Saturation mutagenesis at Ser196 in BaCsn46A from Bacillus Amyloliquefaciens Enhances Enzyme Activity and Thermostability

Saturation mutagenesis at Ser196 in BaCsn46A from Bacillus Amyloliquefaciens Enhances Enzyme Activity and Thermostability | 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 Saturation mutagenesis at Ser196 in BaCsn46A from Bacillus Amyloliquefaciens Enhances Enzyme Activity and Thermostability Wang Yi, Gao Wenjun, Wang Hailong, Xu Kepan, Luo Wen, Guo Jing, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1119314/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 The chitosanase (BaCsn46A) was extracted from Bacillus amyloliquefaciens (GenBank: QEK97559.1) and synthesized after codon optimization. The saturation mutation site was determined by analyzing the sequence and three-dimensional protein model. WT and mutant chitosanase genes were cloned and expressed in E. coli BL21 (DE3). The enzymatic properties of WT and mutants were compared, including the optimal reaction pH, temperature and thermostability. Three mutants S196F, S196Y and S196A with the highest specific enzyme activity were selected for further study. Compared with WT, the specific enzyme activity of S196Y increased by 144.76% (more than other two mutants), and the thermostability was not significantly improved. While the specific enzyme activity of S196A increased by 118.79%, and the thermostability of S196A was much higher than WT. From the perspective of industrial production, S196A is more in line with the requirements of industrial production because of its excellent thermal stability at 60°C. From the results of circular dichroism spectrum, the mutation of chitosanase at Ser196 did not change the secondary protein structure. In addition, CD analysis showed that the secondary structure of WT and mutants did not change significantly, indicating that the improvement of thermostability of S196A was not related to the secondary structure. Molecular Biology General Microbiology Chitosanase Saturated site-directed mutagenesis Enzymatic properties Molecular docking Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Chitosanase (EC 3.2.1.132) is a glycosyl hydrolase and widely distributed in nature. It has been detected among microorganisms, animals and plants [ 1 , 2 ] . According to the current research, the chitosanase activity produced by bacteria is much higher than that of fungi and actinomycetes, and bacteria have the advantage of short fermentation time. By comparison, there is no similarity between the nucleotide sequences of fungal chitosanases and bacterial chitosanases [ 3 ] . According to the similarity of these sequences, chitosanases were categorized to seven glycoside hydrolase (GH) families: GH 2, 5, 7, 8, 46, 75 and 80 [ 4 ] . Most of previous reported fungal chitosanases showed significant similarity with each other and are classified as GH 75 family, while the most bacterial chitosanases belong to GH 46 family [ 3 , 5 ] . Chitosan is an alkaline polysaccharide. Chitosanase can degrade it into chitooligosaccharides (COS) or glucosamine by hydrolyzing the β-1,4-glycosidic linkage [ 6 ] . Chitosan and COS have strong biological activity, however, the water solubility of chitosan is worse than that of COS, which can only be dissolved in diluted acid, therefore COS plays a greater role in practical application. Based on its non-toxic, antibacterial, degradable characteristics, their practical applications involve various fields such as food, agriculture, medicine and environment [ 7 ] . They have a wide range of functions in wound dressing biomaterials [ 8 ] , plant diseases prevention [ 9 ] , inhibiting tumor cells [ 10 ] , adsorbing of heavy metals in wastewater [ 11 ] and so on. It was reported that there were many microorganisms with chitosanase production capability through the traditional screening method in the laboratory, such as Bacillus [ 12 , 13 ] , Pseudomonas [ 14 ] , Matsuebacter chitosanotabidus [ 15 ] , Streptomyces [ 16 ] , Penicillium [ 17 ] , Chaetomium globosum [ 18 ] , etc. However, the directly selected bacteria have low enzyme activity or low enzyme production efficiency, which can not meet industrial requirements. Therefore, the biological transformation of the existing chitosanase by means of molecular biology is a good method for improving the enzyme activity and stability. Until now, many chitosanase genes have been cloned and heterologously expressed. Pichia pastoris is quite effective in folding exogenous proteins and shows a high capacity of extracellular protein production [ 19 ] . As a protein expression system, E. coli is more common than Pichia pastoris , and has the advantages of clear genetic background, low cost, high productivity and compatibility with a variety of antibodies [ 20 ] . Directed evolution and rational design can improve the enzymatic properties, such as protein activity and stability, and the recombination of amino acid sequence is the key step of directed evolution and rational design [ 21 ] . As early as the 1970s, Smith [ 22 ] discovered the technology of oligonucleotide site-directed mutagenesis and won the Nobel Chemistry Prize in 1993. This technology also provided new ideas for future generations and greatly promoted the research progress of protein engineering. On the basis of the derived point saturation mutation technology, it can obtain the other 19 amino acids which respectively replace the amino acids at the target site in a short period of time. There are several methods of point saturation mutation, including oligonucleotide directed mutagenesis [ 22 ] , cassette mutagenesis [ 23 ] , mutagenic oligonucleotide directed PCR amplification [ 24 ] , gene spreading by overlap extension and mutagenic plasma amplification [ 25 ] . In our study, E. coli was used as a heterologous expression vector. Through the calculation and analysis of gene sequence, we determined the mutation site, and compared the three-dimensional model, enzymatic properties and molecular docking between WT chitosanase and mutant chitosanases. Three mutant chitosanases with higher specific enzyme activity than the WT were obtained in the experiment. Among them, the thermostability of S196Y has been greatly improved, which can have a greater application prospect in industry. Materials And Methods Materials Escherichia coli DH5α, BL21(DE3) were purchased from Takara (Dalian, China) and plasmid pET-28a was purchased from Novegen (Darmstadt, Germany). Chitosan (degree of deacetylation over 80%) and D(+)-glucosamine, hydrochloride were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Restriction enzymes Bam H I and Hin d III were purchased from Takara (Nanjing, China).The High Purity Plasmid Small Preparation Kit was purchased from Beijing Bioteke Corporation Co., Ltd, (Beijing, China).The Site-directed Mutagenesis Kit, the Modified Bradford Protein Assay Kit, Ni-NTA Sefinose(TM) Resin 6FF (Settled Resin) and Desalting Gravity Column were purchased from Sangong Biotech (Shanghai) Co., Ltd. (Shanghai, China). The chitosanase gene was from Bacillus amyloliquefaciens (GenBank: QEK97559.1). After optimizing the codon of the gene sequence (BaCsn46A), it was synthesized by Nanjing GenScript Biotechnology Co., Ltd. (Nanjing, China). Three-dimensional model construction and homologous sequence comparison of chitosanase The protein sequence of Bacillus amyloliquefaciens (BaCsn46A) was submitted to Swiss-Model (https://swissmodel.expasy.org/) [26] online server to predict its three-dimensional structure, and the chitosanase molecular model from Bacillus subtilis MY002 was used as the template (PDB ID : 7C6C. 1. A) [27] . The three-dimensional model was analyzed on PyMOL 2.4.1 (https://pymol.org/2/) [28] . The amino acid sequence was compared and analyzed by DNAMAN 9.0. The molecular docking was performed between chitosanase and substrate chitotetraose by autodock 4.2 (http://autodock.scripps.edu/) [29] . Design and synthesis of primers The primers used for saturated site-directed mutagenesis of Ser196 in BaCsn46A were designed and sent to Sangong Biotech (Shanghai) Co., Ltd. (Shanghai, China) for synthesis. The designed primer sequences were shown in Table 1. Construction of saturated mutation library The saturated mutation library was constructed by whole plasmid PCR. The PCR system includes Pfu DNA polymerase and dNTP. This process takes plasmid pET-28a as template and the sequences in Table 1 as primers respectively. After PCR, Endonuclease Dpn I was added to the product and placed in a 37 °C water bath for 1 h to remove the methylated plasmid. The target plasmid in the PCR product was combined with competent cells made of E. coli DH5α and transformed. The above conversion solution which was added to 1 mL Luria-Bertani (LB) solution was incubated at 37 °C and 160 rpm for 1 h. After the bacterial solution was concentrated, it was evenly distributed on the LB solid medium plate containing kanamycin and incubated overnight at 37 °C and 160 rpm. Transformants were selected from the plate and cultured in LB liquid medium. The plasmids were extracted from the bacterial solution according to the kit, and the nucleic acid gel was run to test whether the PCR was successful. Finally, the bacterial solution was sent to Sangong Biotech (Shanghai) Co., Ltd. (Shanghai, China) for sequencing. Expression and purification of recombinant enzymes The sequenced recombinant enzymes were reincubated in LB liquid medium at 37 °C and 160 rpm for about 12 hours. Thereafter, 1 % of the above bacterial solution was transferred to a unused LB liquid medium and continued to incubate for 4 hours under the same conditions, and then isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to induce protein expression. After being incubated overnight at 16 °C and 160 rpm, the bacterial solution was centrifuged by high speed freezing centrifuge, and the cells were resuspended with buffer M0 (20 mM Tris-HCl solution, 500 mM NaCl solution, 10 % glycerol, pH 8.0, constant volume to 1L). Then, the resuspended solution was ultrasonically broken on ice, centrifuged, and the supernatant was taken out and stored at -20 °C. Ni-NTA column can adsorb proteins with histidine tags and imidazole solutions with different concentration gradients (on the basis of buffer M0, 20-300 mM imidazole solution was added respectively) can be used for elution. Ultimately, the purified chitosanase solution can be stored at -20 °C and used for subsequent determination. SDS–polyacrylamide gel electrophoresis (SDS–PAGE) One volume of protein sample was mixed with 4 times volume of SDS-PAGE loading buffer (2.5 mL 1 M Tris-HCl (pH 6.8), 1 g SDS, 50 mg bromophenol blue, 5 mL glycerol, 0.5 mL β-mercaptoethanol，10 ml in total). The mixture solution was boiled for 5 min and was added to the 12 % SDS-PAGE gel. The electrophoresis apparatus ran at 80V first, and then changed to run at 120V until the end. After electrophoresis, the gel was dyed and decolorizing. Finally, a picture containing protein bands was obtained on electrophoresis gel imaging system. Determination of chitosanase activity and protein content Chitosanase activity was measured by using 3,5-dinitrosalicylic acid (DNS) method with slight modification [30] . The total reaction system of chitosan enzyme was 2 mL, including 50 mM phosphate buffer (pH 6.6), 1 % colloidal chitosan (chitosan is dissolved in dilute acid, w/v) and an appropriate amount of purified chitosanase solution, which is properly diluted. After being mixed completely, the mixture solution was incubated at 50 °C for 10 min. The reaction was stopped by adding 1.5 mL DNS to the above mixture solution and then the mixture solution was incubated in boiling water for 5 min to develop color. Next, the boiled solution was diluted to 25 mL and centrifuged. Finally, the absorbance of the supernatant was measured at 520 nm with those without enzyme as the control. One enzyme activity unit (U) is defined that the volume of enzyme required for producing 1 μmol sugar of glucosamine hydrochloride per minute under the experimental conditions (50 °C, reaction for 10 min). The protein content was determined according to the method in the Modified Bradford Protein Assay Kit. Biochemical characterization of purified chitosanase The chitosanase activity was determined according to the above method. In order to determine the optimum reaction pH value, the purified chitosanase was measured at 50 °C in different buffer of pH 3-7.2 (citric acid-citrate sodium buffer pH 3-6 and phosphate buffer pH 6-7.2). Under the optimum reaction pH condition, the effect of reaction temperature was studied by measuring the activity of chitosanase