Improving the catalytic properties of xylanase from Alteromones Macleadii H35 through evolution analysis

Improving the catalytic properties of xylanase from Alteromones Macleadii H35 through evolution analysis | 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 Improving the catalytic properties of xylanase from Alteromones Macleadii H35 through evolution analysis Caixia Cui, jia xu, Juntao Wu, Ningning Wang, chenyan zhou This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3855763/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Mar, 2024 Read the published version in Applied Biochemistry and Biotechnology → Version 1 posted 5 You are reading this latest preprint version Abstract Endo-1,4-β-xylanase is considered one of the most important xylanolytic enzymes, and in this study, we aimed to improve the catalytic properties of Alteromones Macleadii xylanase (Xyn ZT-2) using an evolution-guided design approach. Analysis of the amino acid sequence revealed that the amino acids located in close proximity to the active site were highly conserved, with only a few amino acid differences. By introducing various mutations, we were able to modify the catalytic performance of the enzyme. Notably, the A152G mutation resulted in a 9.8-fold increase in activity and a 23.2-fold increase in catalytic efficiency. Furthermore, the optimal temperature of A152G was raised to 65°C, which is 20°C higher than that of Xyn ZT-2, and the half-life period of T287S was enhanced by 4.9 times. These findings demonstrate the significance of amino acid evolution in determining the catalytic performance of xylanase. By utilizing an evolution analysis to create a smaller mutation library, we efficiently enhanced the catalytic performance, thus providing a novel strategy for improving enzyme catalytic efficiency. xylanase protein evolution catalytic performance mutation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Hemicellulose is the main component of plant cell walls and is the second most abundant carbohydrate in nature. It is composed of β-1,4-linked D-xylopyranosyl units with branches of α-L-arabinofuranosyl and α-D-glucuronyl residues ( 21 ). Xylan, as one of the most important components of hemicellulose, is of significant interest for the preparation of high-value-added xylose and oligosaccharides ( 19 ), particularly due to its bio-degradation. Endo-β-1,4-Xylanase, a critical enzyme in xylan degradation, has been widely utilized in various industrial productions, including pulp bleaching, bread making, animal feed manufacturing, and as an additive in detergent formulations ( 22 , 1 , 17 , 15 ). The catalytic activity and stability of enzymes are key factors in evaluating their effectiveness; however, most native xylanases exhibit low activity and stability, and are unable to withstand harsh conditions. To obtain enzymes with desired characteristics, directed evolution, and rational or semi-rational design approaches are commonly used ( 14 , 6 ). The use of error-prone PCR has been shown to improve xylanase activity and stability ( 28 ). Based on protein structure analysis, mutations at specific amino acid sites have been demonstrated to significantly improve enzyme stability ( 25 ). For instance, the rational design of Xylanase Hwxyl10A from Hortaea werneckii led to a 17-fold improvement in thermostability at 50°C ( 3 ). The active pocket is a critical site for enzyme catalysis, and although the active site is highly conserved, changes in amino acids near the active site can also have a significant impact on enzyme activity and function, even if this relationship has not been reported ( 30 ). Mutations in the loop region near the active site have attracted significant attention, with mutants such as M137E/N269G demonstrating 2.2-fold higher catalytic efficiency than the wild-type xylanase, as well as greater thermostability ( 29 ). Yang has demonstrated that the mutation of residues C43N around the active center significantly improves activity and stability ( 27 ). Therefore, the mutation of amino acids near the enzyme active site is expected to improve enzymatic properties, while ensuring enzyme activity. However, the direction of mutated amino acids is uncertain, and most beneficial mutations are still screened through saturation mutation, which requires a significant amount of work ( 23 ). Microorganisms that degrade plant cell walls must possess multiple hemicellulose degradation genes, with glycoside hydrolases from family 43 (GH43) typically including various types of glycoside hydrolases ( 13 ). In our previous research, we successfully identified GH43 xylanase XynZT-2 from Alteromones Macleadii H35 ( 24 ). In this study, we aimed to improve the enzymatic properties of XynZT-2 by analyzing protein evolution and constructing a small mutant library through homologous replacement of amino acids near the active site. After screening for activity, kinetic parameters, and stability, we found that A152G and T287S mutations resulted in improved activity and stability. These results represent a significant advancement in understanding the relationship between residues near the active site and the function of xylanases, and may be applicable in the design of other xylanases. 2. Materials and Methods 2.1 Materials Restriction enzymes ( Eco R I and Hind III), DNA polymerase, and T4 DNA ligase were obtained from Zhengzhou Jiushi Biotechnology Co., Ltd (Henan, China). Birchwood xylan and xylose were purchased from Sigma-Aldrich (China). E. coli strains BL21 (DE3) and DH-5α were used for protein overexpression and plasmid propagation, and gene cloning, respectively. The xylanase XynZT-2 was previously reported in our research from Alteromones Macleadii (GenBank: MT814836). All other chemicals used in this study were of analytical grade. 