Enzyme and Microbial Technology最新文献_第7页

Mechanism of tyrosine 69 in the flavin domain modulating electron transfer efficiency in BVU5 azoreductase 黄素区域酪氨酸69调节BVU5偶氮还原酶电子转移效率的机制

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2026-01-01 Epub Date: 2025-09-25 DOI: 10.1016/j.enzmictec.2025.110757

Xuehui Xie , Dongyang Li , Hangmi Zheng , Ziyi Wu , Jiajie Liu , Wanting Li , Songwei Chen , Yiwen Zhang , Daijie Tang , Na Liu , Qingyun Zhang , Ye Chen

The azoreductase BVU5 is a flavoprotein dependent on NAD(P)H/FMN-mediated electron transfer; However, the functional mechanism of key residues within its flavin domain remains unclear. This study identified the conserved residue Tyr⁶⁹ through bioinformatics analysis and constructed Y69F and Y69C mutants. Enzymatic assays demonstrated that mutants exhibited significantly lower decolorization rates than the BVU5 enzyme across 12 dye molecules, including azo, anthraquinone, and triphenylmethane dyes. For example, when decolorizing Reactive Black 5 (RB5) for 2 h: Y69F mutant achieved 60 %-65 % decolorization, Y69C mutant reached 55 %-60 % decolorization, both markedly lower than the 80 %-85 % efficiency of BVU5.The decolorization hierarchy remained azo dyes > triphenylmethane > anthraquinone dyes. Molecular docking revealed that mutations reconfigured FMN-binding patterns. Although Y69F enlarged the substrate-binding pocket, it failed to enhance the degradation efficiency of the bulky dye Chlorazol Black E. This critical contradiction indicates that substrate binding is not the limiting factor. Combined with evidence such as the lighter color of the mutant enzyme solutions, increased A280/A450 ratios, and enzyme dosage experiments, this study confirms that Tyr⁶⁹ is a key residue that sustains electron transfer efficiency by maintaining the FMN-binding conformation, thereby determining the decolorization performance. Consequently, electron transfer efficiency, rather than substrate binding, is the primary mechanism influencing the catalytic function of BVU5.

偶氮还原酶BVU5是一种依赖于NAD(P)H/ fmn介导的电子转移的黄蛋白；然而，其黄素结构域关键残基的功能机制尚不清楚。本研究通过生物信息学分析鉴定了保守残基Tyr69，构建了Y69F和Y69C突变体。酶分析表明，突变体在12种染料分子上的脱色率明显低于BVU5酶，包括偶氮、蒽醌和三苯甲烷染料。例如，当2 h: Y69F突变体对活性黑5 （RB5）脱色时，脱色率为60 %-65 %，Y69C突变体脱色率为55 %-60 %，均明显低于BVU5的80 %-85 %。脱色等级仍为偶氮染料>； 三苯甲烷>； 蒽醌染料。分子对接显示突变重新配置了fmn结合模式。虽然Y69F扩大了底物结合袋，但未能提高对体积较大的染料氯唑黑e的降解效率，这一关键矛盾表明底物结合并不是限制因素。结合突变酶溶液颜色变浅、A280/A450比值增加、酶用量实验等证据，本研究证实Tyr69是通过维持fnm结合构象来维持电子转移效率的关键残基，从而决定了脱色性能。因此，电子转移效率，而不是底物结合，是影响BVU5催化功能的主要机制。

{"title":"Mechanism of tyrosine 69 in the flavin domain modulating electron transfer efficiency in BVU5 azoreductase","authors":"Xuehui Xie , Dongyang Li , Hangmi Zheng , Ziyi Wu , Jiajie Liu , Wanting Li , Songwei Chen , Yiwen Zhang , Daijie Tang , Na Liu , Qingyun Zhang , Ye Chen","doi":"10.1016/j.enzmictec.2025.110757","DOIUrl":"10.1016/j.enzmictec.2025.110757","url":null,"abstract":"<div><div>The azoreductase BVU5 is a flavoprotein dependent on NAD(P)H/FMN-mediated electron transfer; However, the functional mechanism of key residues within its flavin domain remains unclear. This study identified the conserved residue Tyr<sup>69</sup> through bioinformatics analysis and constructed Y69F and Y69C mutants. Enzymatic assays demonstrated that mutants exhibited significantly lower decolorization rates than the BVU5 enzyme across 12 dye molecules, including azo, anthraquinone, and triphenylmethane dyes. For example, when decolorizing Reactive Black 5 (RB5) for 2 h: Y69F mutant achieved 60 %-65 % decolorization, Y69C mutant reached 55 %-60 % decolorization, both markedly lower than the 80 %-85 % efficiency of BVU5.The decolorization hierarchy remained azo dyes > triphenylmethane > anthraquinone dyes. Molecular docking revealed that mutations reconfigured FMN-binding patterns. Although Y69F enlarged the substrate-binding pocket, it failed to enhance the degradation efficiency of the bulky dye Chlorazol Black E. This critical contradiction indicates that substrate binding is not the limiting factor. Combined with evidence such as the lighter color of the mutant enzyme solutions, increased A280/A450 ratios, and enzyme dosage experiments, this study confirms that Tyr<sup>69</sup> is a key residue that sustains electron transfer efficiency by maintaining the FMN-binding conformation, thereby determining the decolorization performance. Consequently, electron transfer efficiency, rather than substrate binding, is the primary mechanism influencing the catalytic function of BVU5.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"192 ","pages":"Article 110757"},"PeriodicalIF":3.7,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145155168","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}

