| 本文已被：浏览 22次 下载 0次 |
|
| 耐热微泡菌（Microbulbifer thermotolerans）几丁质酶的基因克隆表达及酶学性质分析 |
|
欧阳玉滢1, 林梅2, 王巧贞2, 卢波2, 吕心悦3, 黄俊2, 周蓉2, 梁士颉2, 覃秋容2, 王青艳2, 黄庶识2, 廖思明2
|
|
|
| (1.广西大学化学化工学院;2.广西科学院;3.南宁学院食品与质量工程学院) |
|
| 摘要: |
| 本研究克隆表达耐热微泡菌Microbulbifer thermotolerans YLW106菌株的几丁质酶基因并探究重组酶的酶学性质。从耐热微泡菌Microbulbifer thermotolerans YLW106菌株中克隆几丁质酶基因Chi2375，构建重组质粒pET30a-Chi2375并在大肠杆菌Escherichia coli BL21(DE3)中诱导表达；将重组蛋白经镍柱、Q柱和分子筛纯化后，进行酶学性质研究。实验结果表明，以胶体几丁质为底物，重组酶Chi2375的最适反应温度Topt为55.0℃、最适pH值为8.0、Vmax和Km分别为7.49±0.25U·mg?1、33.75± 1.24 mg·mL?1 。同时Chi2375与胶体几丁质反应的产物为纯度93%以上的几丁二糖，在医药和食品上具有潜在的应用前景。 |
| 关键词: 耐热微泡菌 几丁质酶 克隆表达 纯化 酶学性质 |
| DOI： |
| 投稿时间：2024-02-27修订日期：2024-04-09 |
| 基金项目:国家自然科学基金项目（32060216）,中央引导地方科技发展资金项目(桂科ZY23055011),广西科学院科研发展(2021YFJ1204),广西科技重大专项(桂科AA22096023),广西重大科技创新基地建设项目子课题(2022-36-Z06-01) |
|
| Gene Cloning, Expression and Characterization of chitinase from Microbulbifer thermotolerans YLW106 |
|
Ouyang Yuying1, Lin Mei2, Wang Qiaozhen2, LuBo2, Lv Xinyue3, Huang Jun2, Zhou Rong2, Liang Shijie2, Qin Qiurong2, Wang Qingyan2, Huang Shushi2, Liao Siming2
|
| (1.School of Chemistry and Chemical Engineering, Guangxi University;2.Guangxi Academy of Sciences;3.College of Food and Quality Engineering, Nanning University) |
| Abstract: |
| In this study, the chitinase gene of Microbulbifer thermotolerans YLW106 strain was cloned and expressed, and the enzymatic properties. The chitinase gene Chi2375 was cloned from Microbulbifer thermotolerans YLW106, and there combinant expression plasmid pET30a-Chi2375 was constructed and expressed in Escherichia coli BL21(DE3). The recombinant protein was purified by Ni-Fast, Hiload 16/10 Q Sepharose High Performance and Superdex 75 Increase 10/300 GL, and the enzymatic properties were studied. The results showed that the optimum temperature(Topt) was 55℃ and the optimum pH value was 8.0. Its Vmax and Km values were (7.49±0.25) U·mg?1 and (33.75± 1.24) mg·mL?1 , respectively. Meanwhile, the product of the reaction of Chi2375 with colloidal chitosan is chitosan with more than 90% purity, which has potential applications in medicine and food. |
| Key words: Microbulbifer thermotolerans chitinase expression cloning purification enzymatic properties |