2.2 Genetic phylogenetic analysis The conserved domains of GH43 family proteins were predicted using the Pfam database and Pfam_scan.pl. Homologous sequences with over 75% homology with xynZT-2 were selected by comparing amino acid sequences in NCBI. The evolutionary analysis of the GH43 family was conducted by multi-sequence alignment of amino acid sequences using MEGA software. The mutated amino acids near the conserved active site sequence of Xyn ZT-2 during evolution were searched to identify the mutation sites and evaluate the effect of phylogenetics on xylanase properties. Based on the comparison, five amino acid residues in xylanase Xyn ZT-2 were selected for mutagenesis screening to improve enzyme properties. 2.3 Construction of the plasmids expressing Xyn ZT-2 in E. coli The Xyn ZT-2 gene was synthesized with E. coli codon optimization and introduced into the pET-28a (+) plasmid using restriction enzymes EcoR I and Hind III. Site-directed mutagenesis was performed by PCR using the plasmid pET-28a(+)/Xyn ZT-2 as the template, and the primer pairs used are listed in Table S1 . The Xyn ZT-2 enzyme was then expressed and produced ( 9 ). Enzyme purity was determined by SDS-PAGE, and enzyme concentration was determined using the Bradford assay ( 5 ). 2.4 Activity assay and biochemical characterization Xylanase activity was measured using the 3,5-dinitrosalicylic acid (DNS) reagent, with some modifications based on Bailey et al. (1992). Specifically, 1.5 mL of 0.5% (w/v) beechwood xylan and 1 mL of enzyme solution were incubated at 45°C and pH 6.0 for 15 min. Then, 2.5 mL of DNS solution was added, and the mixture was boiled for 10 min. Finally, the absorbance was measured at 540 nm. One unit (U) of xylanase activity is defined as the amount of enzyme that releases 1 µmol of reducing sugar per minute. The optimal pH for Xyn ZT-2 and its mutants was determined in pH 3–9 buffers at 45°C. The effect of temperature on xylanase activity was evaluated in each optimized buffer from 30°C to 80°C, with the highest activity defined as 100%. The kinetic parameters were determined by nonlinear regression fitting of the Michaelis Menten equation at the optimized temperature, with substrate concentrations ranging from 1.0 to 40.0 mg/mL. Each experiment was performed with three replicates. Thermal stability was determined by measuring the residual activity of Xyn ZT-2 and mutants after incubation at the optimized temperature for different periods (0, 2, 3, 5, 10, 15, 20, 30, 40, 50, and 60 min), with the initial activity defined as 100%. 3. Results and Discussions 3.1 Selection of mutation sites based on evolution analysis Numerous publications have demonstrated that xylanase characteristics from the GH10 and GH11 families can be improved using protein engineering methods, and few publications have explored improving the catalytic performance of enzymes from the GH43 family using site-directed mutagenesis ( 10 , 20 ). In this work, protein engineering methods were used to improve the catalytic performance of Xyn ZT-2 based on evolutionary analysis. A three-dimensional (3D) model of Xyn ZT-2 was constructed using Swiss-Model ( https://swissmodel.expasy.org/ ) (Fig. 1 ). The Xyn ZT-2 structure contains five β-strands, which is a typical characteristic of the GH43 family. ( 11 ). Asp150, Glu237, and His288 on different β-strands (β3, β4, and β5, respectively) were predicted as the active sites compared to the crystallographic structure of β-xylosidase/α-arabinofuranosidase (PDB code: 5GLM) ( 16 ) (Fig. 1 ). The conserved motifs of GH43 family proteins were predicted using the Pfam database and Pfam_scan.pl (Fig. 2 ). The amino acids Asp150, Glu237, and His288 were found to be highly conserved, and the structural flexibilities of GH43 xylanases were analyzed. The region near the active site of the 10 selected GH43 xylanases was aligned using DNAMAN software (Table S2, Fig. 2 ). After analyzing the amino acid residues, it was found that several essential residues are conserved in all GH43 members, whereas the amino acids near the active site vary. The β3 fold sequence shows that there is a small amount of Thr and Gly at the 148 and 152 sites, respectively, instead of Ser and Ala. The β4 fold sequence indicates that there is a small amount of Gly and Pro at the 238 and 239 sites, respectively, instead of Ala and Ser. The β5 fold sequence shows that there is a small amount of Ser at the 287 site, instead of Thr. The changes in these residues may influence the enzyme properties, particularly the amino acid closest to the active site ( 18 ). Therefore, based on the above analysis, the mutation was constructed, including S148T, A152G, A238G, S239P, and T278S. 3.2 Effects of mutations on enzyme activity and stability The mutants, namely S148T, A152G, A238G, S239P, and T278S, were overexpressed in E. coli BL21 (DE3), and subsequently purified. After purification, the SDS-PAGE analysis confirmed that the enzymes were obtained (Fig. 3 A). The activities of Xyn ZT-2 and the five mutants, S148T, A152G, A238G, S239P, and T278S, were evaluated and found to be 1.067, 0.905, 10.472, 0.755, 1.956, and 1.64 U/mg, respectively (Fig. 3 B). The specific activity of mutant A152G was found to be enhanced, exhibiting a 9.8-fold improvement compared to Xyn ZT-2. However, the specific activity of mutant A238G was lower than that of Xyn ZT-2. These results indicate that the residues in the catalytic pocket play an important role in the activity of Xyn ZT-2 and that the mutant A152G can significantly improve the specific activity of Xyn ZT-2. This study investigated the impact of pH and temperature on the activity of Xyn ZT-2 and its mutants (Fig. 4 ). The optimal pH values were determined as 6.5, 7.0, 6.5, 6.5, 6.5, and 7.0 for Xyn ZT-2, S148T, A152G, A238G, S239P, and