at different temperatures ranging from 35 °C to 70 °C. After knowing the optimum reaction pH and temperature, the specific activity of chitosanase can be calculated. The thermostability and pH stability of chitosanase were determined by measuring its activity after being maintained at 60 °C for 90 minutes and on ice at the optimum pH for 8 hours. The enzymatic properties of wild-type (WT, Ser196) and mutants were compared, and then better mutants were selected. Kinetic parameters assay Under the optimum reaction conditions, 1 % colloidal chitosan (w/v) was diluted into 1 g/L, 2 g/L, 4 g/L, 6 g/L and 8 g/L, different concentrations of colloidal chitosan were used as substrates for the enzymatic hydrolysis reactions. To ensure the similar hydrolysis degree of the substrate, the reaction time was shortened to 5 min. According to the calculated data, the values of maximum enzymatic reaction rate ( V max ) and Michaelis constant ( K m ) were obtained. The affinity of substrate to chitosanase can be judged from the value of K m and the catalytic efficiency of chitosanase is determined by the value of K cat / K m [31] . Determination of chitosanase by circular dichroism (CD) The imidazole in the purified enzyme solution was removed by Desalting Gravity Column. The treated enzyme solutions were sent to Jiangnan University for circular dichroism detection. The circular dichroism results of WT and mutant chitosanases were determined on Chirascan spectropolarimeter (Bio-Logic MOS405, France). Results Construction of saturated mutation library The recombinant chitosanase gene was inserted into plasmid pET-28a and the Ser196 site of the original strain was mutated by site directed mutation PCR. The PCR product eliminated the template plasmid with methylation under the action of Dpn I, only the target plasmid was left. The treated PCR product was analyzed by 0.8% agarose electrophoresis, and the result was shown in Fig. 1a. The band was roughly expressed as 6100 bp, which was consistent with that of the target product. The target plasmid was transformed into E. coli BL21 (DE3) competent cells, and the saturated mutation library could be obtained by resistance screening and sequencing. The sequencing results can be processed and analyzed by DNAMAN 9.0. Heterologous expression and enzyme purification The original strain and mutant strains were heterologously expressed in E. coli BL21 (DE3). The chitosanase is an intracellular enzyme, which can be obtained by ultrasonic crushing on ice. The chitosanases were in the supernatant after centrifugation. And the chitosanases were purified through Ni-NTA column. It can be seen from Fig. 1b that the eluent of chitosanase with M300 is almost a single band, indicating that the chitosanase eluted with imidazole solution at this concentration is relatively pure and can be used for subsequent experiments. It can be known that the molecular mass of the chitosanase is about 31 kDa by calculating the mobility (Fig. 1b). Effect of saturation mutation on activity and stability of chitosanase The enzymatic properties of 20 amino acids at 196 site were determined respectively, including the optimum reaction pH, temperature and stability. After determining the optimal pH and temperature, the enzyme activity and protein content were measured under these conditions, and the final specific enzyme activity was calculated. Table 2 shows that the specific enzyme activity of three mutant strains were superior to that of the original strain by measuring the enzymatic properties of the original strain and different mutant strains. According to the comparison, the three mutants with the highest specific enzyme activity and wild-type were selected for the following analysis. As shown in Fig. 2A, the enzyme activity measured in phosphate solution as buffer is much higher than that in citrate solution. Ultimately, it was found that the optimum reaction pH of WT and S196A was 6.0, and S196F and S196Y had the highest enzyme activity at pH 6.6 and 7.0, respectively. It is well known that reaction temperature is also a major factor affecting enzyme activity. Fig. 2B shows that the optimum reaction temperatures of WT, S196F, S196Y and S196A were 55 °C, 60 °C, 45 °C and 55 °C respectively. It can be seen from the figure that S196Y and S196A had higher relative enzyme activities than WT and S196F at low temperature, indicating that they are more suitable for low temperature reaction than the latter. Interestingly, the enzyme activity of S196F reached the highest at 60 °C, while the relative enzyme activity was only about 33 % at 65 °C, indicating that temperature has a great influence on S196F. It can be seen from Fig. 2C that the enzyme activity of WT, S196F, S196Y and S196A remained at about 100% after being maintained in the buffer with the optimum pH on ice for 8 hours, indicating that the chitosanase has good pH stability. Fig. 2D shows that WT, S196F and S196Y have similar thermostability, while S196A has better thermostability than them. S196A remained 71.31 % and 44.57 % enzyme activity when it was kept at 60 °C for 10 minutes and 30 minutes, respectively. WT, S196F and S196Y kept only 40-50 % and about 15 % at 60 °C for 10 minutes and 30 minutes, respectively. Compared with WT, the specific enzyme activity and thermostability of S196A were improved, which made a certain contribution to the industrial application of chitosanase. Kinetic analysis of chitosanase The kinetic parameters corresponding to different amino acids at 196 position after saturation mutation are shown in Table 3. K m value is inversely proportional to the affinity between enzyme and substrate, and K cat / K m value is directly proportional to the catalytic efficiency of enzyme. It was measured that the V max of WT was 2426.86 µmol/min/mg, K m was 5.31 mg/mL, and K cat / K m was 236.14 ml/mg/min. Compared to WT, mutants S196F and S196A possessed 1.39- and 1.46-fold increase, respectively, in the K m value and mutant S196A possessed 1.89- and 1.29-fold increase in the V max and K cat / K m value. Results of circular dichroism chromatographic Circular dichroism (CD) is an important tool to study the thermodynamic stability of proteins. CD provides structural information regarding the bonds and structures based on their chirality [32] . The scanning of the secondary structure showed that the CD spectrum of the mutant was basically the same as that of WT, indicating that the secondary structure of all mutants was not significantly affected (Fig. 3). Discussion Sequence analysis and determination of mutation sites The sequence of the chitosanase gene (BaCsn46A) extracted from Bacillus amyloliquefaciens (GenBank: QEK97559.1) was analyzed based on GenBank database. The gene contained 729 bp, which encoded 242 amino acids. This protein sequence was compared with the protein sequences of other chitosanases in NCBI database, it was found that the chitosanase had 94.9 % identity with other chitosanases from Bacillus amyloliquefaciens , 94.36 % identity with chitosanases from Bacillus subtilis and 72.27 % identity with chitosanases from Bacillus cereus . Multiple sequence alignment of GH46 family members indicated that the key catalytic residues in BaCsn46A gene are Glu19 and Asp35. According to the previous published study on chitosanase, it is found that these two amino acid residues are the key active sites and they have the characteristics of high conservation [33] . Once they are mutated into other amino acid residues, the chitosanase activity was greatly reduced, indicating that these two amino acid residues are likely to be the necessary amino acids for catalysis [34] . Comparing the chitosanase gene sequence produced by different strains (High enzyme activity) with BaCsn46A, it was found that other strains have a certain degree of conservation at Ser196. BaCsn46A is different from them, therefore it was suspected that Ser196 can improve enzyme activity. The three-dimensional model of BaCsn46A was predicted with Bacillus subtilis MY002 as the template and the two chitosanases shared sequence identity of 90.50 %. The catalytic residues Glu19 and Asp35 are far away from Ser196 in the amino acid sequence, but they are relatively close in the three-dimensional model after the amino acid was folded. As can be seen in Fig 4, there is a hydrogen bond between the Ser196 and Asp154 of WT and mutant chitosanases. In addition, the Ser196 of mutants have one more bond connected to Pro153 than the WT, which may cause the stability of the mutants to be higher than that of the WT. Effect of saturation mutation on enzymatic properties As far as we know, the currently reported highest chitosanase activity in Bacillus amyloliquefaciens is Bacillus amyloliquefaciens ECU08 (2380.5 U/mg), which was expressed in Pichia pastoris [13] . In our experiment, the specific enzyme activity of WT was 1174.82 U/mg and the highest chitosanase activity of mutants obtained was 1700.62 U/mg. This experiment provides a strong basis for the subsequent saturated site-directed mutagenesis to improve the activity of chitosanase. The optimum pH value of most of the reported chitosanase is 4-7, which is weakly acidic [35] . The activity of chitosanase isolated from Bacillus cereus GU-02 was very stable in the range of pH 7-9, and the optimum pH was 9, but decreased sharply at pH 10 [36] . The chitosanases in our study are basically in accord with the report. The kinetic analysis of the mutants revealed that compared with WT, S196Y had higher affinity and lower catalytic efficiency, S196F had lower affinity and catalytic efficiency, S196A had lower affinity and higher catalytic efficiency. Therefore, we concluded that the conformation of enzymes of different mutants were modified partially at the mutation point, thus affecting the binding of substrate and enzyme. In our experiment, it was found there was little chance that the affinity between enzyme and substrate and catalytic activity of enzyme could be improved at the same time. However, compared with WT, the specific enzyme activity and thermostability of S196A were improved, which made a certain contribution to the industrial application of chitosanase. Analysis of molecular docking The docking interaction plots of chitosanase active site and substrate is shown in Fig. 5. The docking sites of WT and mutant chitosanases on the same substrate are slightly different, and the interactions may also change. The Fig. 5 shows that the same seven amino acid residues in WT, S196F, S196Y and S196A can form nine hydrogen bonds with the substrate, including Glu235, Glu203, Ile145, Gly45, Ala44, Asp35 and Gln146. The Glu19 residue of WT, S196F and S196A form ionic bond with the substrate, while only the Glu19 residue of S196Y form hydrogen bond with the substrate. Generally, the increase of the number of hydrogen bonds can improve the rigidity of the three-dimensional structure and improve the stability, however, the number of unfavorable bumps of S196Y did not decrease. Meanwhile, the figure also shows that the number of unfavorable bumps of S196F and S196A is less than that of WT, indicating that the enzyme active center may bind more closely to the substrate. The change of interaction force between enzyme and substrate may not completely explain the difference of their enzymatic properties. Bacillus belongs to GH46 family and is the main microbial source for the production of chitosanase. Compared with other glycoside hydrolases, the catalytic tank of GH46 chitosanases show a highly negative charge. Natural chitosan is composed of cationic polysaccharides, which can better combine with chitosanase. This may be the reason for the high activity of chitosanase produced by Bacillus [13, 37] . The isoelectric points of serine, phenylalanine, tyrosine and alanine are all between 5-6. These amino acids are weakly acidic, which may make the substrate and the enzyme molecule combine better, so as to obtain higher enzyme activity or stability. Lyu (2014) emphasize again the importance of acidic residues in the substrate binding cleft [33] . Analysis of circular dichroism chromatographic The destruction of the hydrogen bond network of chitosanase CsnA from Renibacterium sp. QD1 led to the decline of the thermal properties of the mutants, but did not change the secondary structure of their proteins, indicating that they were independent of the secondary structure [38] , which was consistent with our experimental results. Conclusion In this study, the BaCsn46A gene was derived from Bacillus amyloliquefaciens (GenBank: QEK97559.1) and synthesized by Nanjing GenScript Biotechnology Co., Ltd. (Nanjing, China). The mutant chitosanases were expressed in E. coli BL21 (DE3) and the molecular weight of purified chitosanase was about 31 kDa by SDS-PAGE. Three better mutant chitosanases were selected according to specific enzyme activity. The results revealed that the optimum reaction pH, temperature and thermal stability of WT and mutant chitosanase were different, which showed that different amino acid residues had a great influence on the reaction conditions, and the number of hydrogen bonds and amino acid hydrophobicity also affected the enzyme activity. In addition, CD analysis showed that the secondary structure of WT and mutants did not change significantly, indicating that the improvement of thermostability of S196A was not related to the secondary structure. Declarations Data availability The gene squence of chitosanase (BaCsn46A) was from Bacillus amyloliquefaciens (GenBank: QEK97559.1). The gene sequences of three mutants with higher enzyme activity were submitted to NCBI (S196F GenBank: OL415164, S196Y GenBank: OL415163, S196A GenBank: OL415165). Other data and material for this article are available upon request. Funding This work was supported by the Key Research and Development Program of Shandong Province, China (2019JZZY020605), the Natural Science Foundation of the Jiangsu Higher Education Institution of China (19KJB180001), the National Natural Science Foundation of China (31700075), the Initial Research Funding of Changzhou University (ZMF17020115), the Extracurricular Innovation and Entrepreneurship Fund for College Students of Changzhou University (ZMF19020280). Authors Contribution Wang Yi, Xu Kepan and LuoWen analyzed data and contributed new methods or models. Gao Wenjun and Wang Hailong performed research. Wang Yi wrote the paper. Guo Jing, Hong Tingting and Cai Zhiqiang conceived and designed the sudy. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Conflict of interest The authors declare no competing interests. Disclaimer Any opinions, findings, recommendations, and conclusions in this paper are those of the authors, and do not necessarily reflect the views of Changzhou University. Consent to participate Informed consent was obtained from all individual participants included in the study. Consent to publish Additional informed consent was obtained from all individual participants for whom identifying information is included in this article. References Hoell IA, Vaaje-Kolstad G, Eijsink VGH (2010) Structure and function of enzymes acting on chitin and chitosan. Biotechnol Genet Eng 27(1):331–366 Cheng CY, Chang CH, Wu YJ, Li YK (2006) Exploration of Glycosyl Hydrolase Family 75, a Chitosanase from Aspergillus fumigatus *. J Biol Chem 281(6):3137–3144 Shimosaka M, Sato K, Nishiwaki N, Miyazawa T, Okazaki M (2005) Analysis of essential carboxylic amino acid residues for catalytic activity of fungal chitosanases by site-directed mutagenesis. J Biosci Bioeng 100(5):545–550 Kang BR, Song YS, Jung WJ (2021) Differential expression of bio-active metabolites produced by chitosan polymers-based Bacillus amyloliquefaciens fermentation. Carbohyd Polym 260:117799 Guo N, Sun J, Wang W, Gao L, Liu J, Liu Z, Xue C, Mao X (2019) Cloning, expression and characterization of a novel chitosanase from Streptomyces albolongus ATCC 27414. Food Chem 286:696–702 Cahyaningtyas HAA, Suyotha W, Cheirsilp B, Yano S (2021) Statistical optimization of halophilic chitosanase and protease production by Bacillus cereus HMRSC30 isolated from Terasi simultaneous with chitin extraction from shrimp shell waste. Biocatal Agric Biotechnol 31:101918 Pal P, Pal A, Nakashima K, Yadav BK (2021) Applications of chitosan in environmental remediation: A review. Chemosphere 266:128934 Jafari H, Bernaerts KV, Dodi G, Shavandi A (2020) Chitooligosaccharides for wound healing biomaterials engineering. Mat Sci Eng C 117:111266 Chouhan D, Mandal P (2021) Applications of chitosan and chitosan based metallic nanoparticles in agrosciences-A review. Int J Biol Macromol 166:1554–1569 Pei MJ, Mao J, Xu WL, Zhou YS (2019) Photocrosslinkable chitosan hydrogels and their biomedical applications. J Polym Sci Pol Chem 57(18):1862–1871 and Xiao, P. Upadhyay U, Sreedhar I, Singh SA, Patel CM, Anitha KL (2021) Recent advances in heavy metal removal by chitosan based adsorbents. Carbohyd Polym 251:117000 Pechsrichuang P, Lorentzen SB, Aam BB, Tuveng TR, Hamre AG, Eijsink VGH, Yamabhai M (2018) Bioconversion of chitosan into chito-oligosaccharides (CHOS) using family 46 chitosanase from Bacillus subtilis (BsCsn46A). Carbohyd Polym 186:420–428 Luo S, Qin Z, Chen Q, Fan L, Jiang L, Zhao L (2020) High level production of a Bacillus amlyoliquefaciens chitosanase in Pichia pastoris suitable for chitooligosaccharides preparation. Int J Biol Macromol 149:1034–1041 Ando A, Saito A, Arai S, Usuda S, Furuno M, Kaneko N, Shida O, Nagata Y (2008) Molecular Characterization of a Novel Family-46 Chitosanase from Pseudomonas sp. A-01. BIOSCI BIOTECH BIOCH 72(8):2074–2081 Park Jae K, Shimono K, Ochiai N, Shigeru K, Kurita M, Ohta Y, Tanaka K, Matsuda H, Kawamukai M (1999) Purification, Characterization, and Gene Analysis of a Chitosanase (ChoA) from Matsuebacter chitosanotabidus 3001. J Bacteriol 181(21):6642–6649 Ding M, Zhang T, Sun C, Zhang H, Zhang Y (2020) A Chitosanase mutant from Streptomyces sp. N174 prefers to produce functional chitopentasaccharide. Int J Biol Macromol 151:1091–1098 Nguyen AD, Huang CC, Liang TW, Nguyen VB, Pan PS, Wang SL (2014) Production and purification of a fungal chitosanase and chitooligomers from Penicillium janthinellum D4 and discovery of the enzyme activators. Carbohyd Polym 108:331–337 Shehata AN, Aty A (2015) Improved production and partial characterization of chitosanase from a newly isolated Chaetomium globosum KM651986 and its application for chitosan oligosaccharides. J Chem Pharm Res 7(1):727–740 Yan Q, Hua C, Yang S, Li Y, Jiang Z (2012) High level expression of extracellular secretion of a β-glucosidase gene (PtBglu3) from Paecilomyces thermophila in Pichia pastoris . Protein Expres. Purif. 84(1): 64-72 Banerjee S, Salunkhe SS, Apte-Deshpande AD, Mandi NS, Mandal G, Padmanabhan S (2009) Over-expression of proteins using a modified pBAD24 vector in E. coli expression system. Biotechnol Lett 31(7):1031–1036 Cheng F, Xu J-M, Xiang C, Liu Z-Q, Zhao L-Q, Zheng Y-G (2017) Simple-MSSM: a simple and efficient method for simultaneous multi-site saturation mutagenesis. Biotechnol Lett 39(4):567–575 Zoller MJ, Smith M (1982) Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res 10(20):6487–6500 Kegler-Ebo DM, Polack GW, Dimaio D (1996) Use of codon cassette mutagenesis for saturation mutagenesis. Methods Mol Biol 57:297–310 Chiang LW, Kovari I, Howe MM (1993) Mutagenic oligonucleotide-directed PCR amplification (Mod-PCR): an efficient method for generating random base substitution mutations in a DNA sequence element. PCR Methods Appl 2(3):210–217 Geddie ML, Matsumura I (2004) Rapid Evolution of β-Glucuronidase Specificity by Saturation Mutagenesis of an Active Site Loop*. J Biol Chem 279(25):26462–26468 Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L, Lepore R, Schwede T (2018) SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 46(W1):W296–W303 Li Y, Gou Y, Liu Z, Xie T, Wang G (2021) Structure-based rational design of chitosanase CsnMY002 for high yields of chitobiose. Colloids Surf B 202:111692 O\"Donoghue SI, Goodsell DS, Frangakis AS, Jossinet F, Laskowski RA, Nilges M, Saibil HR, Schafferhans A, Wade RC, Westhof E (2010) Visualization of macromolecular structures. Nat Methods 7(3):S42 Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem 30(16):2785–2791 Fuwei W (2011) Screening, identification, fermentation conditions and application of chitosanase producing bacteria. Shanghai Ocean University Zhang XF, Ai YH, Xu Y, Yu XW (2019) High-level expression of Aspergillus niger lipase in Pichia pastoris : Characterization and gastric digestion in vitro. Food Chem 274:305–313 Kulandaisamy R, Kushwaha T, Kumar V, De S, Kumar S, Upadhyay SK, Kumar M, Inampudi KK (2020) Characterization of active/binding site residues of peptidyl-tRNA hydrolase using biophysical and computational studies. Int J Biol Macromol 159:877–885 Lyu Q, Wang S, Xu W, Han B, Liu W, Jones DNM, Liu W (2014) Structural insights into the substrate-binding mechanism for a novel chitosanase. Biochem J 461(2):335–345 Boucher I, Fukamizo T, Honda Y, Willick GE, Neugebauer WA, Brzezinski R (1995) Site-directed Mutagenesis of Evolutionary Conserved Carboxylic Amino Acids in the Chitosanase from Streptomyces sp. N174 Reveals Two Residues Essential for Catalysis (∗). J Biol Chem 270(52):31077–31082 Thadathil N, Velappan SP (2014) Recent developments in chitosanase research and its biotechnological applications: A review. Food Chem 150:392–399 Goo BG, Park JK (2014) Characterization of an alkalophilic extracellular chitosanase from Bacillus cereus GU-02. J Biosci Bioeng 117(6):684–689 Viens P, Lacombe-Harvey ME, Brzezinski R (2015) Chitosanases from Family 46 of Glycoside Hydrolases: From Proteins to Phenotypes. Mar Drugs 13(11):6566–6587 Han Y, Yu R, Gao P, Lu X, Yu W (2018) The hydrogen-bond network around Glu160 contributes to the structural stability of chitosanase CsnA from Renibacterium sp. QD1. Int J Biol Macromol 109:880–887 Tables Table 1 Primers used for saturated site-directed mutagenesis of Ser196 in BaCsn46A Primer names Primer sequences (5’ to 3’) S196F-F GACCTGATGAACCCG TTC GATGAAGATACCC S196F-R GGGTATCTTCATC GAA CGGGTTCATCAGGTC S196L-F GACCTGATGAACCCG CTC GATGAAGATACCC S196L-R GGGTATCTTCATC GAG CGGGTTCATCAGGTC S196P-F GACCTGATGAACCCG CCG GATGAAGATACCC S196P-R GGGTATCTTCATC CGG CGGGTTCATCAGGTC S196Y-F GACCTGATGAACCCG TAC GATGAAGATACCC S196Y-R GGGTATCTTCATC GTA CGGGTTCATCAGGTC S196C-F CCTGATGAACCCG TGC GATGAAGATACC S196C-R GGTATCTTCATC GCA CGGGTTCATCAGG S196W-F GACCTGATGAACCCG TGG GATGAAGATACCC S196W-R GGGTATCTTCATC CCA CGGGTTCATCAGGTC S196H-F GACCTGATGAACCCG CAC GATGAAGATGCCC S196H-R GGGTATCTTCATC GTG CGGGTTCATCAGGTC S196Q-F GACCTGATGAACCCG CAA GATGAAGATACCC S196Q-R GGGTATCTTCATC TTG CGGGTTCATCAGGTC S196R-F CCTGATGAACCCG AGA GATGAAGATACCCAAG S196R-R CTTGGGTATCTTCATC TCT CGGGTTCATCAGG S196I-F CCTGATGAACCCG ATC GATGAAGATACCC S196I-R GGGTATCTTCATC GAT CGGGTTCATCAGG S196N-F CCTGATGAACCCG AAC GATGAAGATACCC S196N-R GGGTATCTTCATC GTT CGGGTTCATCAGG S196K-F GACCTGATGAACCCG AAG GATGAAGATACCCAAG S196K-R CTTGGGTATCTTCATC CTT CGGGTTCATCAGGTC S196V-F GACCTGATGAACCCG GTC GATGAAGATACCC S196V-R GGGTATCTTCATC GAC CGGGTTCATCAGGTC S196T-F CCTGATGAACCCG ACC GATGAAGATACC S196T-R GGTATCTTCATC GGT CGGGTTCATCAGG S196D-F GACCTGATGAACCCG GAC GATGAAGATACCC S196D-R GGGTATCTTCATC GTC CGGGTTCATCAGGTC S196E-F GACCTGATGAACCCG GAG GATGAAGATACCC S196E-R GGGTATCTTCATC CTC CGGGTTCATCAGGTC S196M-F GACCTGATGAACCCG ATG GATGAAGATACCCAAG S196M-R CTTGGGTATCTTCATC CAT CGGGTTCATCAGGTC S196G-F CCTGATGAACCCG GGC GATGAAGATAC S196G-R GTATCTTCATC GCC CGGGTTCATCAGG Table 2 Specific activities corresponding to different kinds of amino acid on the site 196 (The enzyme activity was measured at the optimum pH and temperature, and the protein content was measured according to the method of the kit). Mutant enzyme Specific activity (U/mg) WT 1174.82 S196F 1240.95 S196Y 1700.62 S196L 887.19 S196T 468.15 S196P 28.40 S196A 1395.59 S196C 807.00 S196Q 866.06 S196N 1087.27 S196G 1017.45 S196M 784.58 S196I 673.24 S196K 810.98 S196E 735.29 S196H 572.50 S196D 717.50 S196R 406.89 S196V 766.50 S196W 1012.48 Table 3 Kinetic parameters corresponding to different amino acids at site196 after saturation mutation. Mutant enzyme V max (µmol/min / mg) K m (mg/mL) K cat (s −1 ) K cat / K m (mL/mg / min) WT 2426.86 5.31 1253.88 236.14 S196F 1779.70 7.40 919.51 124.26 S196Y 764.62 3.58 395.05 110.35 S196L 357.13 5.49 184.52 33.61 S196T 392.58 5.88 202.83 34.50 S196P 35.77 7.10 18.48 2.60 S196A 4584.07 7.75 2368.44 305.60 S196C 2475.40 7.04 1278.96 181.67 S196Q 2426.86 6.04 1253.88 207.60 S196N 1316.70 2.20 680.30 309.23 S196G 4584.07 8.90 2368.44 266.12 S196M 3726.96 8.30 1925.60 232.00 S196I 1490.80 5.59 770.25 137.79 S196K 5640.81 10.83 2914.42 269.11 S196E 13914.01 30.75 7188.91 233.79 S196H 630.98 3.30 326.01 98.79 S196D 876.37 3.33 452.79 135.97 S196R 742.33 4.09 383.54 93.77 S196V 1287.73 3.62 665.33 183.79 S196W 2015.86 5.55 1041.53 187.66 Supplementary Files GrphicalAbstract.jpg Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1119314","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":67703965,"identity":"c252424e-bcd5-48de-b49d-2161691f637b","order_by":0,"name":"Wang Yi","email":"","orcid":"","institution":"Changzhou University - Wujin Campus: Changzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wang","middleName":"","lastName":"Yi","suffix":""},{"id":67703966,"identity":"6cc67690-2dc4-4bd8-a6c4-b59a66769568","order_by":1,"name":"Gao Wenjun","email":"","orcid":"","institution":"Changzhou University - Wujin Campus: Changzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gao","middleName":"","lastName":"Wenjun","suffix":""},{"id":67703967,"identity":"7c70132d-0d4e-4a9c-8897-0456937e1759","order_by":2,"name":"Wang Hailong","email":"","orcid":"","institution":"Changzhou University - Wujin Campus: Changzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wang","middleName":"","lastName":"Hailong","suffix":""},{"id":67703968,"identity":"37ca82a5-64e4-415d-872c-a00c760e9cc1","order_by":3,"name":"Xu Kepan","email":"","orcid":"","institution":"Changzhou University - Wujin Campus: Changzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Kepan","suffix":""},{"id":67703969,"identity":"834cccab-fd5f-4d70-a67e-64459df6fb6e","order_by":4,"name":"Luo Wen","email":"","orcid":"","institution":"Changzhou University - Wujin Campus: Changzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Luo","middleName":"","lastName":"Wen","suffix":""},{"id":67703970,"identity":"832117d3-5dc9-423a-8745-84a6b8c285b1","order_by":5,"name":"Guo Jing","email":"","orcid":"","institution":"Changzhou University - Wujin Campus: Changzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guo","middleName":"","lastName":"Jing","suffix":""},{"id":67703971,"identity":"ea907803-8767-4edd-872c-7cc3f5a1be70","order_by":6,"name":"Hong Tingting","email":"","orcid":"","institution":"Changzhou University - Wujin Campus: Changzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Tingting","suffix":""},{"id":67703972,"identity":"f24b042b-8e0a-4a94-ad80-a11ab8f66ba1","order_by":7,"name":"Zhiqiang Cai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYDACCRBhYAPlsRGvJY1kLQyHSdAiP7t544cfBefl+fvPGDB8KDvMwD+7Ab8WxjnHiiV7DG4bzjhwxoBxxrnDDBJ3DuDXwiyRY8bAY3A7wYCxx4CZt+0wg4FEAn4tbEAtjH8MziUYMPMYMP8lRgsPUAtQ8YEEAzagFkZitEhIpBVLyxgkG844w1ZwsOdcOo/EDQJa5Gckb/z45o8dMMQOb3zwo8xajn8GAS1AYABnHQC5lKB6FC2jYBSMglEwCrACACQtOfzgolpfAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-9180-675X","institution":"Changzhou University - Wujin Campus: Changzhou University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Zhiqiang","middleName":"","lastName":"Cai","suffix":""}],"badges":[],"createdAt":"2021-11-27 11:12:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1119314/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1119314/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":16217611,"identity":"cd6dd402-1edb-4f8d-9e57-bddbca1d6ab9","added_by":"auto","created_at":"2021-12-06 16:43:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":61036,"visible":true,"origin":"","legend":"Results of electrophoresis. a: PCR products after Dpn I treatment. b: Protein electrophoretogram of chitosanase corresponding to WT and different kinds of mutants. Lane 1, standard protein marker; lane 2, wide-type; lane 3, S196T; lane 4 S196F; lane 5, S196Y; lane 6, S196L; lane 7, S196P; lane 8, S196A; lane 9, S196C; lane 10, S196Q; lane 11, S196N; lane 12, S196G; lane 13, S196M; lane 14, S196I; lane 15, S196K; lane 16, S196E; lane 17, S196H; lane 18, S196D; lane 19, S196R; lane 20, S196V; lane 21, S196W.","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1119314/v1/6956d16cdf24073831dadb1c.jpg"},{"id":16218475,"identity":"0ba73b0d-6df9-4485-af6f-bb35c790c420","added_by":"auto","created_at":"2021-12-06 16:49:06","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":103923,"visible":true,"origin":"","legend":"a: Effect of pH on enzyme activity of WT, S196F, S196Y and S196A. Chitosanase was measured at 50 °C in different buffer of pH 3-7.2 (citric acid-citrate sodium buffer pH 3-6 and phosphate buffer pH 6-7.2). b: Effect of temperature (35-70 °C) on enzyme activity of WT, S196F, S196Y and S196A under the optimum pH condition. c: The pH stability of WT, S196F, S196Y and S196A. Chitosanase activity was measured under the optimum conditions after chitosanase was maintained on ice for 8 hours. d: The thermostability of WT, S196F, S196Y and S196A. The activity of chitosanase was measured under the optimum conditions after chitosanase was maintained at 60 °C for 0 min, 10 min, 30 min, 60 min and 90 min.","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1119314/v1/371bb4bb002739d7baa05935.jpg"},{"id":16218275,"identity":"ba5dfe9a-7c65-490f-bb7b-56f06488b991","added_by":"auto","created_at":"2021-12-06 16:46:06","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":77264,"visible":true,"origin":"","legend":"Far-UV CD spectra of wild-type and mutants chitosanase in 50 mM PBS (pH 6.0)","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1119314/v1/234a36c6d5b402564c015f6b.jpg"},{"id":16217609,"identity":"6acbb9b7-9832-4a52-9510-e4ae6d4a694a","added_by":"auto","created_at":"2021-12-06 16:43:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":118450,"visible":true,"origin":"","legend":"Three dimensional schematic diagram of amino acid hydrogen bonding in chitosanase. a: WT. b: S196F. c: S196Y. d: S196A.","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1119314/v1/bbecc63186744913a797403e.jpg"},{"id":16217610,"identity":"741858f0-f879-4cca-8a63-1f4266b0d6ae","added_by":"auto","created_at":"2021-12-06 16:43:06","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":107833,"visible":true,"origin":"","legend":"Molecular docking plan of WT and mutant chitosanases show the interactions between the substrate molecule and the enzyme active center. a: WT. b: S196F. c: S196Y. d: S196A.","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1119314/v1/8d05a3dae5dc5bda122663e0.jpg"},{"id":16392314,"identity":"bd977f2e-724c-4965-acc4-86ee42bc1b08","added_by":"auto","created_at":"2021-12-13 11:53:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":888997,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1119314/v1/a0083311-a0ad-4229-89c5-e8d128c0bc18.pdf"},{"id":16217612,"identity":"d1af8a3f-9f8e-48ab-addc-aa250bea8c6a","added_by":"auto","created_at":"2021-12-06 16:43:06","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":76449,"visible":true,"origin":"","legend":"","description":"","filename":"GrphicalAbstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1119314/v1/7f9c7da1b83dd6640beed30b.jpg"}],"financialInterests":"","formattedTitle":"\u003cp\u003eSaturation mutagenesis at Ser196 in BaCsn46A from \u003cem\u003eBacillus Amyloliquefaciens\u003c/em\u003e Enhances Enzyme Activity and Thermostability\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChitosanase (EC 3.2.1.132) is a glycosyl hydrolase and widely distributed in nature. It has been detected among microorganisms, animals and plants\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. According to the current research, the chitosanase activity produced by bacteria is much higher than that of fungi and actinomycetes, and bacteria have the advantage of short fermentation time. By comparison, there is no similarity between the nucleotide sequences of fungal chitosanases and bacterial chitosanases\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. According to the similarity of these sequences, chitosanases were categorized to seven glycoside hydrolase (GH) families: GH 2, 5, 7, 8, 46, 75 and 80\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Most of previous reported fungal chitosanases showed significant similarity with each other and are classified as GH 75 family, while the most bacterial chitosanases belong to GH 46 family\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Chitosan is an alkaline polysaccharide. Chitosanase can degrade it into chitooligosaccharides (COS) or glucosamine by hydrolyzing the β-1,4-glycosidic linkage\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Chitosan and COS have strong biological activity, however, the water solubility of chitosan is worse than that of COS, which can only be dissolved in diluted acid, therefore COS plays a greater role in practical application. Based on its non-toxic, antibacterial, degradable characteristics, their practical applications involve various fields such as food, agriculture, medicine and environment\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. They have a wide range of functions in wound dressing biomaterials\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, plant diseases prevention\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, inhibiting tumor cells\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, adsorbing of heavy metals in wastewater\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e and so on.\u003c/p\u003e \u003cp\u003eIt was reported that there were many microorganisms with chitosanase production capability through the traditional screening method in the laboratory, such as \u003cem\u003eBacillus\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003eMatsuebacter chitosanotabidus\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003eStreptomyces\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003ePenicillium\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003eChaetomium globosum\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, etc. However, the directly selected bacteria have low enzyme activity or low enzyme production efficiency, which can not meet industrial requirements. Therefore, the biological transformation of the existing chitosanase by means of molecular biology is a good method for improving the enzyme activity and stability. Until now, many chitosanase genes have been cloned and heterologously expressed. \u003cem\u003ePichia pastoris\u003c/em\u003e is quite effective in folding exogenous proteins and shows a high capacity of extracellular protein production\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. As a protein expression system, \u003cem\u003eE. coli\u003c/em\u003e is more common than \u003cem\u003ePichia pastoris\u003c/em\u003e, and has the advantages of clear genetic background, low cost, high productivity and compatibility with a variety of antibodies\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDirected evolution and rational design can improve the enzymatic properties, such as protein activity and stability, and the recombination of amino acid sequence is the key step of directed evolution and rational design\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. As early as the 1970s, Smith\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e discovered the technology of oligonucleotide site-directed mutagenesis and won the Nobel Chemistry Prize in 1993. This technology also provided new ideas for future generations and greatly promoted the research progress of protein engineering. On the basis of the derived point saturation mutation technology, it can obtain the other 19 amino acids which respectively replace the amino acids at the target site in a short period of time. There are several methods of point saturation mutation, including oligonucleotide directed mutagenesis\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e, cassette mutagenesis\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, mutagenic oligonucleotide directed PCR amplification\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, gene spreading by overlap extension and mutagenic plasma amplification\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn our study, \u003cem\u003eE. coli\u003c/em\u003e was used as a heterologous expression vector. Through the calculation and analysis of gene sequence, we determined the mutation site, and compared the three-dimensional model, enzymatic properties and molecular docking between WT chitosanase and mutant chitosanases. Three mutant chitosanases with higher specific enzyme activity than the WT were obtained in the experiment. Among them, the thermostability of S196Y has been greatly improved, which can have a greater application prospect in industry.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003eDH5\u0026alpha;, BL21(DE3) were purchased from\u0026nbsp;Takara (Dalian, China)\u0026nbsp;and\u003cem\u003e\u0026nbsp;\u003c/em\u003eplasmid pET-28a\u003cem\u003e\u0026nbsp;\u003c/em\u003ewas purchased from Novegen (Darmstadt, Germany). Chitosan (degree of deacetylation over 80%) and D(+)-glucosamine, hydrochloride were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Restriction enzymes \u003cem\u003eBam\u003c/em\u003eH I and \u003cem\u003eHin\u003c/em\u003ed III were purchased from Takara (Nanjing, China).The High Purity Plasmid Small Preparation Kit was purchased from Beijing Bioteke Corporation Co., Ltd, (Beijing, China).The Site-directed Mutagenesis Kit, the Modified Bradford Protein Assay Kit, Ni-NTA Sefinose(TM) Resin 6FF (Settled Resin) and Desalting Gravity Column were purchased from Sangong Biotech (Shanghai) Co., Ltd. (Shanghai, China). The chitosanase gene was from \u003cem\u003eBacillus amyloliquefaciens\u0026nbsp;\u003c/em\u003e(GenBank: QEK97559.1).