed in the Bacillus subtilis WB800 system. This current study aims to improve the organic solvent stability of BLk through directed evolution using error-prone PCR. The properties of the obtained mutants, including thermostability and optimum temperature, were analyzed. Additionally, the study discusses the possible intrinsic molecular reasons behind these properties. The findings from this research provide valuable insights for expanding the application of keratinase and enhancing the solvent stability of related enzymes. Materials and Methods 2.1. Chemicals and strains The strain Bacillus subtilis WB800 and pHY300 were used as host and vector for gene cloning and expression. The restriction enzymes were purchased from Takara (Dalian, China), DNA polymerases and ClonExpress Ultra One Step Cloning Kit were obtained from vazyme (Nanjing, China). All other chemicals in this study were Sigma Chemical Co. (Shanghai, China) and Sangon Biotech (Shanghai, China). 2.2. Random mutagenesis library generation The recombinant Bacillus subtilis WB800 and Escherichia coli DH 5ɑ carrying PHY300-BLk plasmid were preserved in our laboratory. The mutant library was constructed using error-prone PCR method. The PCR was performed in a 50 µL reaction system containing 0.3 mM dNTP, 0.1 ng/uL of the template DNA, 0.01 mM MnCl 2 , 0.05 U/µL Taq DNA polymerase, 0.02 mM primers and 1.5 mM MgCl 2 . The reaction product was digested with Dpn I at 37°C for 1 h and then inserted into vector PHY300 using homologous recombination method. Recombinant plasmid were transformed into E. coli DH 5ɑ and then submitted into sequencing. Then the verified recombinant plasmid was transformed into B. subtilis WB800 for expression. The primers used in this study are listed in Table S1 . An overnight culture (1 mL) of recombinant B. subtillis WB800 in TB medium containing kanamycin (50 µg/mL) was inoculated into 100 mL Terrific Broth medium containing kanamycin (50 µg/mL) and incubated at 37°C, 180 rpm for 48h. The culture supernatant was obtained by centrifugation and loaded on a HiTrap Phenyl-Sepharose Fast Flow hydrophobic interaction chromatography column. The eluted fractions were collected for activity assay and SDS-PAGE analysis. 2.3. Screen and Assay of protease mutants Protease assay was performed as described previously (Zhu et al. 2016 ). Two milliliters of the sample were added to the solution (2 mL) containing 2% (w/v) casein and 50 mM Tris-HCl buffer (pH 8.0). The mixture was incubated at 40°C for 10 min and terminated by adding 4.0 mL of TCA mixture (containing 0.11 M trichloroacetic acid, 0.22 M sodium acetate and 0.33 M acetic acid). This mixture was further centrifuged at 15,000 ×g for 15 min, and the absorbance of the supernatant was measured at 280 nm against a blank control. One unit (U) of protease activity is defined as the amount of enzyme that hydrolyzes casein and produces 1 µg of tyrosine per min under the assay conditions. In this study, a 50% (v/v) N, N-dimethylformamide (DMF) solution was used to assess the solvent stability of the mutant. The mixtures were incubated in universal bottles with screw caps at 37°C for 2 hours. The initial activity and residual activities were measured using standard assay conditions. The relative residual activity, which indicates stability, was calculated. Mutants that showed significant differences in relative residual activity compared to the WT protease were selected for further analysis. The sequences of these selected mutants were determined. 2.4. The properties of mutants The stability of mutants in organic solvents was assessed using the following method. Protease variants were incubated at a pH of 10.0 and a temperature of 37°C with shaking at 200 rpm in the presence of 50% (v/v) various organic solvents. The remaining activities of the proteases were measured at different time intervals. The half-lives (T 1/2 ) were calculated from the exponential regression curve. Each experiment was performed in triplicate. The optimal temperature for enzymatic activity was determined by increasing the temperature from 20 to 65°C at a pH of 10. The thermostability of the purified enzyme was determined by pre-incubating it for 30 minutes at temperatures ranging from 30 to 60°C. The residual activity was then analyzed using the activity assay method. 2.5. Circular dichroism determination Circular dichroism (CD) curve was performed as our previously described using Chirascan™ CD spectrometer (Leatherhead, UK) (Zhu et al. 2018 ). The sample of 4–6 µM enzyme was infused into 1 mm cuvette. Spectra of sample was measured from 190 to 260 nm using 1nm bandwidth. 2.6. The structural feature of mutants The structure of the keratinase and its mutants was constructed using SWISS-MODEL ( http://swissmodel_expasy.org ) based on the X-ray crystal structure of the protease from Bacillus licheniformis (Protein Data Bank ID: 1A1Y, crystal resolution: 1.05 Å), which share 99.6% identity with keratinase BLk. The structure was visualized and analyzed using Discovery Studio 2019 and PyMOL 2.6 software. 2.7. Molecular Dynamic Simulation Molecular Dynamic (MD) Simulations were performed using Gromacs version 2018 to analyze the factors contributing to improved solvent stability as previous descriptions (Zhu et al. 2020 ). The MD simulations utilized the GROMOS 53A6 force field. The keratinase BLk model was solvated with a 50% (v/v) acetonitrile/water mixture with explicit waters modeled using a simple point charge (SPC) model (Hermans et al. 1984 ). The MD simulations system was neutralized by adding the sodium ions. The electro-neutral systems were then subjected to energy minimization using steepest descent method, followed by a 500-ps MD simulations where the heavy atoms and protein were kept fixed. Subsequently, a 20-ns MD simulation was performed on the entire system with a temperature gradient (300 K and 380 K). The LINCS algorithm was used to constrain bond lengths (Hess et al. 1997 ). The cut-off value for the van der Waals interactions was set at 1.0 nm, and the electrostatic interactions were calculated using a particle mesh Ewald algorithm. Periodic boundary conditions were applied, and the coordinates were saved for analysis at every 1ps interval. Gromacs tools were utilized to analyzed various characterization parameters such as Root Mean Square Deviation (RMSD) value, Root Mean Square Fluctuation (RMSF) value, Radius of gyration (Rg) value, Radial distribution g(r), and hydrogen bond. PyMOL 2.6 open-source version and Discovery studio 2019 programs were used to analyze the structures of enzyme. Results and Discussion 3.1. Library Generation and Screening The properties of keratinase BLk from Bacillus licheniformis were found to be sensitive to 50% (v/v) DMF, which is toxic to most enzymes. Therefore, 50% (v/v) DMF was chosen as the organic solvent for the typical mutant screening. In this study, random mutant library screening was conducted by increasing the transparent cycle in skim milk plates and assessing the retention of proteolytic activity after incubation of the protease in the presence of 50% (v/v) DMF. Thirteen mutants with significant changes in solvent stability, but no significant differences in proteolytic activity, were obtained from the mutant library, which contained over 2000 clones. As shown in Table 1 , mutants D41A, A24E and A24Q exhibited a remarkable increased in activity after being incubated in 50% DMF for 90 minutes, while the other mutants showed a significant reduction compared to the wild-type protease. Structural analysis revealed that all mutations were located on the surface of the protein, mostly in the loop region of enzyme’s secondary structure (as shown in Figure S1 ). This indicates that surface residues in the loop region play an important role in the solvent tolerance of the enzyme, which is consistent with previous reports (Ogino et al. 2007 ). To further explore the properties of the mutants and understand the possible reasons behind the changes in organic solvent stability, the properties of mutants were submitted to further research. Table 1 Relative residual activities of mutant and wild-type of keratinase Mutants Residual activity (%) a Location of mutation Wild-type 82 D41A 114 Surface/Loop A24E 115 Surface/Loop A24Q 130 Surface/Loop D41V 0 Surface/Loop A24V 12 Surface/Loop A24T/G165D 18 Surface/Loop, Interior/Loop G20D/G46D 20 Surface/Loop, Surface/Sheet A13V/G46V 10 Surface/Helix, Surface/Sheet A18V/G117D 9 Surface/Helix, Surface/Loop A137T 30 Surface/Helix S183N 0 Surface/Loop A214V 31 Surface/Sheet A273T 0 Surface/Helix a The residual activities of keratinase were determined after incubation with 50% DMF for 1.5h, and are shown as a percentage of their initial activity without addition of organic solvent. Values are the average of three independent experiments. 