Enzyme and Microbial Technology最新文献

{"title":"Crystal structure of cyclohexylamine oxidase from Acinetobacter sp. YT−02 reveals key residues for catalytic activity and substrate specificity","authors":"Jing Wu ,&nbsp;Zhenggang Han ,&nbsp;Pengrong Li ,&nbsp;Jing Li ,&nbsp;Yuanyuan Chen ,&nbsp;Shangbo Ning ,&nbsp;Hong-jun Chao ,&nbsp;Xue-wang Gao ,&nbsp;Dazhong Yan","doi":"10.1016/j.enzmictec.2025.110700","DOIUrl":"10.1016/j.enzmictec.2025.110700","url":null,"abstract":"<div><div>Cyclohexylamine oxidase is a member of amine oxidases that catalyzes the conversion of cyclohexylamine to cyclohexanone. In our previous work, the enzymatic activity assay of cyclohexylamine oxidase CHAO_YT-02 indicated that its specific activity towards cyclohexylamine of CHAO_YT-02 was ten times higher than that of its homolog CHAO_IH-35A. In this study, the crystal structure of CHAO_YT-02 was determined by the molecular replacement method at a resolution of 1.49 Å. The atomic structure revealed that the amino acid residues Leu302, Trp70, Phe197, Phe349, and Tyr440 constitute the active center pocket of the enzyme. Amino acid residues Ile180, Leu181, and Trp332 separate the active center pocket and an intermediate pocket. Moreover, a molecular dynamics (MD) simulation and the calculation of the binding free energy were performed to predict substrate entry and product release from cyclohexylamine oxidases. Single-amino acid substitution mutants (W70A, I180A, L181A, I208A, F197A, L302A, W332A, F349A, and Y440A) of CHAO_YT-02 were constructed to investigate the role of these amino acid residues in enzymatic properties and substrate specificity. The results indicated that both the amino acid residues in the active center pocket and gating the two pockets affected the activity or substrate specificity of CHAO_YT-02. This study on the structure and catalytic mechanism of cyclohexylamine oxidase is beneficial to eliminating toxic amine compounds in the environment.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110700"},"PeriodicalIF":3.4,"publicationDate":"2025-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144549870","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The effect of the cspA gene on growth development and butenyl-spinosyn biosynthesis in Saccharopolyspora pogona","authors":"Duo Jin,&nbsp;Shanrui Wang,&nbsp;Wangqiong Chen,&nbsp;Jing Fang,&nbsp;Jie Rang,&nbsp;Liqiu Xia,&nbsp;zirong zhu","doi":"10.1016/j.enzmictec.2025.110701","DOIUrl":"10.1016/j.enzmictec.2025.110701","url":null,"abstract":"<div><div>Cold shock proteins (CSPs) represent a universal class of proteins in microorganisms, rapidly inducible under low temperature conditions. As molecular chaperones for RNA, they bind to single-stranded nucleotides, preventing the formation of complex secondary structures. This facilitates efficient translation and gene expression regulation. This investigation pioneers the study of the <em>cspA</em> gene through metabolic engineering techniques, to uncover its critical biological roles in the growth and development of <em>Saccharopolyspora pogona</em> and in butenyl-spinosyn biosynthesis. Employing comparative proteomic and targeted metabolomic analyses, this research elucidates the metabolic pathway alterations prompted by the augmented presence of the cold shock protein CspA. Additionally, it offers initial insights into the regulatory mechanisms by which CspA affects <em>S. pogona</em>'s growth, development, and butenyl-spinosyn production. The outcomes of this study significantly advance our theoretical understanding of the rational optimization of butenyl-spinosyn biosynthetic pathways. They also provide valuable guidance for other actinobacteria aiming to boost their resilience to harsh environments by overexpressing the <em>cspA</em> gene.