\u0026nbsp;After optimizing the codon of the gene sequence (BaCsn46A), it was synthesized by Nanjing GenScript Biotechnology Co., Ltd. (Nanjing, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThree-dimensional model construction and homologous sequence comparison of chitosanase\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protein sequence of \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e (BaCsn46A) was submitted to Swiss-Model (https://swissmodel.expasy.org/)\u003csup\u003e[26]\u003c/sup\u003e online server to predict its three-dimensional structure, and the chitosanase molecular model from \u003cem\u003eBacillus subtilis\u0026nbsp;\u003c/em\u003eMY002 was used as the template (PDB ID : 7C6C. 1. A)\u003csup\u003e[27]\u003c/sup\u003e. The three-dimensional model was analyzed on PyMOL 2.4.1 (https://pymol.org/2/)\u003csup\u003e[28]\u003c/sup\u003e. The amino acid sequence was compared and analyzed by DNAMAN 9.0. The molecular docking was performed between chitosanase and substrate chitotetraose by autodock 4.2 (http://autodock.scripps.edu/)\u003csup\u003e[29]\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDesign and synthesis of primers\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe primers used for saturated site-directed mutagenesis of Ser196 in BaCsn46A were designed and sent to Sangong Biotech (Shanghai) Co., Ltd. (Shanghai, China) for synthesis. The designed primer sequences were shown in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of saturated mutation library\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe saturated mutation library was constructed by whole plasmid PCR. The PCR system includes Pfu DNA polymerase and dNTP. This process takes plasmid pET-28a as template and the sequences in Table 1 as primers respectively. After PCR, Endonuclease \u003cem\u003eDpn\u003c/em\u003e I was added to the product and placed in a 37 \u0026deg;C water bath for 1 h to remove the methylated plasmid. The target plasmid in the PCR product was combined with competent cells made of \u003cem\u003eE. coli\u003c/em\u003e DH5\u0026alpha; and transformed. The above conversion solution which was added to 1 mL Luria-Bertani (LB) solution was incubated at 37 \u0026deg;C and 160 rpm for 1 h. After the bacterial solution was concentrated, it was evenly distributed on the LB solid medium plate containing kanamycin and incubated overnight at 37 \u0026deg;C and 160 rpm. Transformants were selected from the plate and cultured in LB liquid medium. The plasmids were extracted from the bacterial solution according to the kit, and the nucleic acid gel was run to test whether the PCR was successful. Finally, the bacterial solution was sent to Sangong Biotech (Shanghai) Co., Ltd. (Shanghai, China) for sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression and purification of recombinant enzymes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequenced recombinant enzymes were reincubated in LB liquid medium at 37 \u0026deg;C and 160 rpm for about 12 hours. Thereafter, 1 % of the above bacterial solution was transferred to a unused LB liquid medium and continued to incubate for 4 hours under the same conditions, and then \u0026nbsp;isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to induce protein expression. After being incubated overnight at 16 \u0026deg;C and 160 rpm, the bacterial solution was centrifuged by high speed freezing centrifuge, and the cells were resuspended with buffer M0 (20 mM Tris-HCl solution, 500 mM NaCl solution, 10 % glycerol, pH 8.0, constant volume to 1L). Then, the resuspended solution was ultrasonically broken on ice, centrifuged, and the supernatant was taken out and stored at -20 \u0026deg;C. Ni-NTA column can adsorb proteins with histidine tags and imidazole solutions with different concentration gradients (on the basis of buffer M0, 20-300 mM imidazole solution was added respectively) can be used for elution. Ultimately, the purified chitosanase solution can be stored at -20 \u0026deg;C and used for subsequent determination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSDS\u0026ndash;polyacrylamide gel electrophoresis (SDS\u0026ndash;PAGE)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne volume of protein sample was mixed with 4 times volume of SDS-PAGE loading buffer (2.5 mL 1 M Tris-HCl (pH 6.8), 1 g SDS, 50 mg bromophenol blue, 5 mL glycerol, 0.5 mL \u0026beta;-mercaptoethanol，10 ml in total). The mixture solution was boiled for 5 min and was added to the 12 % SDS-PAGE gel. The electrophoresis apparatus ran at 80V first, and then changed to run at 120V until the end. After electrophoresis, the gel was dyed and decolorizing. Finally, a picture containing protein bands was obtained on electrophoresis gel imaging system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of chitosanase activity and protein content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChitosanase activity was measured by using 3,5-dinitrosalicylic acid (DNS) method with slight modification\u003csup\u003e[30]\u003c/sup\u003e. The total reaction system of chitosan enzyme was 2 mL, including 50 mM phosphate buffer (pH 6.6), 1 % colloidal chitosan (chitosan is dissolved in dilute acid, w/v) and an appropriate amount of purified chitosanase solution, which is properly diluted. After being mixed completely, the mixture solution was incubated at 50 \u0026deg;C for 10 min. The reaction was stopped by adding 1.5 mL DNS to the above mixture solution and then the mixture solution was incubated in boiling water for 5 min to develop color. Next, the boiled solution was diluted to 25 mL and centrifuged. Finally, the absorbance of the supernatant was measured at 520 nm with those without enzyme as the control. One enzyme activity unit (U) is defined that the volume of enzyme required for producing 1 \u0026mu;mol sugar of glucosamine hydrochloride per minute under the experimental conditions (50 \u0026deg;C, reaction for 10 min). The protein content was determined according to the method in the Modified Bradford Protein Assay Kit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical characterization of purified chitosanase\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chitosanase activity was determined according to the above method. In order to determine the optimum reaction pH value, the purified chitosanase was measured at 50 \u0026deg;C in different buffer of pH 3-7.2 (citric acid-citrate sodium buffer pH 3-6 and phosphate buffer pH 6-7.2). Under the optimum reaction pH condition, the effect of reaction temperature was studied by measuring the activity of chitosanase at different temperatures ranging from 35 \u0026deg;C to 70 \u0026deg;C. After knowing the optimum reaction pH and temperature, the specific activity of chitosanase can be calculated. The thermostability and pH stability of chitosanase were determined by measuring its activity after being maintained at 60 \u0026deg;C for 90 minutes and on ice at the optimum pH for 8 hours. The enzymatic properties of wild-type (WT, Ser196) and mutants were compared, and then better mutants were selected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKinetic parameters assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder the optimum reaction conditions, 1 % colloidal chitosan (w/v) was diluted into 1 g/L, 2 g/L, 4 g/L, 6 g/L and 8 g/L, different concentrations of colloidal chitosan were used as substrates for the enzymatic hydrolysis reactions. To ensure the similar hydrolysis degree of the substrate, the reaction time was shortened to 5 min. According to the calculated data, the values of maximum enzymatic reaction rate (\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e) and Michaelis constant\u003cem\u003e\u0026nbsp;\u003c/em\u003e(\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e) were obtained. The affinity of substrate to chitosanase can be judged from the value of \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e and the catalytic efficiency of chitosanase is determined by the value of \u003cem\u003eK\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e\u003csup\u003e[31]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of chitosanase by circular dichroism (CD)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe imidazole in the purified enzyme solution was removed by Desalting Gravity Column. The treated enzyme solutions were sent to Jiangnan University for circular dichroism detection. The circular dichroism results of WT and mutant chitosanases were determined on Chirascan spectropolarimeter (Bio-Logic MOS405, France).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eConstruction of saturated mutation library\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe recombinant chitosanase gene was inserted into plasmid pET-28a and the Ser196 site of the original strain was mutated by site directed mutation PCR. The PCR product eliminated the template plasmid with methylation under the action of \u003cem\u003eDpn\u003c/em\u003e I,\u0026nbsp;only the target plasmid was left.\u0026nbsp;The treated PCR product was analyzed by 0.8% agarose electrophoresis, and the result was shown in Fig. 1a.\u0026nbsp;The band was roughly expressed as 6100 bp,\u0026nbsp;which was consistent with that of the target product.\u0026nbsp;The target plasmid was transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) competent cells, and the saturated mutation library could be obtained by resistance screening and sequencing.\u0026nbsp;The sequencing results can be processed and analyzed by\u0026nbsp;DNAMAN 9.0.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHeterologous expression and enzyme purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original strain and mutant strains were heterologously expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3). The chitosanase is an intracellular enzyme, which can be obtained by ultrasonic crushing on ice. The chitosanases were in the supernatant after centrifugation. And the chitosanases were purified through Ni-NTA column. It can be seen from Fig. 1b that the eluent of chitosanase with M300 is almost a single band, indicating that the chitosanase eluted with imidazole solution at this concentration is relatively pure and can be used for subsequent experiments. It can be known that the molecular mass of the chitosanase is about 31 kDa by calculating the mobility (Fig. 1b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of saturation mutation on activity and stability of chitosanase\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe enzymatic properties of 20 amino acids at 196 site were determined respectively, including the optimum reaction pH, temperature and stability. After determining the optimal pH and temperature, the enzyme activity and protein content were measured under these conditions, and the final specific enzyme activity was calculated. Table 2 shows that the specific enzyme activity of three mutant strains were superior to that of the original strain by measuring the enzymatic properties of the original strain and different mutant strains. According to the comparison, the three mutants with the highest specific enzyme activity and wild-type were selected for the following analysis. As shown in Fig. 2A, the enzyme activity measured in phosphate solution as buffer is much higher than that in citrate solution. Ultimately, it was found that the optimum reaction pH of WT and S196A was 6.0, and S196F and S196Y had the highest enzyme activity at pH 6.6 and 7.0, respectively. It is well known that reaction temperature is also a major factor affecting enzyme activity. Fig. 2B shows that the optimum reaction temperatures of WT, S196F, S196Y and S196A were 55 \u0026deg;C, 60 \u0026deg;C, 45 \u0026deg;C and 55 \u0026deg;C respectively. It can be seen from the figure that S196Y and S196A had higher relative enzyme activities than WT and S196F at low temperature, indicating that they are more suitable for low temperature reaction than the latter. Interestingly, the enzyme activity of S196F reached the highest at 60 \u0026deg;C, while the relative enzyme activity was only about 33 % at 65 \u0026deg;C, indicating that temperature has a great influence on S196F.