3.2. The properties of keratinase BLk variants To further investigate solvent stability, we determined the half-life of mutants and wild-type. As shown in Table 2 , three mutants (A24E, A24Q and D41A) exhibited improved stability in all tested solvents except for n-Heptane. Surprisingly, the half-life of D41A, A24E and A24Q increased by 47-, 63- and 6-fold, respectively, in presence of 50% (v/v) acetonitrile compared to the wild-type. Similarly, in presence of 50% (v/v) acetone, the half-life of D41A, A24E and A24Q increased by 22-, 27-, 27-fold, respectively. These mutants demonstrated remarkable tolerance to polar solvents. While the remaining mutants showed lower stability in organic solvents. Figure 1 presents the residual activities of BLk and mutants in the presence of 50% (v/v) acetonitrile over time. The results indicate that amino acid residues at positions 24 and 41 play a significant role in the solvent tolerance of BLk. Previous studies have reported that single residue mutations can improve the organic stability of enzyme, but the significant improvements in solvent stability though single site mutations have rarely been reported (Yamada et al. 2015 ). The mutants obtained in this study are expected to effectively manage keratin in challenging environments. Table 2 Stability of keratinase and mutants in various organic solvents Organic solvent Half-lives a Wild-type D41A A24E A24Q DMF 108.0 ± 3.0h 190.3 ± 2.5 202.8 ± 2.2 247.2 ± 2.5 DMSO > 720.0h > 720.0 > 720.0 > 720.0 Acetonitrile 2.4 ± 0.1h 112.8 ± 0.5 151.2 ± 1.0 146.4 ± 1.2 Acetone 4.6 ± 0.5h 100.8 ± 1.2 122.4 ± 1.0 124.8 ± 1.7 acetic ether 29.0 ± 0.5d 73.0 ± 1.0 69.3 ± 0.4 128.0 ± 2.1 Isoamyl 3.4 ± 0.5d 45.0 ± 0.9 11.3 ± 0.2 31.0 ± 0.1 Toluene 51.0 ± 0.5d 16.0 ± 0.4 12.7 ± 0.1 19.0 ± 0.1 n-Heptane 68.0 ± 0.5d 38.0 ± 0.3 43.3 ± 0.2 18.6 ± 0.1 a The half-lives were calculated from the formula: T 1/2 =0.693t/2.303lg(E o /E). Each value is the average of three independent experiments. The half-lives of keratinase variants in hydrophilic solvents and hydrophobic solvents were separately indicated in hours (h) and days (d). The thermostability of enzymes is an important factor to consider in the industrial application of keratin treatment. This study also investigated the thermostability and optimum temperature of both mutant and wild-type enzyme. As shown in Fig. 2 A, the mutants A24E and A24Q exhibited increased thermostability compared to the WT enzyme. Interestingly, the optimum temperature of A24E and A24Q was also higher (60 °C) than that of the WT protease (Fig. 2 B). However, the mutant D41A showed similar thermostability and optimum temperature to the wild-type BLk. While many studies have reported a significant relationship between organic solvent stability and thermostability, this study did not yield similar results. The mutant D41A showed enhanced organic solvent tolerance, but its thermostability was similar to that of the wild-type protease. These results suggest that solvent tolerance enhancement differs from thermal stability. The proteolytic activities of BLk and its mutants were determined, and kinetic constants including K m and k cat values were analyzed. The results showed no significant difference between them (data not shown). Previously, many studies have demonstrated that stability enhancement often comes at the cost of impaired catalytic activity (Miller 2017 ). However, our study confirmed that the activity can remain unaffected while stability increases. This may be attributed to the fact that the mutation located on the enzyme’s surface has limited contribution to its activity, as mentioned in our previous report (Zhu et al. 2016 ). 3.3 The reason responsible for solvent tolerance. To investigate the secondary structure of enzymes effected by mutation, the Circular dichroism (CD) curve of WT and mutants were determined, as shown in Figure S2, all enzymes presented similar trend line, indicating the mutation had no significant effect on the secondary structure of BLk. A significant difference of solvent stability can be observed in present of 50% (v/v) acetonitrile. Therefore, the solvent mixture of 50% (v/v) acetonitrile was selected as the solvent for MD simulation. To rationalize the reason behind the enhanced solvent stability of the enzyme, 20-ns MD simulations were performed. The Root Mean Square Deviation (RMSD) measures the extent of conformational alteration during the procession of MD simulation and reflects the rigidity of the protein, therefore it is a crucial indicator for evaluating the stability of protein. In this study, the RMSD values of the variants were analyzed, as shown in Fig. 3 . The mutant A24E, A24Q and D41A in 50% (v/v) acetonitrile exhibit lower RMSD values compared to the wild-type enzyme, indicating that the structure of the mutants is more stable than that of the wild-type enzyme. The Root Mean Square Fluctuation (RMSF) value is measure of flexibility of each residue in the target protein. As shown in Fig. 4 , the RMSF value for WT enzyme is obviously higher compared to the other three mutants, particularly in the N-terminal, C-terminal and 155–165 amino acid loop region. The average RMSF values for WT, A24E, A24Q and D41A were 0.096, 0.068, 0.075 and 0.076 nm, respectively. The trend of solvent stability for the BLk variants is A24E > A24Q > D41A > WT, which align with previous experimental findings. Hydrogen bonds (H-bonds) are considered the most important non-covalent interactions. Numerous studies have reported that enhanced stability is always accompanied by the formation of new H-bonds, which is beneficial for stabilization (Pace et al. 2014 ). In our study, new H-bonds were also formed in the mutant. Two new H-bonds were formed between the γ-COOH of Glu24, α-NH2 of Lys22 and β-OH of Ser86 for A24E. Additionally, only one new H-bond was formed between the side chain carbonyl oxygen of Gln24 and β-OH of Ser86 in A24Q, as shown in Fig. 5 . To evaluate the stability of these newly formed H-bonds, the occupancy rates were calculated and are presented in Table 3 . All occupancy rates of the formed H-bonds were over 70%, indicating that these H-bonds can stably exist in mutant’s structure. We speculate that the stable H-bonds promote the formation of an intramolecular interaction network in the mutant, thereby providing structural rigidity to maintain the native conformation of the enzyme. Consequently, the screened mutants A24E and A24Q exhibit the enhanced solvent stability compared to the wildtype BLk. Table 3 Occupancy and distance of hydrogen bond data collected along MD simulations mutants Donor H-X Acceptor Occupancy (%) Distance (Å) A24E Asp24.OE1 Lys22.N 73.6 2.9 ± 0.2 Asp24.OE2 Ser.OG 71.9 2.3 ± 0.2 A24Q Asp24.OE2 Ser.OG 73.1 2.8 ± 0.3 Compared to the wild-type enzyme, the residue Ala41 in the mutant enzyme exhibits a decreased number of hydrogen bonds. In the wild type enzyme, hydrogen bond mainly occurs between Asp41 and either the α-amino or α-carboxy group of the protein backbone. Structurally, residue Asp41 is partially buried and surrounded by a large number of