T278S, respectively. This finding indicates that the xylanase has higher activity in a neutral and slightly acidic environment, and the residue mutant has a negligible impact on the optimal pH. This may be because the mutated amino acids are mostly weakly acidic or neutral. In terms of the optimal temperature, Xyn ZT-2, S148T, S239P, and T278S mutants had an optimal temperature of 45°C, while A152G and A238G mutants had optimal temperatures of 65°C and 40°C, respectively. These results suggest that the S148T, S239P, and T278S mutants did not alter its optimal temperature. However, the A238G mutation decreased the optimal temperature by 5°C, while the A152G mutation increased it by 20°C. This indicates that the A152G mutation significantly enhances the optimal temperature change of XynZT-2. Point mutations can drive genetic diversity to adapt to the environment, and such mutations can be beneficial, neutral, or harmful, resulting in higher enzyme activity or new functions ( 8 , 2 ). The A152G and A238G mutants both mutate from alanine to glycine. The optimal temperature for A152G is higher than that of XynZT-2, while that for A238G is lower than that of XynZT-2. These findings illustrate that the same mutation can have beneficial or harmful effects on the activity of homologous enzymes. In summary, these results indicate that mutations affect the characteristics of XynZT-2, and the A152G mutation significantly enhances its optimal temperature. 3.3 Effect of mutations on catalytic efficiency The kinetic parameters for Xyn ZT-2 and its mutants were determined and are presented in Table 1 . The Km values for S148T, A152G, A238G, S239P, and T278S were 48.1, 0.6, 16.5, 46.0, and 30.9 g/L, respectively, while that of Xyn ZT-2 was 28.2 g/L. A152G and A238G displayed reduced Km values compared to Xyn ZT-2, with A152G showing only 2.1% of the Km of Xyn ZT-2. This indicates a significant improvement in the affinity between mutant A152G and xylan. The kcat of S148T and S239P was found to be improved compared to that of Xyn ZT-2. The kcat/Km value of A152G was 23.2-fold higher than that of Xyn ZT-2, indicating the importance of residue site Ala152 around the active site Asp150 in the catalytic efficiency of Xyn ZT-2. These results demonstrate that specific amino acid site mutations can effectively improve the catalytic performance of xylanase ( 4 ). The changes in the catalytic properties of enzymes during their adaptation to the environment are diverse, with mutations being either beneficial, neutral, or harmful. They may also promote higher enzyme activity or new functions ( 7 , 8 ). Table 1 Kinetic parameters of the mutants. Enzymes Km (g/L) kcat (/s) kcat/Km (g/L/s) Xyn ZT-2 28.2 ± 2.9 11.1 ± 1.6 0.4 S148T 48.1 ± 2.3 17.3 ± 0.5 0.4 A152G 0.6 ± 0.1 5.3 ± 0.1 9.3 A238G 16.5 ± 0.8 3.3 ± 0.21 0.2 S239P 46.0 ± 2.2 22.7 ± 0.6 0.5 T287S 30.9 ± 4.5 10.9 ± 1.6 0.4 3.4 Thermal stability of Xyn ZT-2 and mutations The thermal stability of Xyn ZT-2 and its mutations were studied under optimized reaction conditions. As shown in Fig. 5 , the thermal stability of S148T, A152G, and T278S was improved compared to that of Xyn ZT-2. After 20 minutes of incubation, the activity retention of S148T, A152G, and T278S was 76.5%, 64.9%, and 88.3%, respectively. In contrast, Xyn ZT-2's activity remained at only 31% after incubating at 45°C and pH 6.0 for 20 min. The half-life period of T287S was found to be 4.9 times longer than that of Xyn ZT-2, possibly due to the smaller size of Val compared to Ile, leading to improved enzyme stability ( 12 ). These results demonstrate that mutations can improve the thermal stability of Xyn ZT-2, although other mutations can decrease stability. The activity of A23G and S239P was sharply reduced, reaching almost zero after only 10 minutes of incubation under optimized conditions. These results illustrate the changes in thermal stability that occur during enzyme evolution. Mutations near the active site of amino acids may cause a loss of enzyme activity, while reasonable mutations can significantly improve enzyme activity and stability. Replacing homologous amino acids from enzymes with desired functions is a useful and effective strategy for enhancing enzyme catalytic efficiency or stability ( 26 ). Nevertheless, many enzymes still have unidentified catalytic functions and efficiency. One approach to studying enzyme catalytic performance is to analyze protein evolution in enzyme families and establish smaller databases to obtain expected results from previously unreported sequences. Conclusion In this study, we constructed a small library to explore the relationship between xylanase catalytic performance and protein evolution. The mutation sites were selected based on the sequence homology of the protein family. Homologous amino acids were used to replace the residues close to the active site, namely S148, A152, A238, S239, and T278. We then determined the catalytic properties of the wild Xyn ZT-2 and the five mutants. Our findings showed that A152 and T287S are key residues in xylanase activity and stability, with the A152G mutant exhibiting 9.8-fold higher catalytic activity and 23.2-fold higher catalytic efficiency, while the T287S mutant exhibited 4.9 times improved thermal stability. Our results indicate that the characteristics of xylanase change during the evolutionary process, and rational mutations of amino acids near the active site can be beneficial for improving enzyme properties. The approach of constructing a mutation library provides a feasible strategy to enhance enzymatic properties. Declarations Statements & Declarations Funding This work was supported by the Scientific Research Foundation of Xinxiang Medical University (XYBSKYZZ201717) and the Key Technologies R&D Program of Henan Province (232102311146, 212102210652). Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions Caixia Cui: Project administration, Writing – review and editing, and Supervision. Jia Xu: Data curation. Juntao Wu: Methodology. Ningning Wang: Methodology. Chenyan Zhou: Project administration and Supervision. All authors read and approved the final version of the manuscript. Ethical Approval This study did not require ethics approval. Consent to Participate This study did not involve human subjects. Consent to Publish All authors agreed to submit our work for its publication. 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Docking-guided rational engineering of a macrolide glycosyltransferase glycodiversifies epothilone B. Communications biology , 5 , 100–110. Supplementary Files SI.docx Cite Share Download PDF Status: Published Journal Publication published 28 Mar, 2024 Read the published version in Applied Biochemistry and Biotechnology → Version 1 posted Editorial decision: Resubmit revised form; Major revisions required 21 Feb, 2024 Reviewers agreed at journal 03 Feb, 2024 Reviewers invited by journal 15 Jan, 2024 Editor invited by journal 12 Jan, 2024 First submitted to journal 11 Jan, 2024 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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College","correspondingAuthor":false,"prefix":"","firstName":"jia","middleName":"","lastName":"xu","suffix":""},{"id":267287352,"identity":"f48e7103-faeb-4623-93af-5c5aa5aa88c8","order_by":2,"name":"Juntao Wu","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Juntao","middleName":"","lastName":"Wu","suffix":""},{"id":267287353,"identity":"062ef6ef-526a-4da1-999b-ceb981364fa1","order_by":3,"name":"Ningning Wang","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ningning","middleName":"","lastName":"Wang","suffix":""},{"id":267287354,"identity":"5987e227-65a5-4f20-a373-e4b004af700f","order_by":4,"name":"chenyan zhou","email":"","orcid":"","institution":"Xinxiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"chenyan","middleName":"","lastName":"zhou","suffix":""}],"badges":[],"createdAt":"2024-01-12 05:20:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3855763/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3855763/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12010-024-04936-0","type":"published","date":"2024-03-28T15:01:27+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49740635,"identity":"231aa9f0-e9a2-4895-adc7-7de25363fdb9","added_by":"auto","created_at":"2024-01-17 09:09:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":139278,"visible":true,"origin":"","legend":"\u003cp\u003e3D model structure and active site of Xyn ZT-2.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3855763/v1/9fbf919192ab4a6523ccaafa.png"},{"id":49740633,"identity":"75658af0-f8a0-4d8b-9271-a0e1d8b3f415","added_by":"auto","created_at":"2024-01-17 09:09:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":270896,"visible":true,"origin":"","legend":"\u003cp\u003eSequence logo of sequence alignment of predicted active site regions of GH43 xylanases and multiple sequence alignment of the active site regions of Xyn ZT-2 and 10 GH43 xylanases.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3855763/v1/6b809a3b22d887ec711bc485.png"},{"id":49740634,"identity":"a67b9a6d-abb8-47dc-b732-594f9a79e898","added_by":"auto","created_at":"2024-01-17 09:09:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":101319,"visible":true,"origin":"","legend":"\u003cp\u003e(A) SDS-PAGE of Xyn ZT-2 and purified mutants, (B) specific activities of Xyn ZT-2 and mutants.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3855763/v1/712ad628f72ed220d12f16e3.png"},{"id":49741091,"identity":"d5cc72fa-0d7e-4d03-8967-ac36b7c0d12e","added_by":"auto","created_at":"2024-01-17 09:17:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":140188,"visible":true,"origin":"","legend":"\u003cp\u003eOptimal reaction pH and temperature of Xyn ZT-2 and mutants.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3855763/v1/e173591930d07c20e4214a2c.png"},{"id":49740631,"identity":"f8b38065-61b9-49e4-b6cd-7b4553a4e508","added_by":"auto","created_at":"2024-01-17 09:09:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":105031,"visible":true,"origin":"","legend":"\u003cp\u003eThermal stability of Xyn ZT-2 and mutants.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3855763/v1/7324694ecfd738563eb4190e.png"},{"id":53869917,"identity":"3a4e7ac7-fa0d-4321-90d6-d385ebcae510","added_by":"auto","created_at":"2024-04-01 15:12:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":742842,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3855763/v1/c1b17c39-9405-45c2-b61d-702074f535e7.pdf"},{"id":49740637,"identity":"c4016c9e-4987-41f2-94ee-5abf518a1e5c","added_by":"auto","created_at":"2024-01-17 09:09:55","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":21755,"visible":true,"origin":"","legend":"","description":"","filename":"SI.docx","url":"https://assets-eu.researchsquare.com/files/rs-3855763/v1/5b716779bee49d249a2d881c.docx"}],"financialInterests":"","formattedTitle":"Improving the catalytic properties of xylanase from Alteromones Macleadii H35 through evolution analysis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHemicellulose is the main component of plant cell walls and is the second most abundant carbohydrate in nature. It is composed of β-1,4-linked D-xylopyranosyl units with branches of α-L-arabinofuranosyl and α-D-glucuronyl residues (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Xylan, as one of the most important components of hemicellulose, is of significant interest for the preparation of high-value-added xylose and oligosaccharides (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), particularly due to its bio-degradation. Endo-β-1,4-Xylanase, a critical enzyme in xylan degradation, has been widely utilized in various industrial productions, including pulp bleaching, bread making, animal feed manufacturing, and as an additive in detergent formulations (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The catalytic activity and stability of enzymes are key factors in evaluating their effectiveness; however, most native xylanases exhibit low activity and stability, and are unable to withstand harsh conditions.