\u003c/p\u003e\n\u003cp\u003eIt can be seen from Fig. 2C that the enzyme activity of WT, S196F, S196Y and S196A remained at about 100% after being maintained in the buffer with the optimum pH on ice for 8 hours, indicating that the chitosanase has good pH stability. Fig. 2D shows that WT, S196F and S196Y have similar thermostability, while S196A has better thermostability than them. S196A remained 71.31 % and 44.57 % enzyme activity when it was kept at 60 \u0026deg;C for 10 minutes and 30 minutes, respectively. WT, S196F and S196Y kept only 40-50 % and about 15 % at 60 \u0026deg;C for 10 minutes and 30 minutes, respectively. Compared with WT, the specific enzyme activity and thermostability of S196A were improved, which made a certain contribution to the industrial application of chitosanase.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKinetic analysis of chitosanase\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe kinetic parameters corresponding to different amino acids at 196 position after saturation mutation are shown in Table 3. \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e value is inversely proportional to the affinity between enzyme and substrate, and \u003cem\u003eK\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e value is directly proportional to the catalytic efficiency of enzyme. It was measured that the \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e of WT was 2426.86 \u0026micro;mol/min/mg, \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e was 5.31 mg/mL, and \u003cem\u003eK\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e was 236.14 ml/mg/min. Compared to WT, mutants S196F and S196A possessed 1.39- and 1.46-fold increase, respectively, in the \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e value and mutant S196A possessed 1.89- and 1.29-fold increase in the \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e and \u003cem\u003eK\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e value.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults of circular dichroism chromatographic\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCircular dichroism (CD) is an important tool to study the thermodynamic stability of proteins. CD provides structural information regarding the bonds and structures based on their chirality\u003csup\u003e[32]\u003c/sup\u003e. The scanning of the secondary structure showed that the CD spectrum of the mutant was basically the same as that of WT, indicating that the secondary structure of all mutants was not significantly affected (Fig. 3).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003eSequence analysis and determination of mutation sites\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequence of the chitosanase gene (BaCsn46A) extracted from \u003cem\u003eBacillus amyloliquefaciens\u0026nbsp;\u003c/em\u003e(GenBank: QEK97559.1) was analyzed based on GenBank database. The gene contained 729 bp, which encoded 242 amino acids. This protein sequence was compared with the protein sequences of other chitosanases in NCBI database, it was found that the chitosanase had 94.9 % identity with other chitosanases from \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e, 94.36 % identity with chitosanases from \u003cem\u003eBacillus subtilis\u003c/em\u003e and 72.27 % identity with chitosanases from \u003cem\u003eBacillus cereus\u003c/em\u003e. Multiple sequence alignment of GH46 family members indicated that the key catalytic residues in BaCsn46A gene are Glu19 and Asp35. According to the previous published study on chitosanase, it is found that these two amino acid residues are the key active sites and they have the characteristics of high conservation\u003csup\u003e[33]\u003c/sup\u003e. Once they are mutated into other amino acid residues, the chitosanase activity was greatly reduced, indicating that these two amino acid residues are likely to be the necessary amino acids for catalysis\u003csup\u003e[34]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eComparing the chitosanase gene sequence produced by different strains (High enzyme activity) with BaCsn46A, it was found that other strains have a certain degree of conservation at Ser196. BaCsn46A is different from them, therefore it was suspected that Ser196 can improve enzyme activity. The three-dimensional model of BaCsn46A was predicted with \u003cem\u003eBacillus subtilis\u003c/em\u003e MY002 as the template and the two chitosanases shared sequence identity of 90.50 %. The catalytic residues Glu19 and Asp35 are far away from Ser196 in the amino acid sequence, but they are relatively close in the three-dimensional model after the amino acid was folded. As can be seen in Fig 4, there is a hydrogen bond between the Ser196 and Asp154 of WT and mutant chitosanases. In addition, the Ser196 of mutants have one more bond connected to Pro153 than the WT, which may cause the stability of the mutants to be higher than that of the WT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of saturation mutation on enzymatic properties\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs far as we know, the currently reported highest chitosanase activity in \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e is \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e ECU08 (2380.5 U/mg), which was expressed in \u003cem\u003ePichia pastoris\u003c/em\u003e\u003csup\u003e[13]\u003c/sup\u003e. In our experiment, the specific enzyme activity of WT was 1174.82 U/mg and the highest chitosanase activity of mutants obtained was 1700.62\u0026nbsp;U/mg. This experiment provides a strong basis for the subsequent saturated site-directed mutagenesis to improve the activity of chitosanase. The optimum pH value of most of the reported chitosanase is 4-7, which is weakly acidic\u003csup\u003e[35]\u003c/sup\u003e. The activity of chitosanase isolated from \u003cem\u003eBacillus cereus\u003c/em\u003e GU-02 was very stable in the range of pH 7-9, and the optimum pH was 9, but decreased sharply at pH 10\u003csup\u003e[36]\u003c/sup\u003e. The chitosanases in our study are basically in accord with the report. The kinetic analysis of the mutants revealed that compared with WT, S196Y had higher affinity and lower catalytic efficiency, S196F had lower affinity and catalytic efficiency, S196A had lower affinity and higher catalytic efficiency. Therefore, we concluded that the conformation of enzymes of different mutants were modified partially at the mutation point, thus affecting the binding of substrate and enzyme. In our experiment, it was found there was little chance that the affinity between enzyme and substrate and catalytic activity of enzyme could be improved at the same time. However, compared with WT, the specific enzyme activity and thermostability of S196A were improved, which made a certain contribution to the industrial application of chitosanase.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of molecular docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe docking interaction plots of chitosanase active site and substrate is shown in Fig. 5. The docking sites of WT and mutant chitosanases on the same substrate are slightly different, and the interactions may also change. The Fig. 5 shows that the same seven amino acid residues in WT, S196F, S196Y and S196A can form nine hydrogen bonds with the substrate, including Glu235, Glu203, Ile145, Gly45, Ala44, Asp35 and Gln146. The Glu19 residue of WT, S196F and S196A form ionic bond with the substrate, while only the Glu19 residue of S196Y form hydrogen bond with the substrate. Generally, the increase of the number of hydrogen bonds can improve the rigidity of the three-dimensional structure and improve the stability, however, the number of unfavorable bumps of S196Y did not decrease. Meanwhile, the figure also shows that the number of unfavorable bumps of S196F and S196A is less than that of WT, indicating that the enzyme active center may bind more closely to the substrate. The change of interaction force between enzyme and substrate may not completely explain the difference of their enzymatic properties.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBacillus\u003c/em\u003e belongs to GH46 family and is the main microbial source for the production of chitosanase. Compared with other glycoside hydrolases, the catalytic tank of GH46 chitosanases show a highly negative charge. Natural chitosan is composed of cationic polysaccharides, which can better combine with chitosanase. This may be the reason for the high activity of chitosanase produced by \u003cem\u003eBacillus\u003c/em\u003e\u003csup\u003e[13, 37]\u003c/sup\u003e. The isoelectric points of serine, phenylalanine, tyrosine and alanine are all between 5-6. These amino acids are weakly acidic, which may make the substrate and the enzyme molecule combine better, so as to obtain higher enzyme activity or stability. Lyu (2014) emphasize again the importance of acidic residues in the substrate binding cleft\u003csup\u003e[33]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of circular dichroism chromatographic\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe destruction of the hydrogen bond network of chitosanase CsnA from \u003cem\u003eRenibacterium\u003c/em\u003e sp. QD1 led to the decline of the thermal properties of the mutants, but did not change the secondary structure of their proteins, indicating that they were independent of the secondary structure\u003csup\u003e[38]\u003c/sup\u003e, which was consistent with our experimental results.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, the BaCsn46A gene was derived from \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e (GenBank: QEK97559.1) and synthesized by Nanjing GenScript Biotechnology Co., Ltd. (Nanjing, China). The mutant chitosanases were expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) and the molecular weight of purified chitosanase was about 31 kDa by SDS-PAGE. Three better mutant chitosanases were selected according to specific enzyme activity. The results revealed that the optimum reaction pH, temperature and thermal stability of WT and mutant chitosanase were different, which showed that different amino acid residues had a great influence on the reaction conditions, and the number of hydrogen bonds and amino acid hydrophobicity also affected the enzyme activity. In addition, CD analysis showed that the secondary structure of WT and mutants did not change significantly, indicating that the improvement of thermostability of S196A was not related to the secondary structure.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe gene squence of chitosanase (BaCsn46A) was from \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e (GenBank: QEK97559.1). The gene sequences of three mutants with higher enzyme activity were submitted to NCBI (S196F GenBank: OL415164, S196Y GenBank: OL415163, S196A GenBank: OL415165). Other data and material for this article are available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Key Research and Development Program of Shandong Province, China (2019JZZY020605), the Natural Science Foundation of the Jiangsu Higher Education Institution of China (19KJB180001), the National Natural Science Foundation of China (31700075), the Initial Research Funding of Changzhou University (ZMF17020115), the Extracurricular Innovation and Entrepreneurship Fund for College Students of Changzhou University (ZMF19020280).