引用次数: 0

Homologous expression and characterization of Coniochaeta ligniaria glycoside hydrolase family 115 α-glucuronidase 木质素螺毛菌糖苷水解酶家族115 α-葡萄糖醛酸酶的同源表达与鉴定

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2026-01-01 Epub Date: 2025-09-09 DOI: 10.1016/j.enzmictec.2025.110750

Michael J. Bowman, Nancy N. Nichols

Generation of fermentable sugars from biomass is necessary for microbial production of bioproducts. Due to its inherent complexity, xylan hydrolysis to monosaccharides requires several different enzymatic activities. To completely depolymerize glucuronoarabinoxylan from biomass, α-glucuronidase activity is necessary. The ascomycete fungus Coniochaeta ligniaria has the ability to degrade xylan and is an unutilized source of biomass degrading enzymes. Therefore, a gene encoding a putative Coniochaeta ligniaria GH115 α-glucuronidase was cloned and expressed homologously in Coniochaeta ligniaria as a native secreted protein. Culture supernatants were concentrated and purified by a combination of ultrafiltration, anion-exchange, and size-exclusion chromatography. The purified protein behaved as dominantly dimeric complexes as determined by size-exclusion chromatography. The expressed protein liberated: 4-O-methyl glucuronic acid from beech xylan, birch xylan, and beech-derived glucuronoxylooligosaccharides as the sole product of hydrolysis; and 4-O-methyl glucuronic acid and glucuronic acid from oat spelt xylan. The expressed α-1,2-glucuronidase had greater activity on glucuronoxylooligosaccharides than on full length beech xylan. The expressed α-1,2-glucuronidase, herein designated ClAgu115, had K_m values of: 1.3 mM; 1.2 mM; and 1.0 mM for beech xylan, GH10-hydrolyzed glucuronooligosaccharides, and GH11-hydrolyzed glucuronooligosaccharides, respectively. The measured kinetic constants show that the enzyme prefers an oligosaccharide substrate with the 4-O-methyl glucuronic acid on the non-reducing end. The enzyme had activity between pH 3.0–6.0 and temperatures 10°C-60°C, with optima at pH 4.3 and 40°C. The expression and characterization of ClAgu115 expands the repertoire of fungal GH115 enzymes for use in biomass conversion.

从生物质中产生可发酵糖是微生物生产生物产品所必需的。由于其固有的复杂性，木聚糖水解成单糖需要几种不同的酶活性。为了从生物质中完全解聚葡萄糖醛酸阿拉伯木聚糖，α-葡萄糖醛酸酶活性是必需的。子囊菌木质素Coniochaeta ligniaria具有降解木聚糖的能力，是未利用的生物质降解酶的来源。因此，我们克隆了一个推测为木质素毛刀菌GH115 α-葡糖醛酸酶的基因，并将其作为天然分泌蛋白在木质素毛刀菌中同源表达。培养上清通过超滤、阴离子交换和尺寸排除层析的组合进行浓缩和纯化。纯化后的蛋白主要表现为二聚体复合物，通过尺寸排除层析确定。所表达的蛋白作为水解的唯一产物，从山毛榉木聚糖、桦木聚糖和山毛榉衍生的低聚葡萄糖醛酸中释放出4- o -甲基葡萄糖醛酸；4- o -甲基葡萄糖醛酸和4- o -甲基葡萄糖醛酸来自燕麦斯佩尔木聚糖。α-1,2-葡萄糖醛酸酶对低聚葡萄糖醛酸的活性高于对全长山毛榉木聚糖的活性。表达的α-1,2-葡糖醛酸酶，本文命名为ClAgu115，其Km值为：1.3 mM； 1.2毫米;山毛榉木聚糖、gh10水解的低聚葡萄糖醛酸糖和gh11水解的低聚葡萄糖醛酸糖分别为1.0 mM。测定的动力学常数表明，酶倾向于低聚糖底物，在非还原端有4- o -甲基葡萄糖醛酸。该酶在pH 3.0 ~ 6.0、温度10℃~ 60℃范围内具有活性，pH 4.3和温度40℃时活性最高。ClAgu115的表达和鉴定扩大了真菌GH115酶用于生物质转化的范围。

{"title":"Homologous expression and characterization of Coniochaeta ligniaria glycoside hydrolase family 115 α-glucuronidase","authors":"Michael J. Bowman, Nancy N. Nichols","doi":"10.1016/j.enzmictec.2025.110750","DOIUrl":"10.1016/j.enzmictec.2025.110750","url":null,"abstract":"<div><div>Generation of fermentable sugars from biomass is necessary for microbial production of bioproducts. Due to its inherent complexity, xylan hydrolysis to monosaccharides requires several different enzymatic activities. To completely depolymerize glucuronoarabinoxylan from biomass, α-glucuronidase activity is necessary. The ascomycete fungus <em>Coniochaeta ligniaria</em> has the ability to degrade xylan and is an unutilized source of biomass degrading enzymes. Therefore, a gene encoding a putative <em>Coniochaeta ligniaria</em> GH115 α-glucuronidase was cloned and expressed homologously in <em>Coniochaeta ligniaria</em> as a native secreted protein<em>.</em> Culture supernatants were concentrated and purified by a combination of ultrafiltration, anion-exchange, and size-exclusion chromatography. The purified protein behaved as dominantly dimeric complexes as determined by size-exclusion chromatography. The expressed protein liberated: 4-<em>O</em>-methyl glucuronic acid from beech xylan, birch xylan, and beech-derived glucuronoxylooligosaccharides as the sole product of hydrolysis; and 4-<em>O</em>-methyl glucuronic acid and glucuronic acid from oat spelt xylan. The expressed α-1,2-glucuronidase had greater activity on glucuronoxylooligosaccharides than on full length beech xylan. The expressed α-1,2-glucuronidase, herein designated <em>Cl</em>Agu115, had <em>K</em><sub><em>m</em></sub> values of: 1.3 mM; 1.2 mM; and 1.0 mM for beech xylan, GH10-hydrolyzed glucuronooligosaccharides, and GH11-hydrolyzed glucuronooligosaccharides, respectively. The measured kinetic constants show that the enzyme prefers an oligosaccharide substrate with the 4-<em>O</em>-methyl glucuronic acid on the non-reducing end. The enzyme had activity between pH 3.0–6.0 and temperatures 10°C-60°C, with optima at pH 4.3 and 40°C. The expression and characterization of <em>Cl</em>Agu115 expands the repertoire of fungal GH115 enzymes for use in biomass conversion.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"192 ","pages":"Article 110750"},"PeriodicalIF":3.7,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145046785","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}