\u003c/p\u003e \u003cp\u003eTo obtain enzymes with desired characteristics, directed evolution, and rational or semi-rational design approaches are commonly used (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). The use of error-prone PCR has been shown to improve xylanase activity and stability (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Based on protein structure analysis, mutations at specific amino acid sites have been demonstrated to significantly improve enzyme stability (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). For instance, the rational design of Xylanase Hwxyl10A from \u003cem\u003eHortaea werneckii\u003c/em\u003e led to a 17-fold improvement in thermostability at 50\u0026deg;C (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The active pocket is a critical site for enzyme catalysis, and although the active site is highly conserved, changes in amino acids near the active site can also have a significant impact on enzyme activity and function, even if this relationship has not been reported (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Mutations in the loop region near the active site have attracted significant attention, with mutants such as M137E/N269G demonstrating 2.2-fold higher catalytic efficiency than the wild-type xylanase, as well as greater thermostability (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Yang has demonstrated that the mutation of residues C43N around the active center significantly improves activity and stability (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Therefore, the mutation of amino acids near the enzyme active site is expected to improve enzymatic properties, while ensuring enzyme activity. However, the direction of mutated amino acids is uncertain, and most beneficial mutations are still screened through saturation mutation, which requires a significant amount of work (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMicroorganisms that degrade plant cell walls must possess multiple hemicellulose degradation genes, with glycoside hydrolases from family 43 (GH43) typically including various types of glycoside hydrolases (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). In our previous research, we successfully identified GH43 xylanase XynZT-2 from \u003cem\u003eAlteromones Macleadii\u003c/em\u003e H35 (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). In this study, we aimed to improve the enzymatic properties of XynZT-2 by analyzing protein evolution and constructing a small mutant library through homologous replacement of amino acids near the active site. After screening for activity, kinetic parameters, and stability, we found that A152G and T287S mutations resulted in improved activity and stability. These results represent a significant advancement in understanding the relationship between residues near the active site and the function of xylanases, and may be applicable in the design of other xylanases.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eRestriction enzymes (\u003cem\u003eEco\u003c/em\u003eR I and \u003cem\u003eHind\u003c/em\u003e III), DNA polymerase, and T4 DNA ligase were obtained from Zhengzhou Jiushi Biotechnology Co., Ltd (Henan, China). Birchwood xylan and xylose were purchased from Sigma-Aldrich (China). \u003cem\u003eE. coli\u003c/em\u003e strains BL21 (DE3) and DH-5α were used for protein overexpression and plasmid propagation, and gene cloning, respectively. The xylanase XynZT-2 was previously reported in our research from \u003cem\u003eAlteromones Macleadii\u003c/em\u003e (GenBank: MT814836). All other chemicals used in this study were of analytical grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Genetic phylogenetic analysis\u003c/h2\u003e \u003cp\u003eThe conserved domains of GH43 family proteins were predicted using the Pfam database and Pfam_scan.pl. Homologous sequences with over 75% homology with xynZT-2 were selected by comparing amino acid sequences in NCBI. The evolutionary analysis of the GH43 family was conducted by multi-sequence alignment of amino acid sequences using MEGA software. The mutated amino acids near the conserved active site sequence of Xyn ZT-2 during evolution were searched to identify the mutation sites and evaluate the effect of phylogenetics on xylanase properties. Based on the comparison, five amino acid residues in xylanase Xyn ZT-2 were selected for mutagenesis screening to improve enzyme properties.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Construction of the plasmids expressing Xyn ZT-2 in \u003cem\u003eE. coli\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe Xyn ZT-2 gene was synthesized with \u003cem\u003eE. coli\u003c/em\u003e codon optimization and introduced into the pET-28a (+) plasmid using restriction enzymes \u003cem\u003eEcoR\u003c/em\u003e I and \u003cem\u003eHind\u003c/em\u003e III. Site-directed mutagenesis was performed by PCR using the plasmid pET-28a(+)/Xyn ZT-2 as the template, and the primer pairs used are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The Xyn ZT-2 enzyme was then expressed and produced (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Enzyme purity was determined by SDS-PAGE, and enzyme concentration was determined using the Bradford assay (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Activity assay and biochemical characterization\u003c/h2\u003e \u003cp\u003eXylanase activity was measured using the 3,5-dinitrosalicylic acid (DNS) reagent, with some modifications based on Bailey et al. (1992). Specifically, 1.5 mL of 0.5% (w/v) beechwood xylan and 1 mL of enzyme solution were incubated at 45\u0026deg;C and pH 6.0 for 15 min. Then, 2.5 mL of DNS solution was added, and the mixture was boiled for 10 min. Finally, the absorbance was measured at 540 nm. One unit (U) of xylanase activity is defined as the amount of enzyme that releases 1 \u0026micro;mol of reducing sugar per minute.