生物学杂志

生物学杂志 ›› 2021, Vol. 38 ›› Issue (4): 12-.doi: 10. 3969 / j. issn. 2095-1736. 2021. 04. 012

• 研究报告 • 上一篇    下一篇

谷氨酸棒杆菌漆酶基因NCgl0908的克隆、表达及功能

  

  1. 1.江南大学粮食发酵工艺与技术国家工程实验室,无锡214122;2.江南大学工业生物技术教育部重点实验室,无锡214122;3.江南大学生物工程学院,无锡214122

  • 出版日期:2021-08-18 发布日期:2021-08-18
  • 通讯作者: 白仲虎,教授,研究方向为生物医药蛋白的高效表达及发酵过程研究,E-mail: baizhonghu@jiangnan.edu.cn
  • 作者简介:司雅楠,硕士研究生,研究方向为生物化学与分子生物学,E-mail:978386343@qq.com
  • 基金资助:
    基金项目:国家自然科学基金项目(编号21808082,21878124)

Cloning,expression and functional study of laccasegene NCgl0908 from Corynebacteriumglutamicum #br#

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  1. 1. National Engineering Laboratory of Cereal Fermentation Technology, Jiangnan University, Wuxi 214112, China; 2. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; 3. School of Biotechnology, Jiangnan University, Wuxi 214122, China
  • Online:2021-08-18 Published:2021-08-18

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摘要： 漆酶(Laccase)是多铜氧化酶家族中的主要成员,能够催化多种酚类和芳香类化合物的氧化,因此具有很大的工业应摘用潜要力。虽然对漆酶的研究及应用已获得一定进展,但主要集中在植物和真菌漆酶,对细菌漆酶的研究较少。研究从谷氨酸棒杆菌(Corynebacteriumglutamicum)中分离到一个漆酶基因NCgl0908,其全长1542bp,编码513个氨基酸。将其氨基酸序列与几种已鉴定的细菌和真菌漆酶进行序列比对,发现它们的4个铜离子结合位点的氨基酸高度保守,推测NCgl0908是一个新的漆酶基因。把NCgl0908基因克隆入表达载体pColdⅡ,并在大肠杆菌BL21中诱导表达。利用镍柱亲和层析法纯化表达产物,并以2,2′-联氮-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)作为底物检测其漆酶活性以及酶动力学参数,发现其漆酶活性为25. 4 U/ mL,Km为2. 22×10-4mol / L,Vmax为2. 432×10-6mol / (L·min)。

关键词: 谷氨酸棒杆菌, 细菌漆酶, 胞外表达, 酶动力学参数, 大肠杆菌中图分类号

Abstract: Laccase is a main member of the multi-copper oxidase family, which can catalyze the oxidation of a variety of phenols andaromatic compounds, so it has great industrial application potential. Although some progress has been made in the research and appli-cation of laccase, it is mainly focused on plant and fungal laccase, and less research on bacterial laccase. In this study, a laccase geneNCgl0908was isolated fromCorynebacteriumglutamicum, with a total length of 1 542 bp and encoding 513 amino acids. Its amino acid sequence was compared with several identified bacterial and fungal laccases, and the four copper ion binding sites were found to behighly conserved. Therefore,NCgl0908is speculated as a new laccase gene.NCgl0908was cloned into the expression vector pColdⅡ and induced to express inE.coliBL21. The expression product was purified by nickel column affinity chromatography. Its laccase activi-ty and enzyme kinetic parameters were measured by using ABTS as a substrate, and the result showed that the laccase activity was 25. 4U / mL,Kmwas 2.22 ×10-4mol /L, andVmaxwas 2.432 ×10-6mol /(L·min).

Key words: Corynebacteriumglutamicum, bacterial laccase, extracellular expression, enzyme kinetic parameters;Escherichiacoli

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