引用次数: 0

De Novo enzyme design of a minimal and soluble carbonic anhydrase from Mesorhizobium loti assisted by molecular dynamics simulation 在分子动力学模拟的辅助下，从loti中分离出一种最小的可溶碳酸酐酶

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2026-01-01 Epub Date: 2025-09-12 DOI: 10.1016/j.enzmictec.2025.110751

Jiun-Jang Juo, I.-Son Ng

Carbonic anhydrases (CAs) are critical biocatalysts in the carbon capture and utilization due to their remarkable efficiency in converting carbon dioxide into bicarbonate. Among all, the CA from Mesorhizobium loti (MlCA) exhibits the highest catalytic activity. However, the poor expression and low solubility in Escherichia coli significantly restricts its application. To overcome the challenges, we employed a protein minimization strategy to improve both enzymatic expression and solubility. Enhanced expressions were observed in genetic constructs with short N-terminal tags which MlCA expression is strongly affected by mRNA secondary structure near the start codon. For solubility issue, a de novo protein design workflow guided by molecular dynamics simulations was developed. The process consists of four stages: (1) size-constrained de novo design considered using AlphaFold and RFdiffusion, (2) sequence recovery replying on solubleMPNN and ESMfold, (3) In silico screening by FoldX, SASA and molecular dynamics evaluation, and (4) experimental validation. This enabled the generation of compact, stable, and catalytically active MlCA variants with 28 % reduction in protein size among 500 candidates. Finally, the most promising design, dM22, of minimal CA showed improved solubility from 16.1 % to 61.2 % in B7G, a GroELS integrated BL21(DE3) strain.

碳酸酐酶（carbon anhydrase, CAs）具有将二氧化碳转化为碳酸氢盐的显著效率，是碳捕获和利用中的关键生物催化剂。其中，loti中根瘤菌（MlCA）的CA表现出最高的催化活性。但其在大肠杆菌中的低表达和低溶解度严重限制了其应用。为了克服这些挑战，我们采用了蛋白质最小化策略来提高酶的表达和溶解性。在具有短n端标签的遗传结构中，MlCA的表达受到起始密码子附近mRNA二级结构的强烈影响。针对溶解度问题，建立了以分子动力学模拟为指导的蛋白质从头设计流程。该过程包括四个阶段：(1)使用AlphaFold和RFdiffusion考虑尺寸约束的从头设计，(2)依靠solubleMPNN和ESMfold进行序列恢复，(3)通过FoldX， SASA和分子动力学评估进行硅筛选，(4)实验验证。这使得在500个候选基因中产生紧凑、稳定和催化活性的MlCA变异，其蛋白质大小减少了28% %。最后，最有希望的设计dM22，最小CA的设计在GroELS整合BL21（DE3）菌株B7G中的溶解度从16.1 %提高到61.2 %。

{"title":"De Novo enzyme design of a minimal and soluble carbonic anhydrase from Mesorhizobium loti assisted by molecular dynamics simulation","authors":"Jiun-Jang Juo, I.-Son Ng","doi":"10.1016/j.enzmictec.2025.110751","DOIUrl":"10.1016/j.enzmictec.2025.110751","url":null,"abstract":"<div><div>Carbonic anhydrases (CAs) are critical biocatalysts in the carbon capture and utilization due to their remarkable efficiency in converting carbon dioxide into bicarbonate. Among all, the CA from <em>Mesorhizobium loti</em> (MlCA) exhibits the highest catalytic activity. However, the poor expression and low solubility in <em>Escherichia coli</em> significantly restricts its application. To overcome the challenges, we employed a protein minimization strategy to improve both enzymatic expression and solubility. Enhanced expressions were observed in genetic constructs with short N-terminal tags which MlCA expression is strongly affected by mRNA secondary structure near the start codon. For solubility issue, a <em>de novo</em> protein design workflow guided by molecular dynamics simulations was developed. The process consists of four stages: (1) size-constrained <em>de novo</em> design considered using AlphaFold and RFdiffusion, (2) sequence recovery replying on solubleMPNN and ESMfold, (3) <em>In silico</em> screening by FoldX, SASA and molecular dynamics evaluation, and (4) experimental validation. This enabled the generation of compact, stable, and catalytically active MlCA variants with 28 % reduction in protein size among 500 candidates. Finally, the most promising design, dM22, of minimal CA showed improved solubility from 16.1 % to 61.2 % in B7G, a GroELS integrated BL21(DE3) strain.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"192 ","pages":"Article 110751"},"PeriodicalIF":3.7,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145057156","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}

引用次数: 0

Innovative nanosensing methods for lysozyme identification 溶菌酶鉴定的创新纳米传感方法

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2026-01-01 Epub Date: 2025-09-02 DOI: 10.1016/j.enzmictec.2025.110745

