Enzyme and Microbial Technology最新文献_第2页

Integrated bio-nano remediation of produced water using Alcanivorax borkumensis and GQDs-modified ZnO/La₂O₃ nanocomposites 硼砂和gqds改性ZnO/La₂O₃纳米复合材料对采出水的综合生物纳米修复

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-31 DOI: 10.1016/j.enzmictec.2025.110810

Tahseen Hameed Khlaif

This study presents a novel, synergistic bioremediation platform for petroleum-contaminated produced water, utilizing Alcanivorax borkumensis in conjunction with a hybrid nanocomposite of graphene quantum dots (GQDs), zinc oxide (ZnO), and lanthanum oxide (La₂O₃). The nanocomposite was synthesized via ultrasonic-assisted co-precipitation and hydrothermal methods, exhibiting high surface area and photocatalytic potential. The bacterium was isolated from oil-field equipment and identified based on 16S rRNA sequencing, showing selective affinity for hydrocarbons in saline conditions. Integrated systems were optimized by evaluating nanocomposite dose, contact time, and light intensity. Results revealed a clear synergy: GQDs enhanced bacterial adhesion and minimized cytotoxicity, while ZnO/La₂O₃ contributed to robust radical formation under light exposure. Combined systems achieved over 98 % total petroleum hydrocarbon (TPH) and 99 % polycyclic aromatic hydrocarbon (PAH) removal, outperforming individual components. Material characterizations (BET, FTIR, XRD, SEM) confirmed the composite’s stability and biointerface compatibility. Comparative trials across bacteria-only, nanomaterial-only, and hybrid systems validated the superiority of the bio-nanocomposite configuration. The approach merges enzymatic and photocatalytic degradation in a biologically resilient matrix, providing a scalable, environmentally sound strategy for industrial water treatment. These findings offer a comprehensive framework for future application of microbe–nanomaterial hybrids in complex pollutant removal.

这项研究提出了一种新的、协同的石油污染采出水生物修复平台，利用Alcanivorax borkumensis与石墨烯量子点（GQDs）、氧化锌（ZnO）和氧化镧（La₂O₃）的混合纳米复合材料结合。通过超声辅助共沉淀法和水热法合成了具有高比表面积和光催化潜力的纳米复合材料。该细菌从油田设备中分离得到，经16S rRNA测序鉴定，在盐水条件下对烃类具有选择性亲和力。通过评价纳米复合材料的剂量、接触时间和光强对集成系统进行了优化。结果显示了明显的协同作用：GQDs增强了细菌的粘附性并最小化了细胞毒性，而ZnO/La₂O₃有助于在光照下形成强大的自由基。联合系统的总石油烃（TPH）去除率超过98% %，多环芳烃（PAH）去除率超过99% %，优于单个组分。材料表征（BET， FTIR， XRD， SEM）证实了复合材料的稳定性和生物界面相容性。仅细菌、仅纳米材料和混合系统的对比试验验证了生物纳米复合结构的优越性。该方法将酶和光催化降解结合在一个生物弹性基质中，为工业水处理提供了一种可扩展的、环保的策略。这些发现为未来微生物-纳米复合材料在复杂污染物去除中的应用提供了一个全面的框架。

{"title":"Integrated bio-nano remediation of produced water using Alcanivorax borkumensis and GQDs-modified ZnO/La₂O₃ nanocomposites","authors":"Tahseen Hameed Khlaif","doi":"10.1016/j.enzmictec.2025.110810","DOIUrl":"10.1016/j.enzmictec.2025.110810","url":null,"abstract":"<div><div>This study presents a novel, synergistic bioremediation platform for petroleum-contaminated produced water, utilizing <em>Alcanivorax borkumensis</em> in conjunction with a hybrid nanocomposite of graphene quantum dots (GQDs), zinc oxide (ZnO), and lanthanum oxide (La₂O₃). The nanocomposite was synthesized via ultrasonic-assisted co-precipitation and hydrothermal methods, exhibiting high surface area and photocatalytic potential. The bacterium was isolated from oil-field equipment and identified based on 16S rRNA sequencing, showing selective affinity for hydrocarbons in saline conditions. Integrated systems were optimized by evaluating nanocomposite dose, contact time, and light intensity. Results revealed a clear synergy: GQDs enhanced bacterial adhesion and minimized cytotoxicity, while ZnO/La₂O₃ contributed to robust radical formation under light exposure. Combined systems achieved over 98 % total petroleum hydrocarbon (TPH) and 99 % polycyclic aromatic hydrocarbon (PAH) removal, outperforming individual components. Material characterizations (BET, FTIR, XRD, SEM) confirmed the composite’s stability and biointerface compatibility. Comparative trials across bacteria-only, nanomaterial-only, and hybrid systems validated the superiority of the bio-nanocomposite configuration. The approach merges enzymatic and photocatalytic degradation in a biologically resilient matrix, providing a scalable, environmentally sound strategy for industrial water treatment. These findings offer a comprehensive framework for future application of microbe–nanomaterial hybrids in complex pollutant removal.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"195 ","pages":"Article 110810"},"PeriodicalIF":3.7,"publicationDate":"2025-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145881384","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

Naringinase: A comprehensive review on its characteristics, production, and biotechnological applications 柚皮苷酶的特性、生产及生物技术应用综述。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-27 DOI: 10.1016/j.enzmictec.2025.110808

Nidhi S. Hallikeri , Deepak A. Yaraguppi , Bhavya S. Gangadharappa , Nitin Mantri