hydrophobic amino acids, as shown in Fig. 6 A. In contrast, the side chain of residue Ala is just a methyl group, allowing for more flexibility in interactions with the loop region or Ca ion, thereby promoting local stability. Therefore, we hypothesize that the mutation Asp41 → Ala41 reduces the repulsion effect caused by Asp41. Furthermore, the β-COOH of Asp41 is likely to be deprotonated and charged at physiological conditions due to the pKa of its β-COOH below 6.0. It is generally assumed that charged residues are not favorable at interior sites for protein stability (Isom et al. 2010 ). To further investigate the mechanism behind the stability enhancement mediated by the D41A mutation, we analyze the Radius of gyration (Rg) value of the residues group within a 3.5 Å range of Asp41. As shown in Fig. 6 B, The Rg value of D41A mutant was significantly lower than that of the wild-type BLk. This result indicates that the specified region near the mutation site 41 in the D41A mutant is more compact than the counterpart of WT, and thus stabilized the entire enzyme in the presence of 50% (v/v) acetonitrile. The radial distribution g(r) value represents the distribution of solvent molecules around the protein pericore. This value indicates the penetration of acetonitrile molecules into the intramolecular space of the protein molecules, as shown in Fig. 7 . The density of acetonitrile molecules within the core of all mutants is lower than that of the wild-type enzyme. Among the mutants, A24E shows the lowest penetration of acetonitrile molecules, while A24Q and D41A exhibit a similar trend in the g(r) value. These results suggest that the mutants have a more compact structure due to increased interactions within the molecule, such as hydrogen bonds, hydrophobic effects and other non-covalent bonding effects. Additionally, the results further support the hypothesis that the D41A mutation improves stability by enhancing the hydrophobic effect discussed above. Conclusion Recently, keratinase has gained attention due to its application as the key enzyme in keratin hydrolysis. In this study, directed evolution was used to improve the organic solvent stability of keratinase. Three mutants, A24E, A24Q and D41A, were screened and showed 47-, 63- and 61-fold improvement, respectively, compared to the wild-type in the presence of 50% (v/v) acetonitrile. However, only mutants A24E and A24Q exhibited higher thermostability according to the properties analysis. This result indicates that the protein’s organic solvent tolerance is different from its thermostability. Furthermore, the possible mechanism responsible for solvent stability was rationalized. The increased hydrogen bond and hydrophilic interaction within the structure of the enzyme lead to a more compact structure, reducing the penetration of organic solvents. This lays the foundation for improving solvent tolerance. The mutants obtained in this study would be more suitable for industrial applications. Declarations Funding This research was supported by the National Natural Science Foundation of China (32201979), Scientific Research Project of Anhui province (2022AH051682), Anhui University scientific research project outstanding youth project (2023AH030109). Anhui Scientific Research and Innovation Team of Quality Evaluation and Improvement of Traditional Chinese Medicine (2022AH010090). CRediT authorship contribution statement Fucheng Zhu : Designed experiment and Writing-Original draft preparation; Zixu Yan and Jingli Dai : performed experiment, Guosi Li and Peipei Wei : Methodology, Software; Qilin Xu : Visualization, Investigation; Yunfeng Ma and Jingbo Ma : collected and analyzed the data; Naidong Chen and Yongjun Zang : Supervision, Writing - review & editing. Data Availability The data presented in current study are available from the corresponding author on reasonable request. Declaration of competing interests The authors declare that they have no conflicts of interest. 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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-3464492","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":241473856,"identity":"dcde46ea-1163-4cc2-be4a-cdf483a9b62d","order_by":0,"name":"Fucheng Zhu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYBAC9gYGNhBtx8/ffODAhx9EaOE5ANGSLDnjWOLBmT0kaGHccCDH+DAHGzFaJJKfPfi4o5aZ4cCZD4cZeBjk+cUOENKSZm4488xxPsbm3g2HCywYDGfOTsCvxV4ih02at+0YMzPD2Q2HZ/AwJBjcJqCFB6Tlb9sxxjaGnAeHediI1cLYVsPYw5DDQKQWnmdmkr1tB5IlJI4ZAANZgrBfeNiTn0n8bKuzsz/f/PjDhx828vzSBLRAwWEYQ4Io5SBQR7TKUTAKRsEoGIEAABXURa7NEHuCAAAAAElFTkSuQmCC","orcid":"","institution":"West Anhui University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Fucheng","middleName":"","lastName":"Zhu","suffix":""},{"id":241473857,"identity":"e72aa015-1589-4856-adbc-994743b55fa9","order_by":1,"name":"Zixu Yan","email":"","orcid":"","institution":"West Anhui University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zixu","middleName":"","lastName":"Yan","suffix":""},{"id":241473858,"identity":"5f7886d0-5e88-4cd0-b2bf-c6fd9f682ab4","order_by":2,"name":"Jingli Dai","email":"","orcid":"","institution":"West Anhui University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jingli","middleName":"","lastName":"Dai","suffix":""},{"id":241473859,"identity":"5405d558-ec90-46c5-8388-b0cf7b6579ee","order_by":3,"name":"Guosi Li","email":"","orcid":"","institution":"West Anhui University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guosi","middleName":"","lastName":"Li","suffix":""},{"id":241473860,"identity":"4a361c9a-7f44-48f6-90f7-d11c808a1525","order_by":4,"name":"Qiling Xu","email":"","orcid":"","institution":"West Anhui University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qiling","middleName":"","lastName":"Xu","suffix":""},{"id":241473861,"identity":"5b1558a1-652f-4896-9f3c-6390cf0b8207","order_by":5,"name":"Yunfeng Ma","email":"","orcid":"","institution":"Anhui Anlito Biological Technology Co, LTD，Anhui Huoshan Economic and Technological Development Zone P.R.C. Lu'an city","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yunfeng","middleName":"","lastName":"Ma","suffix":""},{"id":241473862,"identity":"ae320fb7-8b43-482f-adb6-8852be2cf9fc","order_by":6,"name":"Jingbo Ma","email":"","orcid":"","institution":"West Anhui University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jingbo","middleName":"","lastName":"Ma","suffix":""},{"id":241473863,"identity":"db607984-8266-42dd-ac3d-fd9a384b6741","order_by":7,"name":"Naidong Chen","email":"","orcid":"","institution":"West Anhui University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Naidong","middleName":"","lastName":"Chen","suffix":""},{"id":241473864,"identity":"1fa990fc-fe32-43e7-8484-f28b338634a7","order_by":8,"name":"Yongjun Zang","email":"","orcid":"","institution":"West Anhui University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yongjun","middleName":"","lastName":"Zang","suffix":""}],"badges":[],"createdAt":"2023-10-19 02:29:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3464492/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3464492/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":45168392,"identity":"f5194e51-039f-4f9a-94ff-73a6f517f11d","added_by":"auto","created_at":"2023-10-24 17:23:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10880,"visible":true,"origin":"","legend":"\u003cp\u003eStability of keratinase variants in presence of 50% (v/v) acetonitrile\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3464492/v1/ff481399a15f3dc5cb0431a3.png"},{"id":45168390,"identity":"8b92d473-17ec-4a10-8cd1-5a2975ee65f2","added_by":"auto","created_at":"2023-10-24 17:23:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":91714,"visible":true,"origin":"","legend":"\u003cp\u003eA: Thermal stability of keratinase variants in different temperature; B: the optimum temperature of keratinase variants\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3464492/v1/c4502a44309af335147db06d.png"},{"id":45168387,"identity":"783d711b-f912-4f20-b818-b937af2c06a8","added_by":"auto","created_at":"2023-10-24 17:23:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":22097,"visible":true,"origin":"","legend":"\u003cp\u003eRoot Mean Square Deviation (RMSD) of keratinase variants analyzed by MD simulation.