Mohammad Darvishi , Seyyed Mohammad Mousavinia , Reza Soleimany , Mohammad Mahdi Heidari , Mohsen Mohammadi , Samad Rastmanesh , Ahmad Mobed

Lysozyme (LYZ) is a critical enzyme recognized for its significant antimicrobial properties, playing an integral role in the immune response and being implicated in various diseases, including infections and inflammatory conditions. Traditional detection methods for LYZ, such as enzyme-linked immunosorbent assays (ELISA) and spectrophotometric techniques, often encounter limitations regarding sensitivity, specificity, and time efficiency. In light of these challenges, there has been substantial advancement in the development of novel biosensor technologies over the past two decades, particularly those that incorporate nanomaterials. These innovative biosensors demonstrate enhanced performance, facilitating rapid and accurate detection of LYZ at low concentrations. This paper aims to provide a comprehensive overview of recently developed biosensors specifically for LYZ, highlighting their design, functionality, and applications in clinical diagnostics and research. Unlike previous reviews, we place a distinct emphasis on the clinical importance of LYZ and its role in various diseases, thereby elucidating its significance in health and disease contexts. Additionally, we will explore the implications of advanced detection methods, particularly those utilizing nanomaterials, for enhancing our understanding of LYZ-related pathologies. By focusing on nanoscale detection techniques, which have not been thoroughly addressed in existing literature, we underscore the transformative potential of these biosensors in LYZ detection. This approach not only contributes to improved disease management but also informs therapeutic strategies, setting our review apart from prior works.

溶菌酶（LYZ）是一种重要的酶，因其显著的抗菌特性而被公认，在免疫反应中起着不可或缺的作用，并与包括感染和炎症在内的各种疾病有关。传统的LYZ检测方法，如酶联免疫吸附试验（ELISA）和分光光度法技术，经常遇到灵敏度、特异性和时间效率方面的限制。鉴于这些挑战，在过去的二十年里，新型生物传感器技术的发展取得了实质性的进步，特别是那些结合纳米材料的技术。这些创新的生物传感器表现出更高的性能，有助于在低浓度下快速准确地检测LYZ。本文旨在全面概述最近开发的LYZ生物传感器，重点介绍它们的设计，功能和在临床诊断和研究中的应用。与以前的综述不同，我们特别强调LYZ的临床重要性及其在各种疾病中的作用，从而阐明其在健康和疾病环境中的重要性。此外，我们将探讨先进的检测方法的意义，特别是那些利用纳米材料，以提高我们对lyz相关病理的理解。通过关注纳米级检测技术，这在现有文献中尚未得到彻底解决，我们强调了这些生物传感器在LYZ检测中的变革潜力。这种方法不仅有助于改善疾病管理，而且为治疗策略提供信息，使我们的综述与先前的工作不同。

{"title":"Innovative nanosensing methods for lysozyme identification","authors":"Mohammad Darvishi , Seyyed Mohammad Mousavinia , Reza Soleimany , Mohammad Mahdi Heidari , Mohsen Mohammadi , Samad Rastmanesh , Ahmad Mobed","doi":"10.1016/j.enzmictec.2025.110745","DOIUrl":"10.1016/j.enzmictec.2025.110745","url":null,"abstract":"<div><div>Lysozyme (LYZ) is a critical enzyme recognized for its significant antimicrobial properties, playing an integral role in the immune response and being implicated in various diseases, including infections and inflammatory conditions. Traditional detection methods for LYZ, such as enzyme-linked immunosorbent assays (ELISA) and spectrophotometric techniques, often encounter limitations regarding sensitivity, specificity, and time efficiency. In light of these challenges, there has been substantial advancement in the development of novel biosensor technologies over the past two decades, particularly those that incorporate nanomaterials. These innovative biosensors demonstrate enhanced performance, facilitating rapid and accurate detection of LYZ at low concentrations. This paper aims to provide a comprehensive overview of recently developed biosensors specifically for LYZ, highlighting their design, functionality, and applications in clinical diagnostics and research. Unlike previous reviews, we place a distinct emphasis on the clinical importance of LYZ and its role in various diseases, thereby elucidating its significance in health and disease contexts. Additionally, we will explore the implications of advanced detection methods, particularly those utilizing nanomaterials, for enhancing our understanding of LYZ-related pathologies. By focusing on nanoscale detection techniques, which have not been thoroughly addressed in existing literature, we underscore the transformative potential of these biosensors in LYZ detection. This approach not only contributes to improved disease management but also informs therapeutic strategies, setting our review apart from prior works.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"192 ","pages":"Article 110745"},"PeriodicalIF":3.7,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144989476","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}

引用次数: 0

Corrigendum to “Influence of a rumen ciliate-derived xylanase on the gut microbiota composition: A potential enzyme for prebiotic applications” [Enzyme Microb. Technol. 190 (2025) 110683] “瘤胃纤毛虫衍生的木聚糖酶对肠道微生物群组成的影响：一种潜在的益生元应用酶”的更正[微生物酶]。技术通报，190 (2025)110683 [j]。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2026-01-01 Epub Date: 2025-09-10 DOI: 10.1016/j.enzmictec.2025.110749

Weide Su , Huiying Luo , Gaoxiang Ai , Qipeng Wei , Zhiheng Zou , Xiaolian Chen , Chuanhui Xu , Jiang Chen , Pingwen Xiong , Wenjing Song , Qiongli Song

引用次数: 0

Metabolic engineering of Bacillus subtilis for enhanced p-Coumaric acid production and antimicrobial applications 枯草芽孢杆菌代谢工程提高对香豆酸产量及其抗菌应用。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2026-01-01 Epub Date: 2025-10-13 DOI: 10.1016/j.enzmictec.2025.110762