Naringinase is a bifunctional enzyme exhibiting both α-L-rhamnosidase and β-D-glucosidase activity, primarily utilized for the hydrolysis of bitter compounds like naringin in citrus products. This enzymatic action yields naringenin, a bioactive flavonoid known for its significant antioxidant, anti-inflammatory, and cardioprotective properties. Given its dual enzymatic function and substrate specificity, naringinase has garnered substantial interest across the food, pharmaceutical, and broader biotechnological sectors. This comprehensive review critically examines recent advancements concerning naringinase, focusing on its microbial production, optimization strategies in fermentation, purification methods, and various immobilization technologies. Aspergillus niger and Penicillium decumbens are identified as the predominant microbial sources for naringinase production, with solid-state fermentation demonstrating economic and cost-effectiveness over submerged fermentation. The application of diverse immobilization techniques, including adsorption, covalent binding, and encapsulation, has been shown to significantly enhance the enzyme's catalytic stability, reusability, and overall efficiency in applications such as juice debittering and the development of functional foods. While advanced biocatalysis approaches like recombinant expression and enzyme engineering offer promising improvements in catalytic properties, the persistent challenges related to production scalability and purification costs warrant further assessment. The integration of sustainable solid-state fermentation with cutting-edge nanomaterials holds considerable promise, opening new avenues for naringinase applications in environmental biotechnology and personalized medicine.

柚皮苷酶是一种具有α- l -鼠李糖苷酶和β- d -葡萄糖苷酶活性的双功能酶，主要用于柑橘产品中柚皮苷等苦味化合物的水解。这种酶的作用产生柚皮素，一种具有生物活性的类黄酮，以其显著的抗氧化、抗炎和心脏保护特性而闻名。鉴于其双重酶功能和底物特异性，柚皮苷酶在食品、制药和更广泛的生物技术领域获得了极大的兴趣。本文综述了柚皮苷酶的最新进展，重点介绍了柚皮苷酶的微生物生产、发酵优化策略、纯化方法和各种固定化技术。黑曲霉和躺倒青霉被认为是柚皮苷酶生产的主要微生物来源，固态发酵比水下发酵更经济、更经济。各种固定技术的应用，包括吸附、共价结合和包封，已被证明可以显著提高酶的催化稳定性、可重复使用性和整体效率，如果汁脱脂和功能食品的开发。虽然重组表达和酶工程等先进的生物催化方法有望改善催化性能，但与生产可扩展性和纯化成本相关的持续挑战需要进一步评估。可持续固态发酵与尖端纳米材料的结合具有相当大的前景，为柚皮苷酶在环境生物技术和个性化医疗中的应用开辟了新的途径。

{"title":"Naringinase: A comprehensive review on its characteristics, production, and biotechnological applications","authors":"Nidhi S. Hallikeri , Deepak A. Yaraguppi , Bhavya S. Gangadharappa , Nitin Mantri","doi":"10.1016/j.enzmictec.2025.110808","DOIUrl":"10.1016/j.enzmictec.2025.110808","url":null,"abstract":"<div><div>Naringinase is a bifunctional enzyme exhibiting both α-<span>L</span>-rhamnosidase and β-<span>D</span>-glucosidase activity, primarily utilized for the hydrolysis of bitter compounds like naringin in citrus products. This enzymatic action yields naringenin, a bioactive flavonoid known for its significant antioxidant, anti-inflammatory, and cardioprotective properties. Given its dual enzymatic function and substrate specificity, naringinase has garnered substantial interest across the food, pharmaceutical, and broader biotechnological sectors. This comprehensive review critically examines recent advancements concerning naringinase, focusing on its microbial production, optimization strategies in fermentation, purification methods, and various immobilization technologies. <em>Aspergillus niger</em> and <em>Penicillium decumbens</em> are identified as the predominant microbial sources for naringinase production, with solid-state fermentation demonstrating economic and cost-effectiveness over submerged fermentation. The application of diverse immobilization techniques, including adsorption, covalent binding, and encapsulation, has been shown to significantly enhance the enzyme's catalytic stability, reusability, and overall efficiency in applications such as juice debittering and the development of functional foods. While advanced biocatalysis approaches like recombinant expression and enzyme engineering offer promising improvements in catalytic properties, the persistent challenges related to production scalability and purification costs warrant further assessment. The integration of sustainable solid-state fermentation with cutting-edge nanomaterials holds considerable promise, opening new avenues for naringinase applications in environmental biotechnology and personalized medicine.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"195 ","pages":"Article 110808"},"PeriodicalIF":3.7,"publicationDate":"2025-12-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145877903","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 activity and stability of thermostable endoglucanase Dictyoglomus turgidum DtCelA with multiple strategies 多种策略增强热稳定性内切葡聚糖酶（Dictyoglomus turgidum DtCelA）活性和稳定性。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-27 DOI: 10.1016/j.enzmictec.2025.110809

Xiaotong Kang , Hang Yuan , Weimeng Li, Yun Hu, Fubao Sun

Thermophilic endoglucanases are of big promise in enzymatic degradation of lignocellulosic substrate, yet their limited catalytic efficiency remains a major barrier to industrial application. This study focused on the improvement of activity and stability of thermostable endoglucanase DtCelA from Dictyoglomus turgidum with a three-stage cascade engineering strategy that integrated model-guided site-directed mutagenesis, disulfide bond introduction, and carbohydrate-binding module (CBM) fusion. Among the variants, the triple mutant S25D/T265C/A305C fused with CBM2 (S25D/T265C/A305C-A-CBM2) exhibited a 90.56 % increase in specific activity toward CMC relative to the wild type, together with a 2.97-fold longer half-life at 75 °C and a 5.3-fold longer at 80 °C. This mutant also displayed 3.0- and 5.4-fold higher hydrolytic activity against Avicel and konjac glucomannan, respectively. On natural lignocellulosic substrates including filter paper, sugarcane bagasse, and corn stover, the mutant also showed potential application. The mutated endoglucanase DtCelA was powerful with enhanced catalytic performance and thermostability after multicascade engineering, which can provide a robust framework for the rational design of cellulases that are optimized for high-temperature biorefinery process.

