摘要
为明确黑芝麻多酚氧化酶的酶学性质,利用大肠杆菌Escherichia coli原核表达了黑芝麻多酚氧化酶(Black sesame polyphenol oxidase,BsPPO)。将合成的基因构建至pMAL-c5x载体,并在大肠杆菌中进行表达,对重组蛋白进行分离纯化及融合标签切除,获得的BsPPO蛋白用于酶学性质探究。结果表明,合成的Bsppo基因1 752 bp,编码585个氨基酸,理论蛋白分子量为65.3 kDa;构建的pMAL-c5x-Bsppo重组质粒在大肠杆菌Escherichia coli BL21(DE3)中可溶表达了MBP-BsPPO蛋白;酶切去除MBP融合标签后对BsPPO进行了酶学性质研究,结果表明BsPPO的最适温度和pH分别为25℃和4.0,在低温和弱酸性环境中有较好的稳定性。短时间低强度的光照和Cu;可激活BsPPO的活性,Zn;和Ca;能抑制其活性。BsPPO可催化单酚、二酚以及三酚类化合物,对L-酪氨酸以及香草酸表现出较高的催化活性,此外BsPPO还对黑芝麻中含有的2-甲氧基肉桂酸、吲哚3-羧酸和根皮素表现出良好的催化活性。研究结果为黑芝麻多酚氧化酶酶学特性的明确奠定了理论基础。
To investigate the enzyme properties of the black sesame polyphenol oxidase (BsPPO),a synthesized Bsppo gene was cloned into the vector pMAL-c5x and expressed in E.coli.Subsequently,the MBP fusion label in the recombinant protein was removed by protease digestion after affinity purification.The synthesized Bsppo gene contained 1 752 bp which encodes585 amino acids with a deduced molecular weight of 65.3 kDa.Transformation of the recombinant vector into E.coli BL21(DE3) resulted in soluble expression of the fusion protein MBP-BsPPO.The enzymatic properties of the recombinant BsPPO was investigated after MBP fusion tag excision followed by affinity purification.The results demonstrated that the optimal temperature and pH for BsPPO was 25℃ and 4.0,respectively.BsPPO exhibited a good stability under low temperature and acidic environment.Low-intensity short-term light exposure increased the activity of BsPPO.Cu;could improve the activity of BsPPO while Zn;and Ca;showed the opposite effect.BsPPO could catalyze the oxidation of monophenols,diphenols,and triphenols,and exhibited good catalytic activity on l-tyrosine and vanillic acid.Moreover,BsPPO exhibited high catalytic activity on black sesame metabolites,including 2-methoxy cinnamic acid,indole-3-carboxylic acid and phloretin.These results may serve as a basis for further characterization of BsPPO.
作者
叶彩燕
刘光
杜念
张瑞芬
刘磊
贾栩超
张名位
Caiyan Ye;Guang Liu;Nian Du;Ruifen Zhang;Lei Liu;Xuchao Jia;Mingwei Zhang(College of Food Science,South China Agricultural University,Guangzhou 510640,Guangdong,China;Key Laboratory of Functional Foods,Ministry of Agriculture and Rural Affairs,Guangdong Key Laboratory of Agricultural Products Processing,Sericultural&Agri-Food Research Institute Guangdong Academy of Agricultural Sciences,Guangzhou 510610,Guangdong,China)
出处
《生物工程学报》
CAS
CSCD
北大核心
2021年第12期4395-4405,共11页
Chinese Journal of Biotechnology
基金
广东特支计划项目(No.2019BT02N112)
广东省农业科学院十四五学科团队建设项目(No.202108TD)
科技创新战略专项资金-高水平农科院建设(Nos.R2018PY-JC002,R2017YJ-YB2006,R2018QD-081)资助。
关键词
黑芝麻多酚氧化酶
大肠杆菌
原核表达
亲和纯化
酶学性质
black sesame polyphenol oxidase
Escherichia coli
prokaryotic expression
affinity purification
enzymatic properties