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWang Yi, Xu Kepan and LuoWen analyzed data and contributed new methods or models. Gao Wenjun and Wang Hailong performed research. Wang Yi wrote the paper. Guo Jing, Hong Tingting and Cai Zhiqiang conceived and designed the sudy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclaimer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAny opinions, findings, recommendations, and conclusions in this paper are those of the authors, and do not necessarily reflect the views of Changzhou University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional informed consent was obtained from all individual participants for whom identifying information is included in this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHoell IA, Vaaje-Kolstad G, Eijsink VGH (2010) Structure and function of enzymes acting on chitin and chitosan. Biotechnol Genet Eng 27(1):331\u0026ndash;366\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng CY, Chang CH, Wu YJ, Li YK (2006) Exploration of Glycosyl Hydrolase Family 75, a Chitosanase from \u003cem\u003eAspergillus fumigatus\u003c/em\u003e*. J Biol Chem 281(6):3137\u0026ndash;3144\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShimosaka M, Sato K, Nishiwaki N, Miyazawa T, Okazaki M (2005) Analysis of essential carboxylic amino acid residues for catalytic activity of fungal chitosanases by site-directed mutagenesis. J Biosci Bioeng 100(5):545\u0026ndash;550\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang BR, Song YS, Jung WJ (2021) Differential expression of bio-active metabolites produced by chitosan polymers-based \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e fermentation. Carbohyd Polym 260:117799\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo N, Sun J, Wang W, Gao L, Liu J, Liu Z, Xue C, Mao X (2019) Cloning, expression and characterization of a novel chitosanase from \u003cem\u003eStreptomyces albolongus\u003c/em\u003e ATCC 27414. Food Chem 286:696\u0026ndash;702\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCahyaningtyas HAA, Suyotha W, Cheirsilp B, Yano S (2021) Statistical optimization of halophilic chitosanase and protease production by \u003cem\u003eBacillus cereus\u003c/em\u003e HMRSC30 isolated from Terasi simultaneous with chitin extraction from shrimp shell waste. Biocatal Agric Biotechnol 31:101918\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePal P, Pal A, Nakashima K, Yadav BK (2021) Applications of chitosan in environmental remediation: A review. Chemosphere 266:128934\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJafari H, Bernaerts KV, Dodi G, Shavandi A (2020) Chitooligosaccharides for wound healing biomaterials engineering. Mat Sci Eng C 117:111266\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChouhan D, Mandal P (2021) Applications of chitosan and chitosan based metallic nanoparticles in agrosciences-A review. Int J Biol Macromol 166:1554\u0026ndash;1569\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePei MJ, Mao J, Xu WL, Zhou YS (2019) Photocrosslinkable chitosan hydrogels and their biomedical applications. J Polym Sci Pol Chem 57(18):1862\u0026ndash;1871\u003cb\u003eand\u003c/b\u003eXiao, P.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUpadhyay U, Sreedhar I, Singh SA, Patel CM, Anitha KL (2021) Recent advances in heavy metal removal by chitosan based adsorbents. Carbohyd Polym 251:117000\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePechsrichuang P, Lorentzen SB, Aam BB, Tuveng TR, Hamre AG, Eijsink VGH, Yamabhai M (2018) Bioconversion of chitosan into chito-oligosaccharides (CHOS) using family 46 chitosanase from \u003cem\u003eBacillus subtilis\u003c/em\u003e (BsCsn46A). Carbohyd Polym 186:420\u0026ndash;428\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo S, Qin Z, Chen Q, Fan L, Jiang L, Zhao L (2020) High level production of a \u003cem\u003eBacillus amlyoliquefaciens\u003c/em\u003e chitosanase in \u003cem\u003ePichia pastoris\u003c/em\u003e suitable for chitooligosaccharides preparation. Int J Biol Macromol 149:1034\u0026ndash;1041\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndo A, Saito A, Arai S, Usuda S, Furuno M, Kaneko N, Shida O, Nagata Y (2008) Molecular Characterization of a Novel Family-46 Chitosanase from \u003cem\u003ePseudomonas\u003c/em\u003e sp. A-01. BIOSCI BIOTECH BIOCH 72(8):2074\u0026ndash;2081\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark Jae K, Shimono K, Ochiai N, Shigeru K, Kurita M, Ohta Y, Tanaka K, Matsuda H, Kawamukai M (1999) Purification, Characterization, and Gene Analysis of a Chitosanase (ChoA) from \u003cem\u003eMatsuebacter chitosanotabidus\u003c/em\u003e 3001. J Bacteriol 181(21):6642\u0026ndash;6649\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing M, Zhang T, Sun C, Zhang H, Zhang Y (2020) A Chitosanase mutant from \u003cem\u003eStreptomyces\u003c/em\u003e sp. N174 prefers to produce functional chitopentasaccharide. Int J Biol Macromol 151:1091\u0026ndash;1098\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen AD, Huang CC, Liang TW, Nguyen VB, Pan PS, Wang SL (2014) Production and purification of a fungal chitosanase and chitooligomers from \u003cem\u003ePenicillium janthinellum\u003c/em\u003e D4 and discovery of the enzyme activators. Carbohyd Polym 108:331\u0026ndash;337\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShehata AN, Aty A (2015) Improved production and partial characterization of chitosanase from a newly isolated \u003cem\u003eChaetomium globosum\u003c/em\u003e KM651986 and its application for chitosan oligosaccharides. J Chem Pharm Res 7(1):727\u0026ndash;740\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan Q, Hua C, Yang S, Li Y, Jiang Z (2012) High level expression of extracellular secretion of a β-glucosidase gene (PtBglu3) from \u003cem\u003ePaecilomyces thermophila\u003c/em\u003e in \u003cem\u003ePichia pastoris\u003c/em\u003e. Protein Expres. Purif. 84(1): 64-72\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBanerjee S, Salunkhe SS, Apte-Deshpande AD, Mandi NS, Mandal G, Padmanabhan S (2009) Over-expression of proteins using a modified pBAD24 vector in \u003cem\u003eE. coli\u003c/em\u003e expression system. Biotechnol Lett 31(7):1031\u0026ndash;1036\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng F, Xu J-M, Xiang C, Liu Z-Q, Zhao L-Q, Zheng Y-G (2017) Simple-MSSM: a simple and efficient method for simultaneous multi-site saturation mutagenesis. Biotechnol Lett 39(4):567\u0026ndash;575\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZoller MJ, Smith M (1982) Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res 10(20):6487\u0026ndash;6500\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKegler-Ebo DM, Polack GW, Dimaio D (1996) Use of codon cassette mutagenesis for saturation mutagenesis. Methods Mol Biol 57:297\u0026ndash;310\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChiang LW, Kovari I, Howe MM (1993) Mutagenic oligonucleotide-directed PCR amplification (Mod-PCR): an efficient method for generating random base substitution mutations in a DNA sequence element. PCR Methods Appl 2(3):210\u0026ndash;217\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeddie ML, Matsumura I (2004) Rapid Evolution of β-Glucuronidase Specificity by Saturation Mutagenesis of an Active Site Loop*. J Biol Chem 279(25):26462\u0026ndash;26468\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L, Lepore R, Schwede T (2018) SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 46(W1):W296\u0026ndash;W303\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Gou Y, Liu Z, Xie T, Wang G (2021) Structure-based rational design of chitosanase CsnMY002 for high yields of chitobiose. Colloids Surf B 202:111692\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO\\\"Donoghue SI, Goodsell DS, Frangakis AS, Jossinet F, Laskowski RA, Nilges M, Saibil HR, Schafferhans A, Wade RC, Westhof E (2010) Visualization of macromolecular structures. Nat Methods 7(3):S42\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem 30(16):2785\u0026ndash;2791\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFuwei W (2011) Screening, identification, fermentation conditions and application of chitosanase producing bacteria. Shanghai Ocean University\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang XF, Ai YH, Xu Y, Yu XW (2019) High-level expression of \u003cem\u003eAspergillus niger\u003c/em\u003e lipase in \u003cem\u003ePichia pastoris\u003c/em\u003e: Characterization and gastric digestion in vitro. Food Chem 274:305\u0026ndash;313\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKulandaisamy R, Kushwaha T, Kumar V, De S, Kumar S, Upadhyay SK, Kumar M, Inampudi KK (2020) Characterization of active/binding site residues of peptidyl-tRNA hydrolase using biophysical and computational studies. Int J Biol Macromol 159:877\u0026ndash;885\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLyu Q, Wang S, Xu W, Han B, Liu W, Jones DNM, Liu W (2014) Structural insights into the substrate-binding mechanism for a novel chitosanase. Biochem J 461(2):335\u0026ndash;345\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoucher I, Fukamizo T, Honda Y, Willick GE, Neugebauer WA, Brzezinski R (1995) Site-directed Mutagenesis of Evolutionary Conserved Carboxylic Amino Acids in the Chitosanase from \u003cem\u003eStreptomyces\u003c/em\u003e sp. N174 Reveals Two Residues Essential for Catalysis (\u0026lowast;). J Biol Chem 270(52):31077\u0026ndash;31082\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThadathil N, Velappan SP (2014) Recent developments in chitosanase research and its biotechnological applications: A review. Food Chem 150:392\u0026ndash;399\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoo BG, Park JK (2014) Characterization of an alkalophilic extracellular chitosanase from \u003cem\u003eBacillus cereus\u003c/em\u003e GU-02. J Biosci Bioeng 117(6):684\u0026ndash;689\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eViens P, Lacombe-Harvey ME, Brzezinski R (2015) Chitosanases from Family 46 of Glycoside Hydrolases: From Proteins to Phenotypes. Mar Drugs 13(11):6566\u0026ndash;6587\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan Y, Yu R, Gao P, Lu X, Yu W (2018) The hydrogen-bond network around Glu160 contributes to the structural stability of chitosanase CsnA from \u003cem\u003eRenibacterium\u003c/em\u003e sp. QD1. Int J Biol Macromol 109:880\u0026ndash;887\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\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\u003ePrimers used for saturated site-directed mutagenesis of Ser196 in BaCsn46A\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimer names\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimer sequences (5\u0026rsquo; to 3\u0026rsquo;)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196F-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGACCTGATGAACCCG\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTTC\u003c/span\u003eGATGAAGATACCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196F-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGGTATCTTCATC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGAA\u003c/span\u003eCGGGTTCATCAGGTC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196L-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGACCTGATGAACCCG\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCTC\u003c/span\u003eGATGAAGATACCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196L-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGGTATCTTCATC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGAG\u003c/span\u003eCGGGTTCATCAGGTC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196P-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGACCTGATGAACCCG\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCCG\u003c/span\u003eGATGAAGATACCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196P-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGGTATCTTCATC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCGG\u003c/span\u003eCGGGTTCATCAGGTC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196Y-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGACCTGATGAACCCG\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTAC\u003c/span\u003eGATGAAGATACCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196Y-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGGTATCTTCATC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGTA\u003c/span\u003eCGGGTTCATCAGGTC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196C-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCTGATGAACCCG\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTGC\u003c/span\u003eGATGAAGATACC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196C-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGTATCTTCATC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGCA\u003c/span\u003eCGGGTTCATCAGG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196W-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGACCTGATGAACCCG\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTGG\u003c/span\u003eGATGAAGATACCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196W-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGGTATCTTCATC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCCA\u003c/span\u003eCGGGTTCATCAGGTC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196H-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGACCTGATGAACCCG\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCAC\u003c/span\u003eGATGAAGATGCCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196H-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGGTATCTTCATC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGTG\u003c/span\u003eCGGGTTCATCAGGTC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196Q-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGACCTGATGAACCCG\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCAA\u003c/span\u003eGATGAAGATACCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196Q-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGGTATCTTCATC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTTG\u003c/span\u003eCGGGTTCATCAGGTC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196R-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCTGATGAACCCG\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eAGA\u003c/span\u003eGATGAAGATACCCAAG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196R-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTTGGGTATCTTCATC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTCT\u003c/span\u003eCGGGTTCATCAGG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196I-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCTGATGAACCCG\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eATC\u003c/span\u003eGATGAAGATACCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196I-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGGTATCTTCATC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGAT\u003c/span\u003eCGGGTTCATCAGG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196N-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCTGATGAACCCG\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eAAC\u003c/span\u003eGATGAAGATACCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196N-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGGTATCTTCATC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGTT\u003c/span\u003eCGGGTTCATCAGG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196K-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGACCTGATGAACCCG\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eAAG\u003c/span\u003eGATGAAGATACCCAAG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196K-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTTGGGTATCTTCATC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCTT\u003c/span\u003eCGGGTTCATCAGGTC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196V-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGACCTGATGAACCCG\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGTC\u003c/span\u003eGATGAAGATACCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196V-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGGTATCTTCATC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGAC\u003c/span\u003eCGGGTTCATCAGGTC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196T-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCTGATGAACCCG\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eACC\u003c/span\u003eGATGAAGATACC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196T-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGTATCTTCATC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGGT\u003c/span\u003eCGGGTTCATCAGG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196D-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGACCTGATGAACCCG\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGAC\u003c/span\u003eGATGAAGATACCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196D-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGGTATCTTCATC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGTC\u003c/span\u003eCGGGTTCATCAGGTC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196E-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGACCTGATGAACCCG\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGAG\u003c/span\u003eGATGAAGATACCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196E-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGGTATCTTCATC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCTC\u003c/span\u003eCGGGTTCATCAGGTC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196M-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGACCTGATGAACCCG\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eATG\u003c/span\u003eGATGAAGATACCCAAG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196M-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTTGGGTATCTTCATC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCAT\u003c/span\u003eCGGGTTCATCAGGTC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196G-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCTGATGAACCCG\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGGC\u003c/span\u003eGATGAAGATAC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196G-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGTATCTTCATC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGCC\u003c/span\u003eCGGGTTCATCAGG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\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\u003eSpecific activities corresponding to different kinds of amino acid on the site 196 (The enzyme activity was measured at the optimum pH and temperature, and the protein content was measured according to the method of the kit).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMutant enzyme\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecific activity (U/mg)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1174.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1240.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1700.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e887.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e468.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e28.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1395.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e807.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196Q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e866.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1087.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1017.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e784.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e673.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e810.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e735.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e572.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e717.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e406.89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e766.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1012.48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \n\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\u003eKinetic parameters corresponding to different amino acids at site196 after saturation mutation.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMutant enzyme\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e (\u0026micro;mol/min\u003csup\u003e/\u003c/sup\u003emg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e (mg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e (s\u003csup\u003e\u0026minus;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e (mL/mg\u003csup\u003e/\u003c/sup\u003emin)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2426.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1253.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e236.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1779.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e919.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e124.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e764.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e395.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e110.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e357.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e184.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e33.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e392.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e202.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e34.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e18.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4584.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2368.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e305.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2475.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1278.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e181.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196Q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2426.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1253.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e207.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1316.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e680.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e309.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4584.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2368.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e266.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3726.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1925.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e232.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1490.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e770.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e137.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5640.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2914.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e269.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13914.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7188.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e233.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e630.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e326.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e98.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e876.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e452.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e135.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e742.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e383.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e93.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1287.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e665.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e183.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS196W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2015.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1041.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e187.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\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":"Chitosanase, Saturated site-directed mutagenesis, Enzymatic properties, Molecular docking","lastPublishedDoi":"10.21203/rs.3.rs-1119314/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1119314/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe chitosanase (BaCsn46A) was extracted from \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e (GenBank: QEK97559.1) and synthesized after codon optimization. The saturation mutation site was determined by analyzing the sequence and three-dimensional protein model. WT and mutant chitosanase genes were cloned and expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3). The enzymatic properties of WT and mutants were compared, including the optimal reaction pH, temperature and thermostability. Three mutants S196F, S196Y and S196A with the highest specific enzyme activity were selected for further study. Compared with WT, the specific enzyme activity of S196Y increased by 144.76% (more than other two mutants), and the thermostability was not significantly improved. While the specific enzyme activity of S196A increased by 118.79%, and the thermostability of S196A was much higher than WT. From the perspective of industrial production, S196A is more in line with the requirements of industrial production because of its excellent thermal stability at 60\u0026deg;C. From the results of circular dichroism spectrum, the mutation of chitosanase at Ser196 did not change the secondary protein structure. In addition, CD analysis showed that the secondary structure of WT and mutants did not change significantly, indicating that the improvement of thermostability of S196A was not related to the secondary structure.\u003c/p\u003e","manuscriptTitle":"Saturation mutagenesis at Ser196 in BaCsn46A from Bacillus Amyloliquefaciens Enhances Enzyme Activity and Thermostability","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-12-06 16:43:04","doi":"10.21203/rs.3.rs-1119314/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":"a25f20db-dd9e-4c11-98f7-15ac46edc117","owner":[],"postedDate":"December 6th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":8961889,"name":"Molecular Biology"},{"id":8961890,"name":"General Microbiology"}],"tags":[],"updatedAt":"2021-12-13T11:53:43+00:00","versionOfRecord":[],"versionCreatedAt":"2021-12-06 16:43:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1119314","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1119314","identity":"rs-1119314","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}