Junjie Zhang , Guodong Zhang , Wenhu Zhu , Youzhi Li , Yutuo Wei , Xianwei Fan

p-Coumaric acid (p-CA) is widely utilized in the food, pharmaceutical and other industries, and has traditionally been derived from plant extraction or chemical synthesis. However, p-CA synthesized by the safe B. subtilis remains poorly explored. In this study, we first engineered a recombinant B. subtilis strain (PBK) capable of synthesizing p-Coumaric acid, achieving an initial yield of 3.81 mg L⁻¹. A high-yielding strain PBnprE was then developed through promoter substitution, with a yield reaching 60.92 mg L⁻¹, and the yield of PBnprE was further increased to 304.04 mg L⁻¹ by optimizing fermentation conditions and substrates, showing an 80-fold increase over PBK. The optimized fermentation extract of PBnprE displayed increased antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, alongside enhanced DPPH and ABTS scavenging capabilities. Compared to PBK, the optimized extracts showed 4.81-fold higher in DPPH and 3.47-fold in ABTS scavenging, consistent with improved antioxidant properties driven by the increased presence of p-CA. This study first successfully constructed a high-yield p-CA producing engineered strain in B. subtilis, providing a valuable platform for synthesizing other secondary metabolites.

对香豆酸（p-CA）广泛应用于食品、制药等行业，传统上是通过植物提取或化学合成得到的。然而，利用安全的枯草芽孢杆菌合成p-CA的研究还很少。在这项研究中，我们首先设计了一种重组枯草芽孢杆菌菌株（PBK），能够合成对香豆酸，初始产量为3.81 mg L-1。通过启动子置换培养出高产菌株PBnprE，其产量达到60.92 mg L-1，通过优化发酵条件和底物，PBnprE的产量进一步提高到304.04 mg L-1，比PBK提高了80倍。优化后的PBnprE发酵提取物对金黄色葡萄球菌、铜绿假单胞菌和大肠杆菌的抑菌活性增强，对DPPH和ABTS的清除能力增强。与PBK相比，优化后的提取物对DPPH的清除能力提高了4.81倍，对ABTS的清除能力提高了3.47倍，这与p-CA含量的增加对抗氧化性能的提高是一致的。本研究首次在枯草芽孢杆菌中成功构建了高产p-CA工程菌株，为其他次生代谢产物的合成提供了有价值的平台。

{"title":"Metabolic engineering of Bacillus subtilis for enhanced p-Coumaric acid production and antimicrobial applications","authors":"Junjie Zhang , Guodong Zhang , Wenhu Zhu , Youzhi Li , Yutuo Wei , Xianwei Fan","doi":"10.1016/j.enzmictec.2025.110762","DOIUrl":"10.1016/j.enzmictec.2025.110762","url":null,"abstract":"<div><div><em>p</em>-Coumaric acid (<em>p</em>-CA) is widely utilized in the food, pharmaceutical and other industries, and has traditionally been derived from plant extraction or chemical synthesis. However, <em>p</em>-CA synthesized by the safe <em>B. subtilis</em> remains poorly explored. In this study, we first engineered a recombinant <em>B. subtilis</em> strain (PBK) capable of synthesizing <em>p</em>-Coumaric acid, achieving an initial yield of 3.81 mg L<sup>−1</sup>. A high-yielding strain PBnprE was then developed through promoter substitution, with a yield reaching 60.92 mg L<sup>−1</sup>, and the yield of PBnprE was further increased to 304.04 mg L<sup>−1</sup> by optimizing fermentation conditions and substrates, showing an 80-fold increase over PBK. The optimized fermentation extract of PBnprE displayed increased antibacterial activity against <em>Staphylococcus aureus</em>, <em>Pseudomonas aeruginosa</em>, and <em>Escherichia coli</em>, alongside enhanced DPPH and ABTS scavenging capabilities. Compared to PBK, the optimized extracts showed 4.81-fold higher in DPPH and 3.47-fold in ABTS scavenging, consistent with improved antioxidant properties driven by the increased presence of <em>p</em>-CA. This study first successfully constructed a high-yield <em>p</em>-CA producing engineered strain in <em>B. subtilis</em>, providing a valuable platform for synthesizing other secondary metabolites.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"192 ","pages":"Article 110762"},"PeriodicalIF":3.7,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145312635","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}

引用次数: 0

Rosmarinic acid production using advanced metabolic engineering strategies 利用先进的代谢工程策略生产迷迭香酸

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2026-01-01 Epub Date: 2025-09-08 DOI: 10.1016/j.enzmictec.2025.110748

Beining Wang , Jintao Lu , Ruzhe Zhang , Jung-Kul Lee , Vipin Chandra Kalia , Chunjie Gong

With improved socio-economic conditions and heightened health awareness, modern consumers now prioritize the nutritional and functional attributes of food over basic satiety. Recently, a high value component, rosmarinic acid synthesized in plants have gained attention as functional food ingredients. Traditional strategy of rosmarinic acid production, including chemical synthesis and plant extraction, are limited by environmental concerns, low yields, and high costs. With the development of biotechnology, metabolic engineering is an alternative strategy for the efficient and cost-effective production of rosmarinic acid. This review focuses on metabolic engineering advances featuring three core strategies: dynamic pathway regulation, cofactor recycling, and microbial co-culture systems. These potential innovations hold great promise for significantly enhancing rosmarinic acid yields. In addition, the review evaluates the economic and technical feasibility of large-scale production, emphasizing the addressing of challenges from traditional production methods.

