黄孢原毛平革菌漆酶基因lac1680的克隆、表达及产酶研究  

Cloning,Expression and Enzyme Production of Laccase Gene lac1680 in Phanerochaete chrysosporium

作　　者：陈建军[1] 刘梁涛 曹香林 CHEN Jian-jun;LIU Liang-tao;CAO Xiang-lin(College of Life Sciences, Henan Normal University, Xinxiang 453007;College of Aquaculture, Henan Normal University, Xinxiang 453007)

机构地区：[1]河南师范大学生命科学学院,新乡453007 [2]河南师范大学水产学院,新乡453007

出　　处：《生物技术通报》2018年第4期214-220,共7页Biotechnology Bulletin

基　　金：国家自然科学基金项目(5201049120029);河南省重点科技攻关计划项目(122102210168;152102210081)

摘　　要：以先前筛选到的高产漆酶黄孢原毛平革菌(Phanerochaete chrysosporium)为模板,利用同源克隆技术合成一个全长为1 680 bp的漆酶基因,核苷酸及氨基酸序列比对显示该基因与真菌漆酶基因有较高的同源性,将其命名为lac1680,将该基因连接到构建好的p ET24a载体上,并转入表达菌株BL21 Escherichia coli(DE3)中,经对重组菌预表达的全菌裂解物进行SDS-PAGE检测,获得75 k D目的条带,表明诱导表达成功。随后利用NI-NTA层析柱对洗脱下来的lac1680蛋白进行纯化,纯化回收后,漆酶纯度可达98%以上。通过对比基因工程菌和黄孢原毛平革菌野生菌株不同培养时间产酶活力,结果表明构建好的工程菌活力比原菌酶活力有明显的提高,提高了近39%。In this study,previously screened highly laccase-yielding Phanerochaete chrysosporium as template,homologous cloningtechnology was employed to synthesize a full-length 1680 bp laccase gene. Alignment of nucleotide and amino acid sequences showed that thegene had high homology with fungal laccase gene,named as lac1680. Then the gene was connected to the p ET24 a vector,and transferred tothe expression strain Escherichia coli BL21（DE3）. The whole cell lysates by the pre-expression of the recombinant strain was detected bySDS-PAGE,and 75 k D band was obtained,thus indicating that the gene expressed successfully. NI-NTA column was then used to purify theeluted lac1680 protein,and the purity was over 98%. Comparing the enzyme-producing activities between the gene engineering strain and wild P. chrysosporium strain in different culture time showed that the activity of engineering strain obviously improved by nearly 39% than the wild one.

关 键 词：黄孢原毛平革菌 漆酶基因 异源表达 蛋白纯化 酶活力 

分 类 号：Q78[生物学—分子生物学] Q936