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110701"},"PeriodicalIF":3.4,"publicationDate":"2025-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144471575","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Deciphering key residues governing thermostability in Thermomyces lanuginosus lipase through Gibbs free energy-guided engineering","authors":"Xia Xiang ,&nbsp;Sidi Wan ,&nbsp;Songjing Zhang ,&nbsp;Enheng Zhu ,&nbsp;Xuejun Lin ,&nbsp;Nanyu Han","doi":"10.1016/j.enzmictec.2025.110702","DOIUrl":"10.1016/j.enzmictec.2025.110702","url":null,"abstract":"<div><div>Lipases serve as indispensable biocatalysts in many industrial applications due to their versatile catalytic abilities. To ensure their thermal resilience of the harsh biological treatment in industry, it is crucial to identify key residues which might impact thermostability. Here, computational design was adopted to decode the stability-determining residues in <em>Thermomyces lanuginosus</em> lipase (TLL). Systematic Gibbs free energy profiling of potent TLL single-point mutational candidates predicted proline 256 (P256) as a thermal liability hotspot. Saturation mutagenesis at P256 discovered that among nineteen P256 variants: (1) five P256 variants exhibited increased melting temperature (ΔT_m up to 2°C); (2) six variants displayed an optimum temperature with 5–10°C elevation; (3) five P256 variants retained up to 21 % higher residual activity after incubation at 80°C. Furthermore, both P256E and P256I demonstrated synergistic improvements in biodiesel conversion rates, P256I specifically exhibited long-term and recycling stability. Molecular dynamics simulations revealed that the substitutions in P256A/E/I/K with compensatory main-chain rotational freedom, facilitating hydrogen bonding network with both upstream and downstream residues, thereby preserving local structural stability. This study pioneers the identification of P256 as a critical residue governing TLL thermostability. Furthermore, our Gibbs-guided engineering strategy generates multi-property-enhanced lipase variants, directly addressing industrial demands.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110702"},"PeriodicalIF":3.4,"publicationDate":"2025-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144471581","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bacillus cereus N-acyl homoserine lactonase and penicillin acylase II against Pseudomonas aeruginosa: An In silico and In vitro investigations exploring the effects of gamma radiation on their quorum quenching activity","authors":"Radwa N. Morgan,&nbsp;Reham R. El-Behery","doi":"10.1016/j.enzmictec.2025.110699","DOIUrl":"10.1016/j.enzmictec.2025.110699","url":null,"abstract":"<div><div>This study explored the quorum quenching (QQ) activities of ISM25 strain, sourced from an environmental setting, on pathogenic <em>Pseudomonas aeruginosa</em> isolates. The isolate ISM25 was first screened for the presence of both <em>AiiA</em> acyl homoserine lactonase and penicillin <em>acylase II</em> genes. The in silico investigation focused on examining the physiochemical properties and QQ activities in relation to both short (&lt;C8) and long chain (&gt;C12) homoserine lactones (HSLs) utilizing AutoDock Vina docking tests. Potential interactions between the acylase II enzyme and carbapenem antibiotics were also investigated during the in silico docking tests. Afterwards, the in vitro QQ activity of ISM25 crude protein extract was assessed against the biofilm index, AHLs molecules, and meropenem minimum inhibitory concentrations of pathogenic <em>P. aeruginosa</em> isolates. The findings from the in silico docking analysis were supported by the crude protein extract's capacity to hydrolyze AHLs generated by the pathogenic <em>P. aeruginosa</em> isolate, as well as the reference C12-HSL signal molecule. The notable decrease in biofilm indices (P &lt; 0.05) following exposure to the crude ISM25 extract further corroborated the expression of both QQ enzymes by the ISM25 isolate and their in silico QQ activity. The crude extract from ISM25 lowered the MIC and sensitized <em>P. aeruginosa</em> to meropenem (P &lt; 0.05), suggesting that ISM25 crude extract containing acylase II can be administered in conjunction with meropenem without affecting its efficacy. Finally, exposure of ISM25 to gamma radiation did not impair its QQ activity at doses below 100 Gy, however, QQ activity was nearly abolished at radiation dose ≥ 500 Gy.