随着社会经济条件的改善和健康意识的提高，现代消费者现在优先考虑食品的营养和功能属性，而不是基本的饱腹感。近年来，从植物中合成的高价值成分迷迭香酸作为功能性食品原料受到了广泛关注。传统的迷迭香酸生产策略，包括化学合成和植物提取，受到环境问题、低产量和高成本的限制。随着生物技术的发展，代谢工程是高效、低成本生产迷迭香酸的替代策略。本文综述了代谢工程的三个核心策略：动态途径调控、辅因子循环和微生物共培养系统。这些潜在的创新技术有望显著提高迷迭香酸的产量。此外，本文还评估了大规模生产的经济和技术可行性，强调解决传统生产方法带来的挑战。

{"title":"Rosmarinic acid production using advanced metabolic engineering strategies","authors":"Beining Wang , Jintao Lu , Ruzhe Zhang , Jung-Kul Lee , Vipin Chandra Kalia , Chunjie Gong","doi":"10.1016/j.enzmictec.2025.110748","DOIUrl":"10.1016/j.enzmictec.2025.110748","url":null,"abstract":"<div><div>With improved socio-economic conditions and heightened health awareness, modern consumers now prioritize the nutritional and functional attributes of food over basic satiety. Recently, a high value component, rosmarinic acid synthesized in plants have gained attention as functional food ingredients. Traditional strategy of rosmarinic acid production, including chemical synthesis and plant extraction, are limited by environmental concerns, low yields, and high costs. With the development of biotechnology, metabolic engineering is an alternative strategy for the efficient and cost-effective production of rosmarinic acid. This review focuses on metabolic engineering advances featuring three core strategies: dynamic pathway regulation, cofactor recycling, and microbial co-culture systems. These potential innovations hold great promise for significantly enhancing rosmarinic acid yields. In addition, the review evaluates the economic and technical feasibility of large-scale production, emphasizing the addressing of challenges from traditional production methods.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"192 ","pages":"Article 110748"},"PeriodicalIF":3.7,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145027364","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}

引用次数: 0

Identification of a thermostable GH6 family cellulase from chaetomium thermophilum exhibiting high cellobiose and ionic liquid tolerance 嗜热毛菌GH6家族耐热纤维素酶的鉴定，具有高纤维二糖和离子液体耐受性

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2026-01-01 Epub Date: 2025-09-23 DOI: 10.1016/j.enzmictec.2025.110755

Pooja , Sushant K. Sinha , Supratim Datta

Efficient conversion of lignocellulosic biomass into fermentable sugars requires cellulases that are both thermostable and tolerant to inhibitors such as ionic liquids (ILs). Thermostable enzymes are particularly valuable for industrial applications, as they maintain activity at elevated temperatures for extended periods, improve product yield, and reduce process costs. In this study, we cloned and characterized CtCel6C, a GH6 family cellulase, from the thermophilic fungus Thermochaetoides thermophila (Chaetomium thermophilum). CtCel6C exhibited a specific activity of 28 U/mg on carboxymethyl cellulose (CMC-Na) at 55 °C and pH 6.0, retaining 90 % of its specific activity after 20 h at optimum pH and temperature (55 °C and pH 6.0). CtCel6C demonstrated broad substrate specificity, effectively hydrolyzing oligosaccharides, soluble substrates (CMC and barley β-glucan), and insoluble substrates such as Avicel, consistently producing cellobiose as the sole product and outperforming other GH6 cellobiohydrolases from C. thermophilum. Despite having an open active-site cleft due to a lack of a fifteen-residue stretch in the C-terminal loop, CtCel6C retains the biochemical characteristics of a processive cellobiohydrolase rather than those of an endoglucanase from the GH6 family. The enzyme also exhibited high tolerance to cellobiose, glucose, and to 20 % (v/v) of the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C₂mim][OAc]), in both McIlvaine buffer (pH 6.0) and seawater. The remarkable IL tolerance of CtCel6C makes it a promising candidate for integration into industrial enzyme cocktails for biomass saccharification.

将木质纤维素生物质有效转化为可发酵糖需要既耐热又耐离子液体（ILs）等抑制剂的纤维素酶。耐热酶在工业应用中特别有价值，因为它们在高温下长时间保持活性，提高产品产量，降低工艺成本。本研究从嗜热真菌Thermochaetoides thermophila （Chaetomium thermophilum）中克隆并鉴定了GH6家族纤维素酶CtCel6C。在55℃和pH 6.0条件下，CtCel6C对羧甲基纤维素（CMC-Na）的比活性为28 U/mg，在最佳pH和温度（55℃和pH 6.0）下，20 h后，CtCel6C的比活性仍保持90% %。CtCel6C表现出广泛的底物特异性，可以有效水解低聚糖、可溶性底物（CMC和大麦β-葡聚糖）和不溶性底物（如Avicel），始终如一地产生纤维素二糖作为唯一产物，优于其他来自嗜热c菌的GH6纤维素生物水解酶。尽管由于在c端环中缺乏15个残基延伸而具有开放的活性位点裂缝，但CtCel6C保留了过程纤维素生物水解酶的生化特征，而不是来自GH6家族的内切葡聚糖酶的生化特征。在McIlvaine缓冲液（pH 6.0）和海水中，该酶对纤维素二糖、葡萄糖和20% % （v/v）的离子液体1-乙基-3-甲基咪唑醋酸盐（[C2mim][OAc]）均表现出较高的耐受性。CtCel6C卓越的IL耐受性使其成为整合到生物质糖化工业酶混合物中的有希望的候选者。