\u003c/p\u003e \u003cp\u003eThe optimal pH for Xyn ZT-2 and its mutants was determined in pH 3\u0026ndash;9 buffers at 45\u0026deg;C. The effect of temperature on xylanase activity was evaluated in each optimized buffer from 30\u0026deg;C to 80\u0026deg;C, with the highest activity defined as 100%.\u003c/p\u003e \u003cp\u003eThe kinetic parameters were determined by nonlinear regression fitting of the Michaelis Menten equation at the optimized temperature, with substrate concentrations ranging from 1.0 to 40.0 mg/mL. Each experiment was performed with three replicates.\u003c/p\u003e \u003cp\u003eThermal stability was determined by measuring the residual activity of Xyn ZT-2 and mutants after incubation at the optimized temperature for different periods (0, 2, 3, 5, 10, 15, 20, 30, 40, 50, and 60 min), with the initial activity defined as 100%.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussions","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Selection of mutation sites based on evolution analysis\u003c/h2\u003e\n \u003cp\u003eNumerous publications have demonstrated that xylanase characteristics from the GH10 and GH11 families can be improved using protein engineering methods, and few publications have explored improving the catalytic performance of enzymes from the GH43 family using site-directed mutagenesis (\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e). In this work, protein engineering methods were used to improve the catalytic performance of Xyn ZT-2 based on evolutionary analysis. A three-dimensional (3D) model of Xyn ZT-2 was constructed using Swiss-Model (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org/\u003c/span\u003e\u003c/span\u003e) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The Xyn ZT-2 structure contains five \u0026beta;-strands, which is a typical characteristic of the GH43 family. (\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e). Asp150, Glu237, and His288 on different \u0026beta;-strands (\u0026beta;3, \u0026beta;4, and \u0026beta;5, respectively) were predicted as the active sites compared to the crystallographic structure of \u0026beta;-xylosidase/\u0026alpha;-arabinofuranosidase (PDB code: 5GLM) (\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe conserved motifs of GH43 family proteins were predicted using the Pfam database and Pfam_scan.pl (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The amino acids Asp150, Glu237, and His288 were found to be highly conserved, and the structural flexibilities of GH43 xylanases were analyzed. The region near the active site of the 10 selected GH43 xylanases was aligned using DNAMAN software (Table S2, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). After analyzing the amino acid residues, it was found that several essential residues are conserved in all GH43 members, whereas the amino acids near the active site vary. The \u0026beta;3 fold sequence shows that there is a small amount of Thr and Gly at the 148 and 152 sites, respectively, instead of Ser and Ala. The \u0026beta;4 fold sequence indicates that there is a small amount of Gly and Pro at the 238 and 239 sites, respectively, instead of Ala and Ser. The \u0026beta;5 fold sequence shows that there is a small amount of Ser at the 287 site, instead of Thr. The changes in these residues may influence the enzyme properties, particularly the amino acid closest to the active site (\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e). Therefore, based on the above analysis, the mutation was constructed, including S148T, A152G, A238G, S239P, and T278S.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Effects of mutations on enzyme activity and stability\u003c/h2\u003e\n \u003cp\u003eThe mutants, namely S148T, A152G, A238G, S239P, and T278S, were overexpressed in \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3), and subsequently purified. After purification, the SDS-PAGE analysis confirmed that the enzymes were obtained (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). The activities of Xyn ZT-2 and the five mutants, S148T, A152G, A238G, S239P, and T278S, were evaluated and found to be 1.067, 0.905, 10.472, 0.755, 1.956, and 1.64 U/mg, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). The specific activity of mutant A152G was found to be enhanced, exhibiting a 9.8-fold improvement compared to Xyn ZT-2. However, the specific activity of mutant A238G was lower than that of Xyn ZT-2. These results indicate that the residues in the catalytic pocket play an important role in the activity of Xyn ZT-2 and that the mutant A152G can significantly improve the specific activity of Xyn ZT-2.