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3464492/v1/60e2272b820c8f542ea3617d.png"},{"id":45169306,"identity":"cb13000c-2d91-4248-8f56-39d4ba702bb0","added_by":"auto","created_at":"2023-10-24 17:31:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":18827,"visible":true,"origin":"","legend":"\u003cp\u003eAverage Root Mean Square Float (RMSF) values calculated from the last 1-ns trajectory for the keratinase variants\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3464492/v1/60e78d98585b3f03e786124b.png"},{"id":45168391,"identity":"1809f672-0fa1-433a-9833-4e4b3122f6d9","added_by":"auto","created_at":"2023-10-24 17:23:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":363954,"visible":true,"origin":"","legend":"\u003cp\u003eHydrogen bonds at mutagenesis site 24 in A24E and A24Q mutants. H-bonds are labelled as \u003cem\u003eyellow dotted lines\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3464492/v1/a24f2a63756dd27f142d85b0.png"},{"id":45168394,"identity":"f7122073-ec06-48e2-98d5-5845c8d373ac","added_by":"auto","created_at":"2023-10-24 17:23:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":360759,"visible":true,"origin":"","legend":"\u003cp\u003eA: Structure of selected residues within 3.5 Å range of mutation site 41; B: Radius of gyration of selected region of mutants D41A and the WT enzyme during entire MD simulation in presence of 50% (v/v) acetonitrile\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3464492/v1/f628046e0c51e93122b8711a.png"},{"id":45168386,"identity":"7790d178-0ae4-401f-bd89-edb310fd6a62","added_by":"auto","created_at":"2023-10-24 17:23:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":12681,"visible":true,"origin":"","legend":"\u003cp\u003eSpherically averaged number densities of acetonitrile molecules as a function of distance from around the whole protease\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3464492/v1/88b5a07a83c379d114ca4482.png"},{"id":45259206,"identity":"41dfaf0f-0ff6-404b-b872-9ea20865305f","added_by":"auto","created_at":"2023-10-26 11:52:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1373448,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3464492/v1/c8c14b3f-9c55-4e1d-b164-b20641f0d9bf.pdf"},{"id":45168393,"identity":"7c617d47-194c-4553-81b0-b102d5ff6c33","added_by":"auto","created_at":"2023-10-24 17:23:51","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":429697,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstracts.png","url":"https://assets-eu.researchsquare.com/files/rs-3464492/v1/2cb082487646aacb518afbce.png"},{"id":45168389,"identity":"f9aeeb44-f98f-449d-a892-10177aee5595","added_by":"auto","created_at":"2023-10-24 17:23:51","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":582196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1\u003c/strong\u003e. The primers used in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S1\u003c/strong\u003e. Structure of identified residues located at the keratinase\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S2\u003c/strong\u003e. Far-UV circular dichroism (CD) spectra of BLk variants at 30 °C.\u003c/p\u003e","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-3464492/v1/6f9ae048772dc4579ce58c33.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Directed evolution of a keratinase BLk from Bacillus licheniformis to enhance the solvent tolerance","fulltext":[{"header":"Introduction","content":"\u003cp\u003eKeratin is a fibrous protein that is abundant and insoluble. It is found in mammalian horns, wool, claws, feathers, and beaks. The stability of keratin is attributed to its tight packing, disulfide bonds, and hydrophobic interactions. These properties enable it to resist degradation by proteolytic enzymes such as pepsin and trypsin (Stiborova et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). While chemical or physico-chemical hydrolysis can be used to utilize keratin, these methods consume a significant amount of energy and produce harmful gases, posing a threat to the environment (Dettmer et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).In recent years, keratinase has gained attention in bioremediation as it plays a crucial role in managing keratin resources and degrading keratin waste into amino acids or soluble peptides (Su et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eVarious keratinase producing microorganisms have been reported, including fungi such as \u003cem\u003eAspergillus\u003c/em\u003e and \u003cem\u003eMicrosporum\u003c/em\u003e (Bohacz et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and bacteria such as \u003cem\u003eBacillus\u003c/em\u003e (Benkiar et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Jagadeesan et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), \u003cem\u003ePseudomonas\u003c/em\u003e (Moonnee et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and \u003cem\u003eStreptomyces\u003c/em\u003e (Indhuja et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). These keratinases can be divided into 14 different protease families according to MEROPS database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ebi.ac.uk/merops/\u003c/span\u003e\u003cspan address=\"http://www.ebi.ac.uk/merops/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The unique property of keratinase, which sets it apart from other proteases, is its ability to bind to insoluble protein substrates. However, natural insoluble substrates like poultry feathers often create a harsh environment, posing a challenge for the application of keratinase. Therefore, it is necessary to enhance the stability or activity of keratinase to facilitate its industrial application.\u003c/p\u003e \u003cp\u003eMultiple strategies have been successfully employed to improve the stability of keratinase. Srivastava et al. significantly enhanced the thermostability and activity of keratinase by immobilizing protease on chitosan (Srivastava et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The activity and thermostability of keratinase KerSMD were significantly improved through domain replacement (Fang et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Liu et al. achieved an excellent mutant with increased thermostability and catalytic efficiency through rational design (Liu et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Enzyme engineering has been recognized as an attractive strategy for enhancing the properties of keratinase (Gupta et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Currently, the focus of improving the stability of keratinase lies mainly in enhancing its thermostability. However, there have been few reports on improving its solvent tolerance. It is widely acknowledged that the keratin hydrolysis reaction mixture contains complex ingredients, including surfactants or other organic solvents. Therefore, it is also crucial for the keratinase to maintain high activity in organic solvents in order to effectively apply it in the process of keratin hydrolysis.