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110699"},"PeriodicalIF":3.4,"publicationDate":"2025-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144365395","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Efficient isoprimeverose production using an enzyme cocktail from engineered Aspergillus oryzae and yeast-assisted purification","authors":"Satoshi Wakai ,&nbsp;Nanami Nakashima ,&nbsp;Hiroko Tsutsumi ,&nbsp;Yoji Hata ,&nbsp;Fahmi Baihaqqi ,&nbsp;Akihiko Kondo ,&nbsp;Chiaki Ogino","doi":"10.1016/j.enzmictec.2025.110698","DOIUrl":"10.1016/j.enzmictec.2025.110698","url":null,"abstract":"<div><div><em>Aspergillus oryzae</em> is a filamentous fungus that possesses various types of carbohydrate -degrading enzymes. Among these, isoprimeverose-producing enzyme (IpeA), acts on a key component of the plant cell wall structure, xyloglucan, to catalyze the release of isoprimeverose — a rare disaccharide that is expected to possess valuable prebiotics properties. Despite these expectations, however, a process for the effective production of isoprimeverose from the xyloglucan still requires further development for commercial-level application. A complicating factor for the lack of such a valuable process is that plant-derived xyloglucan is often modified with other sugars such as galactose and arabinose. Therefore, the effective production of isoprimeverose requires a cooperative form of degradation that must utilize different enzymes. In this study, we genetically engineered two <em>A. oryzae</em> strains — one produces IpeA and the other produces endoglucanase. The two strains were cultivated separately, and an enzyme cocktail was prepared using their respective culture supernatants. This enzyme cocktail successfully produced isoprimeverose from tamarind xyloglucan and tamarind seed gum. Approximately 14 g/L of isoprimeverose was obtained, which corresponds to a theoretical conversion rate of over 90 %. Although glucose and galactose remained in the reaction solution after enzymatic degradation, these by-products could be easily removed via treatment with <em>Saccharomyces cerevisiae</em>. Our developed process, which mimics traditional Japanese sake fermentation using <em>A. oryzae</em> and <em>S. cerevisiae</em>, has enabled efficient production of isoprimeverose.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110698"},"PeriodicalIF":3.4,"publicationDate":"2025-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144330453","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparison of novel α-glucosidases in glycoside hydrolase family 97 isolated from Bacteroides thetaiotaomicron","authors":"Dam-Seul Ko ,&nbsp;Hyun-Mo Jeong ,&nbsp;Yu-Jeong Shin,&nbsp;Da-Woon Jeong,&nbsp;Na-Ri Kim,&nbsp;Jae-Hoon Shim","doi":"10.1016/j.enzmictec.2025.110696","DOIUrl":"10.1016/j.enzmictec.2025.110696","url":null,"abstract":"<div><div>In this study, three genes encoding novel Glycoside Hydrolase (GH) 97 enzymes were cloned from <em>Bacteroides thetaiotaomicron</em> and expressed in <em>Escherichia coli</em>. The recombinant enzymes (Bt_4581, Bt_0683, Bt_3163) were purified using Ni-NTA affinity chromatography and subsequently characterized. All three enzymes released glucose from the non-reducing ends of oligosaccharides and displayed metal ion dependency. Among them, Bt_4581 hydrolyzed a wide range of α-glycosidic linkages, while Bt_0683 and Bt_3163 showed narrower substrate specificity. Amino acid sequence analysis indicated that Bt_4581 and Bt_0683 belong to Group 1, whereas Bt_3163 is part of Group 3. Kinetic studies revealed that Bt_4581 preferred maltooligosaccharides with an odd number of glucosyl units. In contrast, Bt_3163 exhibited a preference for α-<em>p</em>NPG, confirming it as the first characterized α-glucosidase in Group 3 of the GH 97 family.