Cloning, purification and characterization of a novel thermostable recombinant tannase from Galactobacillus timonensis.

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology Pub Date : 2024-12-23 DOI:10.1016/j.enzmictec.2024.110575

Jingya Wu, Huan Zeng, Xinyan Zhong, Xi Chen, Peng Zhang, Zeyuan Deng

{"title":"Cloning, purification and characterization of a novel thermostable recombinant tannase from Galactobacillus timonensis.","authors":"Jingya Wu, Huan Zeng, Xinyan Zhong, Xi Chen, Peng Zhang, Zeyuan Deng","doi":"10.1016/j.enzmictec.2024.110575","DOIUrl":null,"url":null,"abstract":"The exorbitant production costs associated with natural tannases pose a significant challenge to their widespread industrial utilization. Microbial expression systems provide a cost-effective method for enzyme production. In this study, a putative gene encoding the subtype B tannase (Gt-Tan) was cloned from Galactobacillus timonensis and expressed heterologously in Escherichia coli BL21 (DE3) cells. The Gt-Tan was purified using metal affinity chromatography and exhibited a monomeric structure with a molecular weight of 55 kDa. Gt-Tan showed optimal activity at a temperature of 50 ℃ and a pH of 6.0. It was also quite thermostable, with approximately 68.3 % and 54.7 % of its maximal activity retained after incubation at 45 ℃ for 2 h and 40 ℃ for 48 h respectively. Addition of Mn2+, Zn2+, Al3+, urea, n-butanol, and dimethylsulfoxide at a low concentration slightly enhanced the activity of Gt-Tan, whereas Cu2+, Fe3+, Fe2+, Co2+, SDS, cetyltrimethylammonium bromide, DTT, Tween 80, and β-mercaptoethanol significantly inhibited its activity. Km and kcat/Km values were estimated to be 0.83 mM and 19.7 s1 mM1 for methyl gallate, 0.67 mM and 65.4 s1 mM1 for propyl gallate, and 0.22 mM and 240.8 s1 mM1 for tannic acid. These results enhanced our understanding of tannase and provided potential sources for applications in the chemical, feed, and food industries.","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"110575"},"PeriodicalIF":3.4000,"publicationDate":"2024-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Enzyme and Microbial Technology","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1016/j.enzmictec.2024.110575","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}

引用次数: 0

Abstract

The exorbitant production costs associated with natural tannases pose a significant challenge to their widespread industrial utilization. Microbial expression systems provide a cost-effective method for enzyme production. In this study, a putative gene encoding the subtype B tannase (Gt-Tan) was cloned from Galactobacillus timonensis and expressed heterologously in Escherichia coli BL21 (DE3) cells. The Gt-Tan was purified using metal affinity chromatography and exhibited a monomeric structure with a molecular weight of 55 kDa. Gt-Tan showed optimal activity at a temperature of 50 ℃ and a pH of 6.0. It was also quite thermostable, with approximately 68.3 % and 54.7 % of its maximal activity retained after incubation at 45 ℃ for 2 h and 40 ℃ for 48 h respectively. Addition of Mn²⁺, Zn²⁺, Al³⁺, urea, n-butanol, and dimethylsulfoxide at a low concentration slightly enhanced the activity of Gt-Tan, whereas Cu²⁺, Fe³⁺, Fe²⁺, Co²⁺, SDS, cetyltrimethylammonium bromide, DTT, Tween 80, and β-mercaptoethanol significantly inhibited its activity. K_m and k_cat/K_m values were estimated to be 0.83 mM and 19.7 s¹ mM¹ for methyl gallate, 0.67 mM and 65.4 s¹ mM¹ for propyl gallate, and 0.22 mM and 240.8 s¹ mM¹ for tannic acid. These results enhanced our understanding of tannase and provided potential sources for applications in the chemical, feed, and food industries.

查看原文本刊更多论文

timmonensis半乳杆菌重组单宁酶的克隆、纯化及特性研究。

与天然鞣酶相关的高昂生产成本对其广泛的工业利用构成了重大挑战。微生物表达系统为酶的生产提供了一种经济有效的方法。本研究从提蒙半乳杆菌中克隆了一个推测为编码B型单宁酶（Gt-Tan）的基因，并在大肠杆菌BL21 （DE3）细胞中异源表达。通过金属亲和层析纯化得到分子量为55 kDa的Gt-Tan单体结构。Gt-Tan在温度50 ℃、pH 6.0时活性最佳。在45 ℃孵育2 h和40 ℃孵育48 h后，其最大活性分别保持在68.3% %和54.7% %左右。低浓度的Mn2+、Zn2+、Al3+、尿素、正丁醇和二甲亚砜对Gt-Tan的活性有微弱的增强作用，而Cu2+、Fe3+、Fe2+、Co2+、SDS、十六烷基三甲基溴化铵、DTT、吐温80和β-巯基乙醇对其活性有显著的抑制作用。没食子酸甲酯的Km和kcat/Km值分别为0.83 mM和19.7 s1 mM1，没食子酸丙酯的Km和kcat/Km值分别为0.67 mM和65.4 s1 mM1，单宁酸的Km和kcat/Km值分别为0.22 mM和240.8 s1 mM1。这些结果增强了我们对单宁酶的理解，并为其在化学、饲料和食品工业中的应用提供了潜在的来源。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Enzyme and Microbial Technology 生物-生物工程与应用微生物

CiteScore

7.60

自引率

5.90%

发文量

142

审稿时长

38 days

期刊介绍： Enzyme and Microbial Technology is an international, peer-reviewed journal publishing original research and reviews, of biotechnological significance and novelty, on basic and applied aspects of the science and technology of processes involving the use of enzymes, micro-organisms, animal cells and plant cells. We especially encourage submissions on: Biocatalysis and the use of Directed Evolution in Synthetic Biology and Biotechnology Biotechnological Production of New Bioactive Molecules, Biomaterials, Biopharmaceuticals, and Biofuels New Imaging Techniques and Biosensors, especially as applicable to Healthcare and Systems Biology New Biotechnological Approaches in Genomics, Proteomics and Metabolomics Metabolic Engineering, Biomolecular Engineering and Nanobiotechnology Manuscripts which report isolation, purification, immobilization or utilization of organisms or enzymes which are already well-described in the literature are not suitable for publication in EMT, unless their primary purpose is to report significant new findings or approaches which are of broad biotechnological importance. Similarly, manuscripts which report optimization studies on well-established processes are inappropriate. EMT does not accept papers dealing with mathematical modeling unless they report significant, new experimental data.