\u003c/p\u003e \u003cp\u003eThere is a correlation between solvent stability and thermostability, although these two types of stability often exhibit different trends. While it is difficult to predict the solvent stability of a protein and identify the protein's hotspots that affect solvent tolerance using molecular simulation methods, it is relatively easier to enhance the thermostability of a protein (Zhu et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additionally, there have been fewer studies on the evolution of protein solvent tolerance, and the mechanisms underlying improved solvent stability are not well understood. Directed evolution, using error-prone polymerase chain reaction (PCR), is a suitable strategy for modifying enzyme tolerance in organic solvents as it does not rely on prior knowledge of the protein's structure and mechanism (McCullum et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In a previous study, we successfully enhanced the solvent stability of metalloprotease PT121 by using error-prone PCR, resulting in 1.2\u0026ndash;3.5-fold increases in half-lives (Zhu et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Koudelakova et al. also employed the same method to engineer an enzyme with significantly improved stability, increasing its half-life in the presence of 40% (v/v) DMSO by 5040-fold (Koudelakova et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). They found that residues located around the access tunnel, which connects the surrounding solvent with the active pocket of the enzyme, played a crucial role in stabilizing the protein. These studies highlight the effectiveness of directed evolution as a strategy for improving protein solvent tolerance.\u003c/p\u003e \u003cp\u003eIn a previous study, a keratinase BLk from \u003cem\u003eBacillus licheniformis\u003c/em\u003e was expressed in the \u003cem\u003eBacillus subtilis\u003c/em\u003e WB800 system. This current study aims to improve the organic solvent stability of BLk through directed evolution using error-prone PCR. The properties of the obtained mutants, including thermostability and optimum temperature, were analyzed. Additionally, the study discusses the possible intrinsic molecular reasons behind these properties. The findings from this research provide valuable insights for expanding the application of keratinase and enhancing the solvent stability of related enzymes.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals and strains\u003c/h2\u003e \u003cp\u003eThe strain \u003cem\u003eBacillus subtilis\u003c/em\u003e WB800 and pHY300 were used as host and vector for gene cloning and expression. The restriction enzymes were purchased from Takara (Dalian, China), DNA polymerases and ClonExpress Ultra One Step Cloning Kit were obtained from vazyme (Nanjing, China). All other chemicals in this study were Sigma Chemical Co. (Shanghai, China) and Sangon Biotech (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Random mutagenesis library generation\u003c/h2\u003e \u003cp\u003eThe recombinant \u003cem\u003eBacillus subtilis\u003c/em\u003e WB800 and \u003cem\u003eEscherichia coli\u003c/em\u003e DH 5ɑ carrying PHY300-BLk plasmid were preserved in our laboratory. The mutant library was constructed using error-prone PCR method. The PCR was performed in a 50 \u0026micro;L reaction system containing 0.3 mM dNTP, 0.1 ng/uL of the template DNA, 0.01 mM MnCl\u003csub\u003e2\u003c/sub\u003e, 0.05 U/\u0026micro;L Taq DNA polymerase, 0.02 mM primers and 1.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e. The reaction product was digested with \u003cem\u003eDpn\u003c/em\u003e I at 37\u0026deg;C for 1 h and then inserted into vector PHY300 using homologous recombination method. Recombinant plasmid were transformed into \u003cem\u003eE. coli\u003c/em\u003e DH 5ɑ and then submitted into sequencing. Then the verified recombinant plasmid was transformed into \u003cem\u003eB. subtilis\u003c/em\u003e WB800 for expression. The primers used in this study are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eAn overnight culture (1 mL) of recombinant \u003cem\u003eB. subtillis\u003c/em\u003e WB800 in TB medium containing kanamycin (50 \u0026micro;g/mL) was inoculated into 100 mL Terrific Broth medium containing kanamycin (50 \u0026micro;g/mL) and incubated at 37\u0026deg;C, 180 rpm for 48h. The culture supernatant was obtained by centrifugation and loaded on a HiTrap Phenyl-Sepharose Fast Flow hydrophobic interaction chromatography column. The eluted fractions were collected for activity assay and SDS-PAGE analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Screen and Assay of protease mutants\u003c/h2\u003e \u003cp\u003eProtease assay was performed as described previously (Zhu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Two milliliters of the sample were added to the solution (2 mL) containing 2% (w/v) casein and 50 mM Tris-HCl buffer (pH 8.0). The mixture was incubated at 40\u0026deg;C for 10 min and terminated by adding 4.0 mL of TCA mixture (containing 0.11 M trichloroacetic acid, 0.22 M sodium acetate and 0.33 M acetic acid). This mixture was further centrifuged at 15,000 \u0026times;g for 15 min, and the absorbance of the supernatant was measured at 280 nm against a blank control. One unit (U) of protease activity is defined as the amount of enzyme that hydrolyzes casein and produces 1 \u0026micro;g of tyrosine per min under the assay conditions.\u003c/p\u003e \u003cp\u003eIn this study, a 50% (v/v) N, N-dimethylformamide (DMF) solution was used to assess the solvent stability of the mutant. The mixtures were incubated in universal bottles with screw caps at 37\u0026deg;C for 2 hours. The initial activity and residual activities were measured using standard assay conditions. The relative residual activity, which indicates stability, was calculated. Mutants that showed significant differences in relative residual activity compared to the WT protease were selected for further analysis. The sequences of these selected mutants were determined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. The properties of mutants\u003c/h2\u003e \u003cp\u003eThe stability of mutants in organic solvents was assessed using the following method. Protease variants were incubated at a pH of 10.0 and a temperature of 37\u0026deg;C with shaking at 200 rpm in the presence of 50% (v/v) various organic solvents. The remaining activities of the proteases were measured at different time intervals. The half-lives (T\u003csub\u003e1/2\u003c/sub\u003e) were calculated from the exponential regression curve. Each experiment was performed in triplicate. The optimal temperature for enzymatic activity was determined by increasing the temperature from 20 to 65\u0026deg;C at a pH of 10. The thermostability of the purified enzyme was determined by pre-incubating it for 30 minutes at temperatures ranging from 30 to 60\u0026deg;C. The residual activity was then analyzed using the activity assay method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Circular dichroism determination\u003c/h2\u003e \u003cp\u003eCircular dichroism (CD) curve was performed as our previously described using Chirascan\u0026trade; CD spectrometer (Leatherhead, UK) (Zhu et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The sample of 4\u0026ndash;6 \u0026micro;M enzyme was infused into 1 mm cuvette. Spectra of sample was measured from 190 to 260 nm using 1nm bandwidth.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. The structural feature of mutants\u003c/h2\u003e \u003cp\u003eThe structure of the keratinase and its mutants was constructed using SWISS-MODEL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://swissmodel_expasy.org\u003c/span\u003e\u003cspan address=\"http://swissmodel_expasy.