\u003c/p\u003e\n \u003cp\u003eThis study investigated the impact of pH and temperature on the activity of Xyn ZT-2 and its mutants (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The optimal pH values were determined as 6.5, 7.0, 6.5, 6.5, 6.5, and 7.0 for Xyn ZT-2, S148T, A152G, A238G, S239P, and T278S, respectively. This finding indicates that the xylanase has higher activity in a neutral and slightly acidic environment, and the residue mutant has a negligible impact on the optimal pH. This may be because the mutated amino acids are mostly weakly acidic or neutral. In terms of the optimal temperature, Xyn ZT-2, S148T, S239P, and T278S mutants had an optimal temperature of 45\u0026deg;C, while A152G and A238G mutants had optimal temperatures of 65\u0026deg;C and 40\u0026deg;C, respectively. These results suggest that the S148T, S239P, and T278S mutants did not alter its optimal temperature. However, the A238G mutation decreased the optimal temperature by 5\u0026deg;C, while the A152G mutation increased it by 20\u0026deg;C. This indicates that the A152G mutation significantly enhances the optimal temperature change of XynZT-2. Point mutations can drive genetic diversity to adapt to the environment, and such mutations can be beneficial, neutral, or harmful, resulting in higher enzyme activity or new functions (\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e). The A152G and A238G mutants both mutate from alanine to glycine. The optimal temperature for A152G is higher than that of XynZT-2, while that for A238G is lower than that of XynZT-2. These findings illustrate that the same mutation can have beneficial or harmful effects on the activity of homologous enzymes. In summary, these results indicate that mutations affect the characteristics of XynZT-2, and the A152G mutation significantly enhances its optimal temperature.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Effect of mutations on catalytic efficiency\u003c/h2\u003e\n \u003cp\u003eThe kinetic parameters for Xyn ZT-2 and its mutants were determined and are presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The Km values for S148T, A152G, A238G, S239P, and T278S were 48.1, 0.6, 16.5, 46.0, and 30.9 g/L, respectively, while that of Xyn ZT-2 was 28.2 g/L. A152G and A238G displayed reduced Km values compared to Xyn ZT-2, with A152G showing only 2.1% of the Km of Xyn ZT-2. This indicates a significant improvement in the affinity between mutant A152G and xylan. The kcat of S148T and S239P was found to be improved compared to that of Xyn ZT-2. The kcat/Km value of A152G was 23.2-fold higher than that of Xyn ZT-2, indicating the importance of residue site Ala152 around the active site Asp150 in the catalytic efficiency of Xyn ZT-2. These results demonstrate that specific amino acid site mutations can effectively improve the catalytic performance of xylanase (\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e). The changes in the catalytic properties of enzymes during their adaptation to the environment are diverse, with mutations being either beneficial, neutral, or harmful. They may also promote higher enzyme activity or new functions (\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e,\u0026nbsp;\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eKinetic parameters of the mutants.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEnzymes\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eKm\u003c/em\u003e (g/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ekcat\u003c/em\u003e (/s)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ekcat/Km\u003c/em\u003e(g/L/s)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eXyn ZT-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS148T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e48.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA152G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA238G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS239P\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e46.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT287S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Thermal stability of Xyn ZT-2 and mutations\u003c/h2\u003e\n \u003cp\u003eThe thermal stability of Xyn ZT-2 and its mutations were studied under optimized reaction conditions. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, the thermal stability of S148T, A152G, and T278S was improved compared to that of Xyn ZT-2. After 20 minutes of incubation, the activity retention of S148T, A152G, and T278S was 76.5%, 64.9%, and 88.3%, respectively. In contrast, Xyn ZT-2\u0026apos;s activity remained at only 31% after incubating at 45\u0026deg;C and pH 6.0 for 20 min. The half-life period of T287S was found to be 4.9 times longer than that of Xyn ZT-2, possibly due to the smaller size of Val compared to Ile, leading to improved enzyme stability (\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e). These results demonstrate that mutations can improve the thermal stability of Xyn ZT-2, although other mutations can decrease stability. The activity of A23G and S239P was sharply reduced, reaching almost zero after only 10 minutes of incubation under optimized conditions. These results illustrate the changes in thermal stability that occur during enzyme evolution. Mutations near the active site of amino acids may cause a loss of enzyme activity, while reasonable mutations can significantly improve enzyme activity and stability. Replacing homologous amino acids from enzymes with desired functions is a useful and effective strategy for enhancing enzyme catalytic efficiency or stability (\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e). Nevertheless, many enzymes still have unidentified catalytic functions and efficiency. One approach to studying enzyme catalytic performance is to analyze protein evolution in enzyme families and establish smaller databases to obtain expected results from previously unreported sequences.