嗜热性内切葡聚糖酶在木质纤维素底物的酶解中具有很大的前景，但其有限的催化效率仍然是工业应用的主要障碍。本研究采用三级级联工程策略，将模型引导的位点定向诱变、二硫键引入和碳水化合物结合模块（CBM）融合结合在一起，重点研究了从瘤胃Dictyoglomus turgidum中提高热稳定型内切葡聚糖酶DtCelA的活性和稳定性。其中，与CBM2融合的三突变体S25D/T265C/A305C- a -CBM2 （S25D/T265C/A305C- a -CBM2）对CMC的比活性比野生型提高了90.56 %，75℃时半衰期延长了2.97倍，80℃时半衰期延长了5.3倍。该突变体对Avicel和魔芋葡甘露聚糖的水解活性分别提高了3.0倍和5.4倍。在包括滤纸、甘蔗渣和玉米秸秆在内的天然木质纤维素基质上，该突变体也显示出潜在的应用前景。突变后的内切葡聚糖酶DtCelA具有较强的催化性能和热稳定性，可为优化高温生物炼制工艺的纤维素酶的合理设计提供坚实的框架。

{"title":"Enhanced activity and stability of thermostable endoglucanase Dictyoglomus turgidum DtCelA with multiple strategies","authors":"Xiaotong Kang , Hang Yuan , Weimeng Li, Yun Hu, Fubao Sun","doi":"10.1016/j.enzmictec.2025.110809","DOIUrl":"10.1016/j.enzmictec.2025.110809","url":null,"abstract":"<div><div>Thermophilic endoglucanases are of big promise in enzymatic degradation of lignocellulosic substrate, yet their limited catalytic efficiency remains a major barrier to industrial application. This study focused on the improvement of activity and stability of thermostable endoglucanase <em>Dt</em>CelA from <em>Dictyoglomus turgidum</em> with a three-stage cascade engineering strategy that integrated model-guided site-directed mutagenesis, disulfide bond introduction, and carbohydrate-binding module (CBM) fusion. Among the variants, the triple mutant S25D/T265C/A305C fused with CBM2 (S25D/T265C/A305C-A-CBM2) exhibited a 90.56 % increase in specific activity toward CMC relative to the wild type, together with a 2.97-fold longer half-life at 75 °C and a 5.3-fold longer at 80 °C. This mutant also displayed 3.0- and 5.4-fold higher hydrolytic activity against Avicel and konjac glucomannan, respectively. On natural lignocellulosic substrates including filter paper, sugarcane bagasse, and corn stover, the mutant also showed potential application. The mutated endoglucanase <em>Dt</em>CelA was powerful with enhanced catalytic performance and thermostability after multicascade engineering, which can provide a robust framework for the rational design of cellulases that are optimized for high-temperature biorefinery process.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"195 ","pages":"Article 110809"},"PeriodicalIF":3.7,"publicationDate":"2025-12-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145862488","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

Enzymatic synthesis of sucrose esters: Advances and challenges in high-efficiency and regioselective catalysis 酶法合成蔗糖酯：高效和区域选择性催化的进展和挑战

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-26 DOI: 10.1016/j.enzmictec.2025.110806

Lei Zhao , Jinghui Lai , Shenghui Zhang , Mengqi Dai , Enxiang Zong , Youqiang Xu , Xiuting Li

As an eco-friendly and non-toxic nonionic surfactant, enzymatically synthesized sucrose esters (SEs) are increasingly valued across the food, cosmetic, and pharmaceutical industries due to their environmentally benign production characteristics. However, current enzymatic synthesis faces several fundamental constraints, like limited diversity of enzyme resources restricting process versatility, insufficient catalytic activity and thermostability of enzymes leading to suboptimal reaction efficiency, and prohibitive enzyme production costs hindering industrial scalability. To address these critical challenges, this review presents a systematic summarize of SEs synthetic technologies and their emerging applications, with particular emphasis on elucidating enzymatic synthetic mechanisms. Due to limited enzyme resources, we innovatively propose a high-throughput bioinformatics-driven enzyme mining platform that employs SEs synthase sequences as molecular probes for screening the SEs synthases, identifying previously uncharacterized enzyme candidates with potentially enhanced catalytic efficiencies. Through systematic bottleneck analysis and a forward-looking perspective, our work establishes a theoretical foundation for developing cost-effective enzymatic processes while providing a valuable repository of potential enzyme candidates for the first time. These findings offer useful guidelines for promoting enzymatic SEs production.

酶促合成蔗糖酯（SEs）作为一种环保无毒的非离子表面活性剂，因其对环境无害的生产特性，在食品、化妆品和制药等行业越来越受到重视。然而，目前的酶合成面临着几个基本的限制，如酶资源的有限多样性限制了工艺的通用性，酶的催化活性和热稳定性不足导致反应效率不理想，酶的生产成本过高阻碍了工业可扩展性。为了解决这些关键的挑战，本文综述了SEs合成技术及其新兴应用的系统总结，特别强调阐明酶合成机制。由于酶资源有限，我们创新地提出了一种高通量生物信息学驱动的酶挖掘平台，该平台采用SEs合酶序列作为分子探针筛选SEs合酶，识别以前未表征的具有潜在增强催化效率的候选酶。通过系统的瓶颈分析和前瞻性的观点，我们的工作为开发具有成本效益的酶促工艺奠定了理论基础，同时首次提供了有价值的潜在候选酶库。这些发现为促进酶促SEs的产生提供了有用的指导。