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) based on the X-ray crystal structure of the protease from \u003cem\u003eBacillus licheniformis\u003c/em\u003e (Protein Data Bank ID: 1A1Y, crystal resolution: 1.05 \u0026Aring;), which share 99.6% identity with keratinase BLk. The structure was visualized and analyzed using Discovery Studio 2019 and PyMOL 2.6 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Molecular Dynamic Simulation\u003c/h2\u003e \u003cp\u003eMolecular Dynamic (MD) Simulations were performed using Gromacs version 2018 to analyze the factors contributing to improved solvent stability as previous descriptions (Zhu et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The MD simulations utilized the GROMOS 53A6 force field. The keratinase BLk model was solvated with a 50% (v/v) acetonitrile/water mixture with explicit waters modeled using a simple point charge (SPC) model (Hermans et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). The MD simulations system was neutralized by adding the sodium ions. The electro-neutral systems were then subjected to energy minimization using steepest descent method, followed by a 500-ps MD simulations where the heavy atoms and protein were kept fixed. Subsequently, a 20-ns MD simulation was performed on the entire system with a temperature gradient (300 K and 380 K). The LINCS algorithm was used to constrain bond lengths (Hess et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). The cut-off value for the van der Waals interactions was set at 1.0 nm, and the electrostatic interactions were calculated using a particle mesh Ewald algorithm. Periodic boundary conditions were applied, and the coordinates were saved for analysis at every 1ps interval. Gromacs tools were utilized to analyzed various characterization parameters such as Root Mean Square Deviation (RMSD) value, Root Mean Square Fluctuation (RMSF) value, Radius of gyration (Rg) value, Radial distribution g(r), and hydrogen bond. PyMOL 2.6 open-source version and Discovery studio 2019 programs were used to analyze the structures of enzyme.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Library Generation and Screening\u003c/h2\u003e \u003cp\u003eThe properties of keratinase BLk from \u003cem\u003eBacillus licheniformis\u003c/em\u003e were found to be sensitive to 50% (v/v) DMF, which is toxic to most enzymes. Therefore, 50% (v/v) DMF was chosen as the organic solvent for the typical mutant screening. In this study, random mutant library screening was conducted by increasing the transparent cycle in skim milk plates and assessing the retention of proteolytic activity after incubation of the protease in the presence of 50% (v/v) DMF. Thirteen mutants with significant changes in solvent stability, but no significant differences in proteolytic activity, were obtained from the mutant library, which contained over 2000 clones. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, mutants D41A, A24E and A24Q exhibited a remarkable increased in activity after being incubated in 50% DMF for 90 minutes, while the other mutants showed a significant reduction compared to the wild-type protease. Structural analysis revealed that all mutations were located on the surface of the protein, mostly in the loop region of enzyme\u0026rsquo;s secondary structure (as shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). This indicates that surface residues in the loop region play an important role in the solvent tolerance of the enzyme, which is consistent with previous reports (Ogino et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). To further explore the properties of the mutants and understand the possible reasons behind the changes in organic solvent stability, the properties of mutants were submitted to further research.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRelative residual activities of mutant and wild-type of keratinase\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMutants\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResidual activity (%) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLocation of mutation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWild-type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD41A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e114\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSurface/Loop\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA24E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSurface/Loop\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA24Q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSurface/Loop\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD41V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSurface/Loop\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA24V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSurface/Loop\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA24T/G165D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSurface/Loop, Interior/Loop\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG20D/G46D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSurface/Loop, Surface/Sheet\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA13V/G46V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSurface/Helix, Surface/Sheet\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA18V/G117D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSurface/Helix, Surface/Loop\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA137T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSurface/Helix\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS183N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSurface/Loop\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA214V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSurface/Sheet\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA273T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSurface/Helix\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003ea\u003c/sup\u003e The residual activities of keratinase were determined after incubation with 50% DMF for 1.5h, and are shown as a percentage of their initial activity without addition of organic solvent. Values are the average of three independent experiments.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. The properties of keratinase BLk variants\u003c/h2\u003e \u003cp\u003eTo further investigate solvent stability, we determined the half-life of mutants and wild-type. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, three mutants (A24E, A24Q and D41A) exhibited improved stability in all tested solvents except for n-Heptane. Surprisingly, the half-life of D41A, A24E and A24Q increased by 47-, 63- and 6-fold, respectively, in presence of 50% (v/v) acetonitrile compared to the wild-type. Similarly, in presence of 50% (v/v) acetone, the half-life of D41A, A24E and A24Q increased by 22-, 27-, 27-fold, respectively. These mutants demonstrated remarkable tolerance to polar solvents. While the remaining mutants showed lower stability in organic solvents. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the residual activities of BLk and mutants in the presence of 50% (v/v) acetonitrile over time. The results indicate that amino acid residues at positions 24 and 41 play a significant role in the solvent tolerance of BLk. Previous studies have reported that single residue mutations can improve the organic stability of enzyme, but the significant improvements in solvent stability though single site mutations have rarely been reported (Yamada et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The mutants obtained in this study are expected to effectively manage keratin in challenging environments.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStability of keratinase and mutants in various organic solvents\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eOrganic solvent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eHalf-lives \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWild-type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD41A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA24E\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA24Q\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e108.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e190.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e202.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e247.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMSO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;720.0h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;720.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;720.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;720.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcetonitrile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e112.