{"title":"Identification of a thermostable GH6 family cellulase from chaetomium thermophilum exhibiting high cellobiose and ionic liquid tolerance","authors":"Pooja , Sushant K. Sinha , Supratim Datta","doi":"10.1016/j.enzmictec.2025.110755","DOIUrl":"10.1016/j.enzmictec.2025.110755","url":null,"abstract":"<div><div>Efficient conversion of lignocellulosic biomass into fermentable sugars requires cellulases that are both thermostable and tolerant to inhibitors such as ionic liquids (ILs). Thermostable enzymes are particularly valuable for industrial applications, as they maintain activity at elevated temperatures for extended periods, improve product yield, and reduce process costs. In this study, we cloned and characterized <em>Ct</em>Cel6C, a GH6 family cellulase, from the thermophilic fungus <em>Thermochaetoides thermophila</em> (<em>Chaetomium thermophilum</em>). <em>Ct</em>Cel6C exhibited a specific activity of 28 U/mg on carboxymethyl cellulose (CMC-Na) at 55 °C and pH 6.0, retaining 90 % of its specific activity after 20 h at optimum pH and temperature (55 °C and pH 6.0). <em>Ct</em>Cel6C demonstrated broad substrate specificity, effectively hydrolyzing oligosaccharides, soluble substrates (CMC and barley β-glucan), and insoluble substrates such as Avicel, consistently producing cellobiose as the sole product and outperforming other GH6 cellobiohydrolases from <em>C. thermophilum</em>. Despite having an open active-site cleft due to a lack of a fifteen-residue stretch in the C-terminal loop, <em>Ct</em>Cel6C retains the biochemical characteristics of a processive cellobiohydrolase rather than those of an endoglucanase from the GH6 family. The enzyme also exhibited high tolerance to cellobiose, glucose, and to 20 % (v/v) of the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C<sub>2</sub>mim][OAc]), in both McIlvaine buffer (pH 6.0) and seawater. The remarkable IL tolerance of <em>Ct</em>Cel6C makes it a promising candidate for integration into industrial enzyme cocktails for biomass saccharification.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"192 ","pages":"Article 110755"},"PeriodicalIF":3.7,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145217523","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}

引用次数: 0

Enhanced synthesis of alanyl-glutamine dipeptide via molecular modification and process optimization of α-amino acid ester acyltransferase EAET α-氨基酸酯酰基转移酶EAET的分子修饰和工艺优化促进丙氨酰-谷氨酰胺二肽的合成。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2026-01-01 Epub Date: 2025-09-22 DOI: 10.1016/j.enzmictec.2025.110754

Jinao Wei , Xiaoxi Liu , Yifei Gao, Jing-wen Yang, Hong-bin Zhang, Xue-qin Hu

This study presents a significant advancement in the enzymatic synthesis of alanyl-glutamine (Ala-Gln) through the development of a high-performance α-ester acyltransferase (EAET) using molecular engineering. We successfully cloned EAET from Bacillus pumilus, which exhibits low sequence identity (<30 %) compared to other reported acyltransferases (AETs). Using AlphaFold3, we constructed a highly reliable structural model of EAET, validated by metrics such as pLDDT, ipTM, and PAE. Through semi-rational design, we introduced the F330Y mutation, which enhanced enzymatic activity by 1.5-fold and extended the half-life at 30°C by 1.3-fold. Molecular dynamics simulations revealed increased flexibility around residue 330 and additional hydrogen bonds in the F330Y mutant, both of which contribute to its improved catalytic efficiency and thermostability. Response surface methodology was employed to optimize enzyme production conditions, resulting in a maximum enzyme activity of 218.20 U/mL[91.3 U/(mL·OD600)]. Systematic optimization of catalytic parameters, including pH, temperature, substrate ratio, concentration, enzyme dosage, and reaction time, enabled an 82.59 % conversion yield at 300 mM substrate concentration. These results not only surpass previous benchmarks but also demonstrate the industrial potential of engineered EAET variants for Ala-Gln synthesis. This study highlights the successful integration of computational tools with molecular engineering to enhance enzyme performance, offering a greener and more sustainable approach for Ala-Gln production.

本研究利用分子工程技术开发了高性能α-酯酰基转移酶（EAET），在酶促合成丙氨酰-谷氨酰胺（Ala-Gln）方面取得了重大进展。我们成功地从矮芽孢杆菌中克隆了EAET，该菌株具有低序列同源性(

{"title":"Enhanced synthesis of alanyl-glutamine dipeptide via molecular modification and process optimization of α-amino acid ester acyltransferase EAET","authors":"Jinao Wei , Xiaoxi Liu , Yifei Gao, Jing-wen Yang, Hong-bin Zhang, Xue-qin Hu","doi":"10.1016/j.enzmictec.2025.110754","DOIUrl":"10.1016/j.enzmictec.2025.110754","url":null,"abstract":"<div><div>This study presents a significant advancement in the enzymatic synthesis of alanyl-glutamine (Ala-Gln) through the development of a high-performance α-ester acyltransferase (EAET) using molecular engineering. We successfully cloned EAET from <em>Bacillus pumilus</em>, which exhibits low sequence identity (<30 %) compared to other reported acyltransferases (AETs). Using AlphaFold3, we constructed a highly reliable structural model of EAET, validated by metrics such as pLDDT, ipTM, and PAE. Through semi-rational design, we introduced the F330Y mutation, which enhanced enzymatic activity by 1.5-fold and extended the half-life at 30°C by 1.3-fold. Molecular dynamics simulations revealed increased flexibility around residue 330 and additional hydrogen bonds in the F330Y mutant, both of which contribute to its improved catalytic efficiency and thermostability. Response surface methodology was employed to optimize enzyme production conditions, resulting in a maximum enzyme activity of 218.20 U/mL[91.3 U/(mL·OD600)]. Systematic optimization of catalytic parameters, including pH, temperature, substrate ratio, concentration, enzyme dosage, and reaction time, enabled an 82.59 % conversion yield at 300 mM substrate concentration. These results not only surpass previous benchmarks but also demonstrate the industrial potential of engineered EAET variants for Ala-Gln synthesis. This study highlights the successful integration of computational tools with molecular engineering to enhance enzyme performance, offering a greener and more sustainable approach for Ala-Gln production.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"192 ","pages":"Article 110754"},"PeriodicalIF":3.7,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145205964","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}