{"title":"Enzymatic synthesis of sucrose esters: Advances and challenges in high-efficiency and regioselective catalysis","authors":"Lei Zhao , Jinghui Lai , Shenghui Zhang , Mengqi Dai , Enxiang Zong , Youqiang Xu , Xiuting Li","doi":"10.1016/j.enzmictec.2025.110806","DOIUrl":"10.1016/j.enzmictec.2025.110806","url":null,"abstract":"<div><div>As an eco-friendly and non-toxic nonionic surfactant, enzymatically synthesized sucrose esters (SEs) are increasingly valued across the food, cosmetic, and pharmaceutical industries due to their environmentally benign production characteristics. However, current enzymatic synthesis faces several fundamental constraints, like limited diversity of enzyme resources restricting process versatility, insufficient catalytic activity and thermostability of enzymes leading to suboptimal reaction efficiency, and prohibitive enzyme production costs hindering industrial scalability. To address these critical challenges, this review presents a systematic summarize of SEs synthetic technologies and their emerging applications, with particular emphasis on elucidating enzymatic synthetic mechanisms. Due to limited enzyme resources, we innovatively propose a high-throughput bioinformatics-driven enzyme mining platform that employs SEs synthase sequences as molecular probes for screening the SEs synthases, identifying previously uncharacterized enzyme candidates with potentially enhanced catalytic efficiencies. Through systematic bottleneck analysis and a forward-looking perspective, our work establishes a theoretical foundation for developing cost-effective enzymatic processes while providing a valuable repository of potential enzyme candidates for the first time. These findings offer useful guidelines for promoting enzymatic SEs production.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"195 ","pages":"Article 110806"},"PeriodicalIF":3.7,"publicationDate":"2025-12-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145881383","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

Efficient production of active recombinant cholesterol oxidase from Rhodococcus erythropolis in Escherichia coli via his-tag assisted refolding strategy for cholesterol oxidation 利用his标签辅助胆固醇氧化的重折叠策略在大肠杆菌中高效生产重组红红球菌活性胆固醇氧化酶

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-24 DOI: 10.1016/j.enzmictec.2025.110805

Meka Saima Perdani , Dwini Normayulisa Putri , Dita Ariyanti , Cintiya Septa Hasannah , Fitri Yuliasari , Ibnu Maulana Hidayatullah , Heri Hermansyah , Yosuke Fukutani , Masafumi Yohda

This study reports the cloning, expression, and characterization of recombinant cholesterol oxidase (ChOx) from Rhodococcus erythropolis in Escherichia coli BL21(DE3). The ChOx gene was inserted into the pET23b(+) vector using primers designed with site-specific modifications that enabled His-tag activation, expression, and purification by nickel-affinity chromatography to yield a protein of ∼55 kDa. A urea-gradient dialysis refolding protocol was employed to recover active enzyme from inclusion bodies. The purified enzyme exhibited a specific activity of 13.68 U/mg, representing a 2.5-fold improvement over previously reported recombinant ChOx yields with a recovery yield of 76.16 %. Cholesterol oxidation assays optimized enzyme concentration, substrate concentration, and temperature. Optimum activity occurred at 0.4 mg/mL enzyme and 20 mM cholesterol, achieving > 80 % substrate conversion, while the enzyme retained stability up to 50 °C. This work demonstrates a His-tag-assisted refolding strategy that efficiently produces active recombinant Rhodococcus erythropolis ChOx (RhoChOx). The optimization of catalytic parameters highlights the enzyme’s robustness under diverse conditions. These findings establish RhoChOx as a promising biocatalyst for industrial cholesterol oxidation and provide a methodological framework applicable to other recombinant oxidases.

本研究报道了红红红球菌重组胆固醇氧化酶（ChOx）在大肠杆菌BL21（DE3）中的克隆、表达和特性分析。将ChOx基因插入pET23b（+）载体，使用经过位点特异性修饰设计的引物，通过镍亲和层析使his标签激活、表达和纯化，产生约55 kDa的蛋白。采用尿素梯度透析复折叠方法从包涵体中回收活性酶。纯化酶的比活性为13.68 U/mg，比先前报道的重组ChOx产量提高2.5倍，回收率为76.16 %。胆固醇氧化试验优化酶浓度、底物浓度和温度。酶的最佳活性为0.4 mg/mL和20 mM胆固醇，达到>； 80 %底物转化率，酶在50°C下保持稳定性。这项工作证明了his标签辅助的重折叠策略，有效地产生活性重组红polis红球菌ChOx （RhoChOx）。催化参数的优化突出了酶在不同条件下的鲁棒性。这些发现确立了RhoChOx作为工业胆固醇氧化生物催化剂的前景，并为其他重组氧化酶提供了适用的方法框架。

{"title":"Efficient production of active recombinant cholesterol oxidase from Rhodococcus erythropolis in Escherichia coli via his-tag assisted refolding strategy for cholesterol oxidation","authors":"Meka Saima Perdani , Dwini Normayulisa Putri , Dita Ariyanti , Cintiya Septa Hasannah , Fitri Yuliasari , Ibnu Maulana Hidayatullah , Heri Hermansyah , Yosuke Fukutani , Masafumi Yohda","doi":"10.1016/j.enzmictec.2025.110805","DOIUrl":"10.1016/j.enzmictec.2025.110805","url":null,"abstract":"<div><div>This study reports the cloning, expression, and characterization of recombinant cholesterol oxidase (ChOx) from Rhodococcus erythropolis in Escherichia coli BL21(DE3). The ChOx gene was inserted into the pET23b(+) vector using primers designed with site-specific modifications that enabled His-tag activation, expression, and purification by nickel-affinity chromatography to yield a protein of ∼55 kDa. A urea-gradient dialysis refolding protocol was employed to recover active enzyme from inclusion bodies. The purified enzyme exhibited a specific activity of 13.68 U/mg, representing a 2.5-fold improvement over previously reported recombinant ChOx yields with a recovery yield of 76.16 %. Cholesterol oxidation assays optimized enzyme concentration, substrate concentration, and temperature. Optimum activity occurred at 0.4 mg/mL enzyme and 20 mM cholesterol, achieving > 80 % substrate conversion, while the enzyme retained stability up to 50 °C. This work demonstrates a His-tag-assisted refolding strategy that efficiently produces active recombinant Rhodococcus erythropolis ChOx (RhoChOx). The optimization of catalytic parameters highlights the enzyme’s robustness under diverse conditions. These findings establish RhoChOx as a promising biocatalyst for industrial cholesterol oxidation and provide a methodological framework applicable to other recombinant oxidases.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"195 ","pages":"Article 110805"},"PeriodicalIF":3.7,"publicationDate":"2025-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145838175","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

