摘要
漆酶是一种含铜的多酚氧化酶,在有机农药残留和木质素的降解方面具有潜在的工业价值。从特异腐质霉Y1(Humicola insolens Y1)中分离到了漆酶基因Lac1,其全长1 803 bp,编码600个氨基酸,与NCBI蛋白数据库比对结果表明,与来源于Chaetomium globosum CBS 148.51(XP_001228806)的多酚氧化酶序列相似性最高为71%,表明是一个新的漆酶基因。将其克隆入毕赤酵母表达载体pPIC9r中,通过PCR和SDS-PAGE检测证实基因已经整合入酵母基因组中且能分泌表达。在3 L发酵罐中,重组Lac1蛋白表达量达到3.79mg/mL发酵液,酶活性达到1.3 U/mL发酵液。酶学性质分析结果显示,漆酶的最适作用温度为65℃,最适反应pH为4.5,且在pH 6～11的范围内酶的相对活力均在70%以上。
Laccase is a copper-containing polyphenol oxidase,which has potential industrial value in the degradation of organic pesticide residues and lignin. In this study,the laccase gene Lac1 was isolated from a Humicola insolens Y1. The 1 803 bp DNA of Lac1 encoded a mature protein with 600 amino acids. The deduced amino acid sequence was aligned with available protein sequences held in the Gen Bank,the results showed that the highest similarity was71% which was the polyphenol oxidase sequence from Chaetomium globosum CBS 148. 51( XP 001228806). The recombinant vector pPIC9 r-Lac1 was constructed and transferred into Pichia pastris to integrate into the yeast genome and the recombinant laccase was secreted expression. It was confirmed by the PCR and SDS-PAGE. In 3 L fermentation,the expression of Lac1 protein reached 3.79 mg/mL,and the enzyme activity reached 1.3 U/mL. The analysis of enzymatic properties showed that the optimum temperature was 65℃,the optimum pH was 4.5,and the relative activity of enzyme was above 70% in pH 6 ~ 11.
作者
张丽洁
徐欣欣
田健
初晓宇
朱宝成
伍宁丰
ZHANG Lijie;XU Xinxin;TIAN Jian;CHU Xiaoyu;ZHU Baocheng;WU Ningfeng(College of Life Sciences,Hebei Agricultural University,Hebei Baoding 071000;Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,China)
出处
《中国农业科技导报》
CAS
CSCD
北大核心
2019年第2期46-53,共8页
Journal of Agricultural Science and Technology
基金
国家863计划项目(2013AA102804)资助
关键词
漆酶
基因克隆
异源表达
毕赤酵母
Laccase
gene cloning
heterologous expression
Pichia pastoris
作者简介
张丽洁,硕士研究生,研究方向为基因工程药物学。E-mail:zhang1013lj@163.com;通信作者:初晓宇,副研究员,博士,主要从事酶工程研究。E-mail:sadylee@163.com;通信作者:朱宝成,教授,博士,主要从事植物基因工程研究。E-mail:zhu2222@126.com.