引用次数: 0

A thermostable cellobiose phosphorylase from Thermoclostridium caenicola and its application to in vitro biotransformation 一种耐热性纤维二糖磷酸化酶及其在体外生物转化中的应用

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2026-01-01 Epub Date: 2025-09-19 DOI: 10.1016/j.enzmictec.2025.110753

Haotian Cheng , Yuan Li , Yi Zhou , Shuke Wu , Yi-Heng P. Job Zhang

Cellobiose phosphorylase (CBP) catalyzes the reversible phosphorolysis of cellobiose to glucose and α-D-glucose-1-phosphate. CBPs play an important role in the in vivo enzymatic utilization of cellulosic materials. Here we discovered a novel CBP from Thermoclostridium caenicola (TcCBP), displaying 50–75 % sequence homology with previously reported CBPs. Recombinant TcCBP was well expressed in E. coli BL21(DE3), with a 1.6-fold increase in soluble expression levels over the widely-used Clostridium thermocellum CBP (CtCBP). This enzyme exhibited broad pH adaptability, maintaining substantial activities across pH 4.0–7.5 in the synthetic direction and pH 5.0–7.5 in the phosphorolytic direction. Compared to CtCBP, TcCBP displayed the superior thermostability and a nearly 100-fold improvement in "Product-to-Enzyme Ratio" (PE value). The biosynthesis of myo-inositol from cellobiose was conducted by using this CBP along with other four thermophilic enzymes (i.e., phosphoglucomutase, inositol 1-phosphate synthase, inositol monophosphatase, and polyphosphate glucokinase) in one pot without step-by-step addition of enzymes. Approximately 96 mM of myo-inositol was produced from 50 mM of cellobiose. These results indicated that this enzyme could be a potential thermophilic enzyme used for the production of value-added biochemicals by in vitro BioTransformation.

纤维素二糖磷酸化酶（CBP）催化纤维素二糖可逆磷酸解生成葡萄糖和α- d -葡萄糖-1-磷酸。CBPs在纤维素材料的体内酶利用中起着重要作用。在这里，我们发现了一个新的CBP从热梭菌caenicola (TcCBP)，显示50 - 75% %序列同源性与先前报道的CBPs。重组TcCBP在大肠杆菌BL21（DE3）中表达良好，可溶性表达量比广泛使用的热胞梭菌CBP （CtCBP）高1.6倍。该酶表现出广泛的pH适应性，在pH 4.0-7.5的合成方向和pH 5.0-7.5的磷酸化方向上保持大量的活性。与CtCBP相比，TcCBP表现出更好的热稳定性，“产物与酶比”（PE值）提高了近100倍。利用该CBP和其他四种嗜热酶（即磷酸葡萄糖化酶、肌醇1-磷酸合成酶、肌醇单磷酸酶和多磷酸葡萄糖激酶）在一个锅中进行了由纤维素糖合成肌醇的生物合成，而无需逐步添加酶。大约96 mM的肌醇由50 mM的纤维二糖产生。这些结果表明，该酶可能是一种潜在的通过体外生物转化生产增值生化产品的嗜热酶。

{"title":"A thermostable cellobiose phosphorylase from Thermoclostridium caenicola and its application to in vitro biotransformation","authors":"Haotian Cheng , Yuan Li , Yi Zhou , Shuke Wu , Yi-Heng P. Job Zhang","doi":"10.1016/j.enzmictec.2025.110753","DOIUrl":"10.1016/j.enzmictec.2025.110753","url":null,"abstract":"<div><div>Cellobiose phosphorylase (CBP) catalyzes the reversible phosphorolysis of cellobiose to glucose and α-D-glucose-1-phosphate. CBPs play an important role in the <em>in vivo</em> enzymatic utilization of cellulosic materials. Here we discovered a novel CBP from <em>Thermoclostridium caenicola</em> (TcCBP), displaying 50–75 % sequence homology with previously reported CBPs. Recombinant TcCBP was well expressed in <em>E. coli</em> BL21(DE3), with a 1.6-fold increase in soluble expression levels over the widely-used <em>Clostridium thermocellum</em> CBP (CtCBP). This enzyme exhibited broad pH adaptability, maintaining substantial activities across pH 4.0–7.5 in the synthetic direction and pH 5.0–7.5 in the phosphorolytic direction. Compared to CtCBP, TcCBP displayed the superior thermostability and a nearly 100-fold improvement in \"Product-to-Enzyme Ratio\" (PE value). The biosynthesis of <em>myo</em>-inositol from cellobiose was conducted by using this CBP along with other four thermophilic enzymes (i.e., phosphoglucomutase, inositol 1-phosphate synthase, inositol monophosphatase, and polyphosphate glucokinase) in one pot without step-by-step addition of enzymes. Approximately 96 mM of <em>myo</em>-inositol was produced from 50 mM of cellobiose. These results indicated that this enzyme could be a potential thermophilic enzyme used for the production of value-added biochemicals by <em>in vitro</em> BioTransformation.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"192 ","pages":"Article 110753"},"PeriodicalIF":3.7,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145109831","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}

引用次数: 0