Cellulose biosynthesis in nature and In Vitro: mechanisms and challenges 纤维素在自然界和体外的生物合成：机制和挑战

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-24 DOI: 10.1016/j.enzmictec.2025.110804

Shijing Sun , Huasha Liang , Zehai Lei , Jialei Xu , Siqi Wang , Zhongyuan Zhao , Tongming Yin

This review systematically compares the biosynthetic mechanisms of cellulose in plants and bacteria, focusing on the structure and function of their distinct enzymatic complexes. In bacteria, cellulose synthesis is driven by the BcsAB transmembrane complex alongside auxiliary proteins like BcsC and BcsD. In plants, rosette-shaped cellulose synthase complexes (CSCs), which are composed of multiple CesA catalytic subunits, synthesize cellulose microfibrils. This process is critically coordinated by auxiliary proteins, such as KOR1 and COBRA-LIKE proteins, which associate with or regulate the CSCs to ensure proper microfibril formation and crystallization. A central challenge in the field is the in vitro production of cellulose I, the native crystalline form with superior mechanical properties. While strategies such as enzyme extraction and heterologous expression in Escherichia coli enable cellulose production, they typically yield the less desirable cellulose II allomorph and face issues of low yield and instability. This review synthesizes current knowledge on key enzymes, in vitro synthesis methods, and resulting cellulose characteristics to identify key obstacles and future pathways toward the efficient bio-manufacturing of cellulose I for sustainable applications.

本文系统地比较了植物和细菌中纤维素的生物合成机制，重点介绍了它们不同的酶复合物的结构和功能。在细菌中，纤维素的合成是由BcsAB跨膜复合体以及BcsC和BcsD等辅助蛋白驱动的。在植物中，由多个纤维素合成酶催化亚基组成的玫瑰状纤维素合成酶复合物（CSCs）可合成纤维素微原纤维。这一过程由辅助蛋白（如KOR1和COBRA-LIKE蛋白）进行关键协调，它们与CSCs相关或调节CSCs以确保适当的微纤维形成和结晶。该领域的一个核心挑战是纤维素I的体外生产，这是一种具有优越机械性能的天然晶体形式。虽然酶提取和在大肠杆菌中异种表达等策略能够生产纤维素，但它们通常会产生不太理想的纤维素II异型，并面临低产量和不稳定的问题。本文综述了目前在关键酶、体外合成方法和纤维素特性方面的知识，以确定纤维素I的高效生物制造和可持续应用的关键障碍和未来途径。

{"title":"Cellulose biosynthesis in nature and In Vitro: mechanisms and challenges","authors":"Shijing Sun , Huasha Liang , Zehai Lei , Jialei Xu , Siqi Wang , Zhongyuan Zhao , Tongming Yin","doi":"10.1016/j.enzmictec.2025.110804","DOIUrl":"10.1016/j.enzmictec.2025.110804","url":null,"abstract":"<div><div>This review systematically compares the biosynthetic mechanisms of cellulose in plants and bacteria, focusing on the structure and function of their distinct enzymatic complexes. In bacteria, cellulose synthesis is driven by the BcsAB transmembrane complex alongside auxiliary proteins like BcsC and BcsD. In plants, rosette-shaped cellulose synthase complexes (CSCs), which are composed of multiple CesA catalytic subunits, synthesize cellulose microfibrils. This process is critically coordinated by auxiliary proteins, such as KOR1 and COBRA-LIKE proteins, which associate with or regulate the CSCs to ensure proper microfibril formation and crystallization. A central challenge in the field is the <em>in vitro</em> production of cellulose I, the native crystalline form with superior mechanical properties. While strategies such as enzyme extraction and heterologous expression in <em>Escherichia coli</em> enable cellulose production, they typically yield the less desirable cellulose II allomorph and face issues of low yield and instability. This review synthesizes current knowledge on key enzymes, <em>in vitro</em> synthesis methods, and resulting cellulose characteristics to identify key obstacles and future pathways toward the efficient bio-manufacturing of cellulose I for sustainable applications.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"195 ","pages":"Article 110804"},"PeriodicalIF":3.7,"publicationDate":"2025-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145881448","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

The potential and innovative applications of CRISPR gene editing technology in enzyme gene development CRISPR基因编辑技术在酶基因开发中的潜力和创新应用。

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-20 DOI: 10.1016/j.enzmictec.2025.110799

Youmin Zhu

The CRISPR gene editing technology is simple in design and highly efficient, making it the most widely used gene editing tool today. At present, CRISPR gene editing technology has shown a certain application value in enzyme development, but its application potential has not been fully developed. CRISPR gene editing technology can not only be used to knockin enzyme genes and knockout genes that are not conducive to enzyme expression, but can also be applied to single-base editing of enzyme genes, tandem sgRNA for multi-enzyme gene editing, sgRNA library for enzyme screening, endogenous enzyme gene modification, transcriptional activation or inhibition of enzyme gene expression, and fluorescence imaging of enzyme genes. Especially, this review innovatively proposes for the first time that CRISPR gene editing technology can be used for site specific fusion of enzyme genes, cell surface display of endogenous enzymes, and knockin of super long DNA for simultaneous expression of multiple enzymes, providing new ideas for maximizing the value of CRISPR gene editing technology in enzyme development in the future.