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e151.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e146.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcetone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e122.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e124.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eacetic ether\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e73.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e69.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e128.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsoamyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eToluene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003en-Heptane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003ea\u003c/sup\u003e The half-lives were calculated from the formula: T\u003csub\u003e1/2\u003c/sub\u003e=0.693t/2.303lg(E\u003csub\u003eo\u003c/sub\u003e/E). Each value is the average of three independent experiments. The half-lives of keratinase variants in hydrophilic solvents and hydrophobic solvents were separately indicated in hours (h) and days (d).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe thermostability of enzymes is an important factor to consider in the industrial application of keratin treatment. This study also investigated the thermostability and optimum temperature of both mutant and wild-type enzyme. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, the mutants A24E and A24Q exhibited increased thermostability compared to the WT enzyme. Interestingly, the optimum temperature of A24E and A24Q was also higher (60 \u0026deg;C) than that of the WT protease (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). However, the mutant D41A showed similar thermostability and optimum temperature to the wild-type BLk. While many studies have reported a significant relationship between organic solvent stability and thermostability, this study did not yield similar results. The mutant D41A showed enhanced organic solvent tolerance, but its thermostability was similar to that of the wild-type protease. These results suggest that solvent tolerance enhancement differs from thermal stability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe proteolytic activities of BLk and its mutants were determined, and kinetic constants including \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e and \u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e values were analyzed. The results showed no significant difference between them (data not shown). Previously, many studies have demonstrated that stability enhancement often comes at the cost of impaired catalytic activity (Miller \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, our study confirmed that the activity can remain unaffected while stability increases. This may be attributed to the fact that the mutation located on the enzyme\u0026rsquo;s surface has limited contribution to its activity, as mentioned in our previous report (Zhu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 The reason responsible for solvent tolerance.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the secondary structure of enzymes effected by mutation, the Circular dichroism (CD) curve of WT and mutants were determined, as shown in Figure S2, all enzymes presented similar trend line, indicating the mutation had no significant effect on the secondary structure of BLk.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA significant difference of solvent stability can be observed in present of 50% (v/v) acetonitrile. Therefore, the solvent mixture of 50% (v/v) acetonitrile was selected as the solvent for MD simulation. To rationalize the reason behind the enhanced solvent stability of the enzyme, 20-ns MD simulations were performed. The Root Mean Square Deviation (RMSD) measures the extent of conformational alteration during the procession of MD simulation and reflects the rigidity of the protein, therefore it is a crucial indicator for evaluating the stability of protein. In this study, the RMSD values of the variants were analyzed, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The mutant A24E, A24Q and D41A in 50% (v/v) acetonitrile exhibit lower RMSD values compared to the wild-type enzyme, indicating that the structure of the mutants is more stable than that of the wild-type enzyme.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Root Mean Square Fluctuation (RMSF) value is measure of flexibility of each residue in the target protein. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the RMSF value for WT enzyme is obviously higher compared to the other three mutants, particularly in the N-terminal, C-terminal and 155\u0026ndash;165 amino acid loop region. The average RMSF values for WT, A24E, A24Q and D41A were 0.096, 0.068, 0.075 and 0.076 nm, respectively. The trend of solvent stability for the BLk variants is A24E\u0026thinsp;\u0026gt;\u0026thinsp;A24Q\u0026thinsp;\u0026gt;\u0026thinsp;D41A\u0026thinsp;\u0026gt;\u0026thinsp;WT, which align with previous experimental findings.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHydrogen bonds (H-bonds) are considered the most important non-covalent interactions. Numerous studies have reported that enhanced stability is always accompanied by the formation of new H-bonds, which is beneficial for stabilization (Pace et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In our study, new H-bonds were also formed in the mutant. Two new H-bonds were formed between the γ-COOH of Glu24, α-NH2 of Lys22 and β-OH of Ser86 for A24E. Additionally, only one new H-bond was formed between the side chain carbonyl oxygen of Gln24 and β-OH of Ser86 in A24Q, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e. To evaluate the stability of these newly formed H-bonds, the occupancy rates were calculated and are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. All occupancy rates of the formed H-bonds were over 70%, indicating that these H-bonds can stably exist in mutant\u0026rsquo;s structure. We speculate that the stable H-bonds promote the formation of an intramolecular interaction network in the mutant, thereby providing structural rigidity to maintain the native conformation of the enzyme. Consequently, the screened mutants A24E and A24Q exhibit the enhanced solvent stability compared to the wildtype BLk.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOccupancy and distance of hydrogen bond data collected along MD simulations\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003emutants\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDonor H-X\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAcceptor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOccupancy (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDistance (\u0026Aring;)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eA24E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAsp24.OE1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLys22.N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e73.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAsp24.OE2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSer.OG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e71.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA24Q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAsp24.OE2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSer.OG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e73.