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110696"},"PeriodicalIF":3.4,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144522232","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Arazyme prevents skin aging through regulation of matrix metalloproteinase and collagen synthesis","authors":"Jong-Hoon Kim ,&nbsp;Hwa Lee ,&nbsp;Kwang-Hee Son ,&nbsp;Tae-Sook Jeong ,&nbsp;Ho-Yong Park","doi":"10.1016/j.enzmictec.2025.110695","DOIUrl":"10.1016/j.enzmictec.2025.110695","url":null,"abstract":"<div><div>Arazyme, an enzyme derived from <em>Serratia proteamaculans</em>, has demonstrated efficacy in enhancing skin barrier function in studies involving skin cell treatments and topical application on animal skin. The objective of this study was to assess the anti-wrinkle and anti-aging effects of Arazyme in skin keratinocytes and fibroblasts subjected to ultraviolet B (UVB) radiation and oxidative stress. Keratinocytes (HaCaT cells) and fibroblasts (CCD-986sk) were exposed to UVB (15 mJ/cm²) radiation or oxidative stress induced by 2 mM 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), followed by treatment with Arazyme (0.1–0.5 μM) for 24 h. The effects of Arazyme were compared to those of individual treatments with papain, trypsin, or retinol, which served as reference compounds. Key parameters examined included the expression of matrix metalloproteinases (<em>MMP-1, MMP-3, and MMP-13</em>), collagen synthesis, and cellular senescence markers (<em>LMNB1, p16, p21</em>, and <em>p53</em>). Additionally, the impact of Arazyme on cellular signaling pathways, including ERK, JNK, and NF-κB, was assessed. Arazyme significantly suppressed UVB-induced expression of MMP-1, MMP-3, and MMP-13 in a dose-dependent manner in HaCaT cells compared to other treatments. In UVB-exposed fibroblasts, Arazyme reduced both mRNA and protein levels of MMPs, while also enhancing procollagen concentration and collagen gene expression. Furthermore, Arazyme inhibited the activation of ERK, JNK, and NF-κB signaling pathways in keratinocytes. In AAPH-stimulated HaCaT cells, Arazyme significantly attenuated the expression of senescence-related markers, including <em>LMNB1, p16, p21</em>, and <em>p53</em>, and decreased the proportion of senescence-positive cells in fibroblasts. Our in vitro findings suggest that Arazyme may help attenuate UVB- and oxidative stress-induced markers of skin aging, indicating its potential as a candidate for further investigation in anti-aging skincare research.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110695"},"PeriodicalIF":3.4,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144321341","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Semi-rational engineering of 3-Ketosteroid-Δ1-dehydrogenase boosts catalytic efficiency and robustness for steroid bioconversion","authors":"Zijuan Tao ,&nbsp;Yusong Zhang ,&nbsp;Yanmei Dai ,&nbsp;Changshun Huang ,&nbsp;Liangli Luo ,&nbsp;Junhao Yue ,&nbsp;Jing'e Yue ,&nbsp;Hanbing Shi ,&nbsp;Zhimin Ou","doi":"10.1016/j.enzmictec.2025.110692","DOIUrl":"10.1016/j.enzmictec.2025.110692","url":null,"abstract":"<div><div>Steroid drugs play a pivotal role in clinical therapeutics, with androsta-1,4-diene-3,17-dione (ADD) serving as a critical intermediate whose efficient biosynthesis relies on the catalytic activity of 3-ketosteroid-Δ1-dehydrogenase (KstD). Building upon the previously engineered KstD₂^ep variant, this study employed semi-rational design strategies to enhance KstD₂’s high-substrate-loading adaptability. Key residues (V332, L334, G534) were systematically identified through homology modeling and molecular docking, followed by constructing a combinatorial mutant library. Through alanine scanning and iterative screening of single/multiple-site mutations, the optimal mutant KstD₂^ep (V332E/L334T/G534V) demonstrated a 1.1-fold enhancement in catalytic efficiency compared to the KstD₂^ep. Molecular dynamics simulations confirmed significantly enhanced structural stability in the mutant. Whole-cell catalytic optimization revealed expanded operational tolerance to temperature fluctuations, pH variations, and co-solvent exposure. Implementing high-density fermentation coupled with fed-batch substrate supplementation, the process achieved a 1.5-fold increase in substrate conversion efficiency, yielding 117.75 g/L ADD. These advancements position the engineered variant as a high-potential candidate for scalable steroid biotransformation, addressing key barriers to enzymatic stability and process efficiency for enzyme-driven biosynthesis of steroidal pharmaceutical intermediates.