CRISPR基因编辑技术设计简单，效率高，是当今应用最广泛的基因编辑工具。目前，CRISPR基因编辑技术在酶的开发中已显示出一定的应用价值，但其应用潜力尚未得到充分开发。CRISPR基因编辑技术不仅可以用于敲入酶基因和敲除不利于酶表达的基因，还可以用于酶基因的单碱基编辑、串联sgRNA用于多酶基因编辑、sgRNA文库用于酶筛选、内源性酶基因修饰、酶基因表达的转录激活或抑制、酶基因的荧光成像等。特别是，本文创新性地首次提出了CRISPR基因编辑技术可用于酶基因的位点特异性融合、内源性酶的细胞表面展示、超长DNA的敲入以同时表达多种酶，为未来最大化CRISPR基因编辑技术在酶开发中的价值提供了新的思路。

引用次数: 0

Levansucrase from Gluconacetobacter diazotrophicus. Key residues involved in levan synthesis 重氮养糖醋杆菌的左旋蔗糖酶。利凡合成中涉及的关键残基

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-12 DOI: 10.1016/j.enzmictec.2025.110802

Ana G. Martínez , Yamira Quintero , Duniesky Martínez , Alexis Musacchio , Odet Céspedes , Carmen Menéndez

Levansucrase from Gluconacetobacter diazotrophicus (LsdA, EC 2.4.1.10) produces short-chain fructooligosaccharides, mainly 1-kestotriose, and levan polysaccharide from sucrose. By random mutagenesis, twenty-five LsdA residues were identified as being crucial for levan synthesis, with the substitution of these residues resulting in a decrease or complete elimination of levan synthesis. Fourteen residues (R171, H172, S228, R229, T243, F304, N306, E314, E327, R334, A369, D398, H419, and G432) were identified within the active site cavity, while eleven were dispersed across the protein. Saturation mutagenesis of H172 and R171 (-1 subsite) showed that the H172E/P mutant primarily exhibited sucrose hydrolysis, while the H172S and R171K-H172S variants were less affected in transfructosylation. Furthermore, HPAEC-PAD analysis revealed that the H172S and R171K-H172S variants synthesized 1-kestotriose, 6-kestotriose, 6G-kestotriose, and 1,1-kestotetraose. These variants had specific activity values similar to those of the native LsdA. However, the synthesis of 1,6-kestotetraose was found to be compromised, indicating a loss of the processive mechanism, suggesting that these variants have lost the ability to elongate via β-(2→6) links.

重氮营养葡萄糖醋杆菌（glucconacetobacter diazotrophicus, LsdA, EC 2.4.1.10）的左旋蔗糖酶（Levansucrase）产生短链低聚果糖，主要是1-酮三糖，并从蔗糖中产生左旋多糖。通过随机诱变，鉴定出25个对levan合成至关重要的LsdA残基，这些残基的替代导致levan合成的减少或完全消除。在活性位点空腔内鉴定出14个残基（R171、H172、S228、R229、T243、F304、N306、E314、E327、R334、A369、D398、H419和G432）， 11个残基分散在整个蛋白中。H172和R171（-1亚位）的饱和诱变表明，H172E/P突变体主要表现为蔗糖水解，而H172S和R171K-H172S突变体在转果糖基化方面受影响较小。此外，HPAEC-PAD分析显示，H172S和R171K-H172S变体合成1-酮三糖、6-酮三糖、6g -酮三糖和1,1-酮四糖。这些变体具有与本地LsdA相似的特定活性值。然而，1,6-酮四糖的合成被发现受到损害，表明过程机制的丧失，这表明这些变体已经失去了通过β-（2→6）连接延长的能力。

{"title":"Levansucrase from Gluconacetobacter diazotrophicus. Key residues involved in levan synthesis","authors":"Ana G. Martínez , Yamira Quintero , Duniesky Martínez , Alexis Musacchio , Odet Céspedes , Carmen Menéndez","doi":"10.1016/j.enzmictec.2025.110802","DOIUrl":"10.1016/j.enzmictec.2025.110802","url":null,"abstract":"<div><div>Levansucrase from <em>Gluconacetobacter diazotrophicus</em> (LsdA, EC 2.4.1.10) produces short-chain fructooligosaccharides, mainly 1-kestotriose, and levan polysaccharide from sucrose. By random mutagenesis, twenty-five LsdA residues were identified as being crucial for levan synthesis, with the substitution of these residues resulting in a decrease or complete elimination of levan synthesis. Fourteen residues (R171, H172, S228, R229, T243, F304, N306, E314, E327, R334, A369, D398, H419, and G432) were identified within the active site cavity, while eleven were dispersed across the protein. Saturation mutagenesis of H172 and R171 (-1 subsite) showed that the H172E/P mutant primarily exhibited sucrose hydrolysis, while the H172S and R171K-H172S variants were less affected in transfructosylation. Furthermore, HPAEC-PAD analysis revealed that the H172S and R171K-H172S variants synthesized 1-kestotriose, 6-kestotriose, 6G-kestotriose, and 1,1-kestotetraose. These variants had specific activity values similar to those of the native LsdA. However, the synthesis of 1,6-kestotetraose was found to be compromised, indicating a loss of the processive mechanism, suggesting that these variants have lost the ability to elongate via β-(2→6) links.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"195 ","pages":"Article 110802"},"PeriodicalIF":3.7,"publicationDate":"2025-12-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145753851","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