\u003c/p\u003e"},{"header":"Conclusion","content":" \u003cp\u003eIn this study, we constructed a small library to explore the relationship between xylanase catalytic performance and protein evolution. The mutation sites were selected based on the sequence homology of the protein family. Homologous amino acids were used to replace the residues close to the active site, namely S148, A152, A238, S239, and T278. We then determined the catalytic properties of the wild Xyn ZT-2 and the five mutants. Our findings showed that A152 and T287S are key residues in xylanase activity and stability, with the A152G mutant exhibiting 9.8-fold higher catalytic activity and 23.2-fold higher catalytic efficiency, while the T287S mutant exhibited 4.9 times improved thermal stability. Our results indicate that the characteristics of xylanase change during the evolutionary process, and rational mutations of amino acids near the active site can be beneficial for improving enzyme properties. The approach of constructing a mutation library provides a feasible strategy to enhance enzymatic properties.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eStatements \u0026amp; Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Scientific Research Foundation of Xinxiang Medical University (XYBSKYZZ201717) and the Key Technologies R\u0026amp;D Program of Henan Province (232102311146, 212102210652).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCaixia Cui: Project administration, Writing \u0026ndash; review and editing, and Supervision.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eJia Xu: Data curation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eJuntao Wu: Methodology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNingning Wang: Methodology.\u003c/p\u003e\n\u003cp\u003eChenyan Zhou: Project administration and Supervision.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not require ethics approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not involve human subjects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agreed to submit our work for its publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data and materials are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlag\u0026ouml;z, D., Varan, N. 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Docking-guided rational engineering of a macrolide glycosyltransferase glycodiversifies epothilone B. \u003cem\u003eCommunications biology\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e, 100\u0026ndash;110.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"applied-biochemistry-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"abab","sideBox":"Learn more about [Applied Biochemistry and Biotechnology](https://www.springer.com/journal/12010)","snPcode":"12010","submissionUrl":"https://submission.nature.com/new-submission/12010/3","title":"Applied Biochemistry and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"xylanase, protein evolution, catalytic performance, mutation","lastPublishedDoi":"10.21203/rs.3.rs-3855763/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3855763/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEndo-1,4-β-xylanase is considered one of the most important xylanolytic enzymes, and in this study, we aimed to improve the catalytic properties of \u003cem\u003eAlteromones Macleadii\u003c/em\u003e xylanase (Xyn ZT-2) using an evolution-guided design approach. Analysis of the amino acid sequence revealed that the amino acids located in close proximity to the active site were highly conserved, with only a few amino acid differences. By introducing various mutations, we were able to modify the catalytic performance of the enzyme. Notably, the A152G mutation resulted in a 9.8-fold increase in activity and a 23.2-fold increase in catalytic efficiency. Furthermore, the optimal temperature of A152G was raised to 65\u0026deg;C, which is 20\u0026deg;C higher than that of Xyn ZT-2, and the half-life period of T287S was enhanced by 4.9 times. These findings demonstrate the significance of amino acid evolution in determining the catalytic performance of xylanase. By utilizing an evolution analysis to create a smaller mutation library, we efficiently enhanced the catalytic performance, thus providing a novel strategy for improving enzyme catalytic efficiency.\u003c/p\u003e","manuscriptTitle":"Improving the catalytic properties of xylanase from Alteromones Macleadii H35 through evolution analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-17 09:09:50","doi":"10.21203/rs.3.rs-3855763/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Resubmit revised form; Major revisions required","date":"2024-02-21T18:30:17+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-02-03T10:41:40+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-15T14:55:41+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Applied Biochemistry and Biotechnology","date":"2024-01-12T09:37:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Applied Biochemistry and Biotechnology","date":"2024-01-11T20:38:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"applied-biochemistry-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"abab","sideBox":"Learn more about [Applied Biochemistry and Biotechnology](https://www.springer.com/journal/12010)","snPcode":"12010","submissionUrl":"https://submission.nature.com/new-submission/12010/3","title":"Applied Biochemistry and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"69f37ca8-c85f-4f64-8f6e-c55577cdc289","owner":[],"postedDate":"January 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-04-01T15:08:53+00:00","versionOfRecord":{"articleIdentity":"rs-3855763","link":"https://doi.org/10.1007/s12010-024-04936-0","journal":{"identity":"applied-biochemistry-and-biotechnology","isVorOnly":false,"title":"Applied Biochemistry and Biotechnology"},"publishedOn":"2024-03-28 15:01:27","publishedOnDateReadable":"March 28th, 2024"},"versionCreatedAt":"2024-01-17 09:09:50","video":"","vorDoi":"10.1007/s12010-024-04936-0","vorDoiUrl":"https://doi.org/10.1007/s12010-024-04936-0","workflowStages":[]},"version":"v1","identity":"rs-3855763","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3855763","identity":"rs-3855763","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}