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eCompared to the wild-type enzyme, the residue Ala41 in the mutant enzyme exhibits a decreased number of hydrogen bonds. In the wild type enzyme, hydrogen bond mainly occurs between Asp41 and either the α-amino or α-carboxy group of the protein backbone. Structurally, residue Asp41 is partially buried and surrounded by a large number of hydrophobic amino acids, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eA. In contrast, the side chain of residue Ala is just a methyl group, allowing for more flexibility in interactions with the loop region or Ca ion, thereby promoting local stability. Therefore, we hypothesize that the mutation Asp41 \u0026rarr; Ala41 reduces the repulsion effect caused by Asp41. Furthermore, the β-COOH of Asp41 is likely to be deprotonated and charged at physiological conditions due to the pKa of its β-COOH below 6.0. It is generally assumed that charged residues are not favorable at interior sites for protein stability (Isom et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). To further investigate the mechanism behind the stability enhancement mediated by the D41A mutation, we analyze the Radius of gyration (Rg) value of the residues group within a 3.5 \u0026Aring; range of Asp41. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, The Rg value of D41A mutant was significantly lower than that of the wild-type BLk. This result indicates that the specified region near the mutation site 41 in the D41A mutant is more compact than the counterpart of WT, and thus stabilized the entire enzyme in the presence of 50% (v/v) acetonitrile.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe radial distribution g(r) value represents the distribution of solvent molecules around the protein pericore. This value indicates the penetration of acetonitrile molecules into the intramolecular space of the protein molecules, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003e. The density of acetonitrile molecules within the core of all mutants is lower than that of the wild-type enzyme. Among the mutants, A24E shows the lowest penetration of acetonitrile molecules, while A24Q and D41A exhibit a similar trend in the g(r) value. These results suggest that the mutants have a more compact structure due to increased interactions within the molecule, such as hydrogen bonds, hydrophobic effects and other non-covalent bonding effects. Additionally, the results further support the hypothesis that the D41A mutation improves stability by enhancing the hydrophobic effect discussed above.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e "},{"header":"Conclusion","content":"\u003cp\u003eRecently, keratinase has gained attention due to its application as the key enzyme in keratin hydrolysis. In this study, directed evolution was used to improve the organic solvent stability of keratinase. Three mutants, A24E, A24Q and D41A, were screened and showed 47-, 63- and 61-fold improvement, respectively, compared to the wild-type in the presence of 50% (v/v) acetonitrile. However, only mutants A24E and A24Q exhibited higher thermostability according to the properties analysis. This result indicates that the protein\u0026rsquo;s organic solvent tolerance is different from its thermostability. Furthermore, the possible mechanism responsible for solvent stability was rationalized. The increased hydrogen bond and hydrophilic interaction within the structure of the enzyme lead to a more compact structure, reducing the penetration of organic solvents. This lays the foundation for improving solvent tolerance. The mutants obtained in this study would be more suitable for industrial applications.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Natural Science Foundation of China (32201979), Scientific Research Project of Anhui province (2022AH051682), Anhui University scientific research project outstanding youth project (2023AH030109). Anhui Scientific Research and Innovation Team of Quality Evaluation and Improvement of Traditional Chinese Medicine (2022AH010090).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFucheng Zhu\u003c/strong\u003e: Designed experiment and Writing-Original draft preparation; \u003cstrong\u003eZixu Yan\u003c/strong\u003e and \u003cstrong\u003eJingli Dai\u003c/strong\u003e: performed experiment, \u003cstrong\u003eGuosi Li\u003c/strong\u003e and \u003cstrong\u003ePeipei Wei\u003c/strong\u003e: Methodology, Software; \u003cstrong\u003eQilin Xu\u003c/strong\u003e: Visualization, Investigation; \u003cstrong\u003eYunfeng Ma\u003c/strong\u003e and \u003cstrong\u003eJingbo Ma\u003c/strong\u003e: collected and analyzed the data; \u003cstrong\u003eNaidong Chen\u003c/strong\u003e and \u003cstrong\u003eYongjun Zang\u003c/strong\u003e: Supervision, Writing - review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data presented in current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBenkiar A, Nadia ZJ, Badis A, Rebzani F, Soraya BT, Rekik H, Naili B, Ferradji FZ, Bejar S, Jaouadi B (2013) Biochemical and molecular characterization of a thermo- and detergent-stable alkaline serine keratinolytic protease from \u003cem\u003eBacillus circulans\u003c/em\u003e strain DZ100 for detergent formulations and feather-biodegradation process. Int Biodeterior Biodegrad 83:129\u0026ndash;138. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ibiod.2013.05.014\u003c/span\u003e\u003cspan address=\"10.1016/j.ibiod.2013.05.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBohacz J, Kornillowicz-Kowalska T, Kitowski I, Ciesielska A (2020) Degradation of chicken feathers by \u003cem\u003eAphanoascus keratinophilus\u003c/em\u003e and \u003cem\u003eChrysosporium tropicum\u003c/em\u003e strains from pellets of predatory birds and its practical aspect. 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Appl Biochem Biotechnol 178(4):725\u0026ndash;738. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s12010-015-1905-7\u003c/span\u003e\u003cspan address=\"10.1007/s12010-015-1905-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"Keratinase BLk, Organic solvent stability, Directed evolution, MD Simulation","lastPublishedDoi":"10.21203/rs.3.rs-3464492/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3464492/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eKeratinase, an important protein used in hair degradation, requires stability enhancement in industrial applications due to the harsh reaction environment for keratin hydrolysis. Previous studies have focused on improving the thermostability of keratinase. In this study, directed evolution was applied to enhance the organic solvent stability of keratinase BLk from \u003cem\u003eBacillus licheniformis\u003c/em\u003e. Three excellent mutants were screened and exhibited significantly improved stability in various solvents, although similar results were not observed in terms of thermostability. The identified mutations were located on the enzyme's surface. The half-life of the D41A, A24E, and A24Q mutants increased by 47-, 63-, and 61-fold, respectively, in the presence of 50% (v/v) acetonitrile compared to the wild-type. Similarly, in the presence of 50% (v/v) acetone, the half-life of these mutants increased by 22-, 27-, and 27-fold compared to the wild-type. Importantly, the proteolytic activity of all selected mutants was similar to that of the parent keratinase BLk. Furthermore, molecular dynamic simulation was employed to analyze the possible reasons for the enhanced solvent stability. The results suggest that increased intramolecular interactions, such as hydrogen bonds and hydrophobic interactions, may contribute to the improved solvent tolerance. The mutants obtained in this study hold significant potential for industrial applications.\u003c/p\u003e","manuscriptTitle":"Directed evolution of a keratinase BLk from Bacillus licheniformis to enhance the solvent tolerance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-24 17:23:46","doi":"10.21203/rs.3.rs-3464492/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":"06c63ff3-2213-4d54-a1a2-5e68828ae57d","owner":[],"postedDate":"October 24th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-10-26T11:44:41+00:00","versionOfRecord":[],"versionCreatedAt":"2023-10-24 17:23:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3464492","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3464492","identity":"rs-3464492","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}