Microbial-enzyme co-fermentation of low-grade tobacco: Metagenomics and metabolomic insights into flavor formation 低品位烟草的微生物-酶共发酵：元基因组学和代谢组学对风味形成的见解

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-11 DOI: 10.1016/j.enzmictec.2025.110803

Ming Shu , Huijie Xue , Yang Yang , Xiao Zhang , Shitou Li , Tengfei Bian , Kailong Yuan , Chunping Xu

Microbial-enzyme co-fermentation effectively enhances the quality of low-grade tobacco leaves quality, but the underlying mechanisms of flavor formation remain unclear. This study investigated the dynamics and relationships of microbial communities and volatile aroma metabolites during low-grade tobacco leaves fermentation through metagenomics and headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS). Results showed that during microbial-enzyme co-fermentation, the tobacco leaves fermented for four days (D4) exhibited the highest levels of total sugars and reducing sugars, the peak total content of aroma metabolites, and the best sensory quality. Pseudomonadota, Bacillota, and Ascomycota were dominant microorganisms during fermentation. During the initial stage (D1–D4), Saccharomyces was the dominant genus, which was subsequently displaced by Pantoea at D5. This microbial succession coincided with a decline in sensory quality, indicating its crucial role in shaping flavor evolution during co-fermentation. During microbial-enzyme co-fermentation process, a total of 46 volatile metabolites were detected in low-grade tobacco leaves. Among them, seven esters with high variable important in projection values and strong microbial correlations were identified as characteristic aroma metabolites, including ethyl phenylacetate, benzyl acetate, phenylethyl acetate, ethyl myristate, ethyl palmitate, ethyl oleate, and methyl linolenate. Gene function annotation revealed carbohydrate metabolism was the most abundant, followed by amino acid metabolism. Spearman correlation analysis elucidated the formation mechanism of characteristic ester metabolites. Specifically, short-chain esters correlated with glycerolipid and amino acid metabolism, while long-chain esters linked to glycolysis and fatty-acid biosynthetic pathways.

微生物-酶共发酵可有效提高低质烟叶品质，但其风味形成机制尚不清楚。本研究采用宏基因组学、顶空固相微萃取-气相色谱-质谱联用技术研究了低度烟叶发酵过程中微生物群落与挥发性香气代谢物的动态变化及其相互关系。结果表明，发酵4 d （D4）烟叶总糖和还原糖含量最高，香气代谢产物总含量最高，感官品质最佳。假单胞菌、杆状杆菌和子囊菌是发酵过程中的优势菌群。在初始阶段（D1-D4）， Saccharomyces是优势属，随后在D5被Pantoea取代。这种微生物演替与感官品质的下降相吻合，表明其在共发酵过程中形成风味演变的关键作用。在微生物-酶共发酵过程中，低品位烟叶共检测到46种挥发性代谢物。其中，7种具有高投影值重要变量和强微生物相关性的酯类被鉴定为特征香气代谢物，包括苯基乙酸乙酯、乙酸苄酯、乙酸苯乙酯、肉豆酸乙酯、棕榈酸乙酯、油酸乙酯和亚麻酸甲酯。基因功能注释显示碳水化合物代谢最丰富，其次是氨基酸代谢。Spearman相关分析阐明了特征性酯代谢产物的形成机制。具体来说，短链酯类与甘油脂和氨基酸代谢相关，而长链酯类与糖酵解和脂肪酸生物合成途径相关。

{"title":"Microbial-enzyme co-fermentation of low-grade tobacco: Metagenomics and metabolomic insights into flavor formation","authors":"Ming Shu , Huijie Xue , Yang Yang , Xiao Zhang , Shitou Li , Tengfei Bian , Kailong Yuan , Chunping Xu","doi":"10.1016/j.enzmictec.2025.110803","DOIUrl":"10.1016/j.enzmictec.2025.110803","url":null,"abstract":"<div><div>Microbial-enzyme co-fermentation effectively enhances the quality of low-grade tobacco leaves quality, but the underlying mechanisms of flavor formation remain unclear. This study investigated the dynamics and relationships of microbial communities and volatile aroma metabolites during low-grade tobacco leaves fermentation through metagenomics and headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS). Results showed that during microbial-enzyme co-fermentation, the tobacco leaves fermented for four days (D4) exhibited the highest levels of total sugars and reducing sugars, the peak total content of aroma metabolites, and the best sensory quality. <em>Pseudomonadota</em>, <em>Bacillota</em>, and <em>Ascomycota</em> were dominant microorganisms during fermentation. During the initial stage (D1–D4), <em>Saccharomyces</em> was the dominant genus, which was subsequently displaced by <em>Pantoea</em> at D5. This microbial succession coincided with a decline in sensory quality, indicating its crucial role in shaping flavor evolution during co-fermentation. During microbial-enzyme co-fermentation process, a total of 46 volatile metabolites were detected in low-grade tobacco leaves. Among them, seven esters with high variable important in projection values and strong microbial correlations were identified as characteristic aroma metabolites, including ethyl phenylacetate, benzyl acetate, phenylethyl acetate, ethyl myristate, ethyl palmitate, ethyl oleate, and methyl linolenate. Gene function annotation revealed carbohydrate metabolism was the most abundant, followed by amino acid metabolism. Spearman correlation analysis elucidated the formation mechanism of characteristic ester metabolites. Specifically, short-chain esters correlated with glycerolipid and amino acid metabolism, while long-chain esters linked to glycolysis and fatty-acid biosynthetic pathways.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"194 ","pages":"Article 110803"},"PeriodicalIF":3.7,"publicationDate":"2025-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145733639","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 unique highly efficient, thermostable and multi-substrate specific galactanase (AtGH53) from Acetivibrio thermocellus cleaving both β (1,4)- and β (1,6)- linked galactans 一种独特的高效、耐热和多底物特异性半乳糖酶（AtGH53），来自热细胞活动弧菌，可切割β(1,4)-和β(1,6)-连接的半乳糖