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110692"},"PeriodicalIF":3.4,"publicationDate":"2025-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144280806","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Expanding fluorescence imaging tools in Staphylococcus aureus: Optimized expression of red fluorescent proteins","authors":"Fuminori Kato","doi":"10.1016/j.enzmictec.2025.110694","DOIUrl":"10.1016/j.enzmictec.2025.110694","url":null,"abstract":"<div><div>Fluorescent proteins (FPs) are widely used as molecular imaging tools for visualizing protein localization, gene expression, and bacterial labeling. However, most commercially available FPs are optimized for expression in human cells or <em>Escherichia coli</em>, and often exhibit poor expression in other bacterial species due to differences in codon usage preference, which critically affects translation efficiency. <em>Staphylococcus aureus</em>, a clinically important Gram-positive bacterium, presents additional challenges for heterologous protein expression due to codon usage bias and gene regulatory mechanisms. While our previous study has developed expression vectors that enable robust expression of GFP and its color variants in <em>S. aureus</em>, expression of red fluorescent proteins (RFPs) remain limited. In this study, I improved the expression of red fluorescent proteins (RFPs)—mCherry, mOrange2, E2-Crimson—and the photoconvertible protein Dendra2 in <em>S. aureus</em>. Codon optimization was performed based on <em>S. aureus</em> codon usage preferences, and RNA secondary structures at the 5′ region were minimized to enhance translation efficiency. The fully optimized mCherry(Sa2) exhibited strong fluorescence at both colony and single-cell levels. Similarly, mOrange2(Sa), E2-Crimson(Sa), and Dendra2(Sa) showed robust expression following host-adapted codon design, and Dendra2(Sa) retained its photoconvertible functionality. These results demonstrate that codon usage adjustment and RNA structure optimization are effective strategies for achieving high-level expression of diverse fluorescent proteins in <em>S. aureus</em> and provide valuable insights into optimizing heterologous protein expression in non-model bacterial systems.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110694"},"PeriodicalIF":3.4,"publicationDate":"2025-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144307024","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterization of a GH10 family thermophilic, alkali- and salt-tolerant xylanase from Xinjiang salt lake","authors":"Rong-Huan Song ,&nbsp;Dan Zhu ,&nbsp;Zhong-qiao Yang ,&nbsp;Jian-Ling Li ,&nbsp;Zheng-Feng Yang ,&nbsp;Zhi-Hua Lv ,&nbsp;Kai-Qing Xie ,&nbsp;Li-Quan Yang ,&nbsp;A. Zhou-Cun ,&nbsp;Peng Sang ,&nbsp;Yi-Rui Yin","doi":"10.1016/j.enzmictec.2025.110693","DOIUrl":"10.1016/j.enzmictec.2025.110693","url":null,"abstract":"<div><div>Xylanase is extensively employed in the food, feed, and paper sectors, with those derived from extreme environments offering distinct advantages. This study identified a novel xylanase gene (designated <em>xynaes</em>) through metagenomic analysis of samples from Aiting Lake, Xinjiang, China. Cloned and expressed in Escherichia coli after PCR amplification. The recombinant protein was purified using Ni-NTA affinity chromatography XynAES demonstrated optimal activity at pH 8.0 and 65 °C, its half-life (T_1/2) was 120 min. XynAES preserved over 80 % residual activity after 12 h in pH 6.0–9.0 buffer. Its activity was enhanced to 132 % and 135 % in the presence of 1 mM Mg²⁺ and Zn²⁺, respectively. Additionally, XynAES maintained over 60 % relative activity in 0–3.0 M NaCl and its Km and Vmax of XynAES were determined to be 3.23 mg/mL and 72.46 μmol/min/mg, respectively. It is worth noting that the main products of XynAES enzymatic hydrolysis of xylan are xylose disaccharides and xylose tetrasaccharides, and XynAES shows obvious activity against the pre-treated wheat bran. In summary, XynAES is a thermophilic, alkali-tolerant, and salt-resistant xylanase, signifying its potential applications in the feed, food baking, paper manufacturing, and prebiotic production industries.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"190 ","pages":"Article 110693"},"PeriodicalIF":3.4,"publicationDate":"2025-06-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144262912","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}