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-12-08 DOI: 10.1016/j.enzmictec.2025.110801

Shreya Biswas , Ardhendu Mandal , Carlos M.G.A. Fontes , Arun Goyal

The gene encoding endo-β-1,4-galactanase, AtGH53 from Acetivibrio thermocellus was cloned, expressed and the encoded soluble protein was biochemically characterized. Purified AtGH53 showed molecular mass of approximately, 36 kDa, an optimum temperature 70°C and half-life of 15 h at 70°C. AtGH53 displayed stability in acidic and alkaline pH ranges, with an optimum pH 7.5. AtGH53 activity increased over 30 % by Ni or Co²⁺ ions. AtGH53 exhibited broad substrate specificity, displaying the highest activity with potato pectic-galactan with V_max of 1432 U.mg⁻¹ and K_M of 1.2 mg.mL⁻¹ . TLC and HPLC analyses of potato galactan hydrolysis by AtGH53 showed initially the endo-lytic cleaving property and later shifting to exo-lytic mode. This was confirmed by the release of β-1,4-linked galacto-oligosaccharides of higher degrees of polymerization (DP>3) from potato galactan in first 2 h, followed by accumulation of galactobiose and galactose up to 24 h. In contrast, hydrolysis of larch-arabinogalactan by AtGH53 (specific activity, 148.6 U.mg⁻¹) resulted in the release of β-1,6-galactobiose from branches of the polymer. This indicated that AtGH53 also exhibits lower efficiency in hydrolyzing β-1,6-galactan of arabinogalactan via an exo-mode of action. The broad substrate specificity, pH stability and thermostability of AtGH53 make it a versatile enzyme for biotechnological applications.

克隆、表达了热细胞活动弧菌内切-β-1,4-半乳糖酶（endo-β-1,4-半乳糖酶）编码基因AtGH53，并对编码的可溶性蛋白进行了生化表征。纯化后的AtGH53分子量约为36 kDa，最适温度为70℃，70℃时半衰期为15 h。AtGH53在酸性和碱性范围内均表现出稳定性，最适pH值为7.5。Ni 或Co2+离子使AtGH53活性增加30% %以上。AtGH53具有广泛的底物特异性，对马铃薯果胶半乳的活性最高，Vmax为1432 U。mg¹ 和KM 1.2 mg. ml⁻¹ 。AtGH53对马铃薯半乳氨酸水解的TLC和HPLC分析显示，半乳氨酸最初具有内解裂解性质，随后转变为外解模式。马铃薯半乳糖在前2 h内释放出较高聚合度的β-1,4-连接半乳糖低聚糖（DP>3），随后在24 h内积累半乳糖和半乳糖，证实了这一点。相比之下，AtGH53水解落叶松阿拉伯半乳聚糖(比活性，148.6 U。Mg−1)导致β-1,6-半乳糖二糖从聚合物的分支中释放。这表明AtGH53通过外显作用模式水解阿拉伯半乳聚糖中的β-1,6-半乳聚糖的效率也较低。AtGH53广泛的底物特异性、pH稳定性和热稳定性使其成为生物技术应用的多功能酶。

{"title":"A unique highly efficient, thermostable and multi-substrate specific galactanase (AtGH53) from Acetivibrio thermocellus cleaving both β (1,4)- and β (1,6)- linked galactans","authors":"Shreya Biswas , Ardhendu Mandal , Carlos M.G.A. Fontes , Arun Goyal","doi":"10.1016/j.enzmictec.2025.110801","DOIUrl":"10.1016/j.enzmictec.2025.110801","url":null,"abstract":"<div><div>The gene encoding endo-<em>β</em>-1,4-galactanase, <em>At</em>GH53 from <em>Acetivibrio thermocellus</em> was cloned, expressed and the encoded soluble protein was biochemically characterized. Purified <em>At</em>GH53 showed molecular mass of approximately, 36 kDa, an optimum temperature 70°C and half-life of 15 h at 70°C. <em>At</em>GH53 displayed stability in acidic and alkaline pH ranges, with an optimum pH 7.5. <em>At</em>GH53 activity increased over 30 % by Ni or Co<sup>2+</sup> ions. <em>At</em>GH53 exhibited broad substrate specificity, displaying the highest activity with potato pectic-galactan with <em>V</em><sub><em>max</em></sub> of 1432 U.mg⁻¹ and <em>K</em><sub><em>M</em></sub> of 1.2 mg.mL⁻¹ . TLC and HPLC analyses of potato galactan hydrolysis by <em>At</em>GH53 showed initially the endo-lytic cleaving property and later shifting to exo-lytic mode. This was confirmed by the release of β-1,4-linked galacto-oligosaccharides of higher degrees of polymerization (DP>3) from potato galactan in first 2 h, followed by accumulation of galactobiose and galactose up to 24 h. In contrast, hydrolysis of larch-arabinogalactan by <em>At</em>GH53 (specific activity, 148.6 U.mg<sup>−1</sup>) resulted in the release of β-1,6-galactobiose from branches of the polymer. This indicated that <em>At</em>GH53 also exhibits lower efficiency in hydrolyzing β-1,6-galactan of arabinogalactan <em>via</em> an exo-mode of action. The broad substrate specificity, pH stability and thermostability of <em>At</em>GH53 make it a versatile enzyme for biotechnological applications.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"194 ","pages":"Article 110801"},"PeriodicalIF":3.7,"publicationDate":"2025-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145733640","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