Coprinopsis cinerea 漆酶基因的克隆及其在毕赤酵母中的表达

 本文通过RT-PCR从Coprinopsis cinerea okayama7#130中克隆得到laccase1，应用SignalP3.0Server软件分析其氨基酸序列后设计不含信号肽序列的新引物扩增得到不含信号肽的漆酶基因1（lac1），并构建重组酵母表达载体pPIC9K-lac1，电击转化毕赤酵母GS1115并用甲醇诱导表达，之后研究重组酶的酶学性质。克隆得到的laccase1全长为1593bp，编码530个氨基酸，其中信号肽包含18个氨基酸。SDS-PAGE显示重组蛋白大小约为65KDa。酶学性质研究表明，该酶最适反应温度为45℃，最适pH为4.3。重组漆酶在45℃保存3h后活性基本保持不变，在pH4.0～10.0的范围内稳定性较好。成功分泌表达的漆酶活力达到1.108U/mL。

Cloning of laccase gene form Coprinopsis cinerea and study on its expression in Pichia pastoris

The laccase gene 1 had been cloned by RT-PCR from Coprinopsis cinerea okayama7#130, through analyzing of SignalP3.0 Server, a new primer had been designed that it didn’t have signal peptide sequence for laccase1(lac1) and construction of Pichia pastoris expression plasmid pPIC9K-lac1, then transformed into the Pichia pastoris GS115 and induction expression, studied properties of recombinant enzyme at last. The full length of laccase 1was 1593bp, which coded 530 amino acids and included 18 amino acids of Signal peptide sequence. SDS-PAGE show that obvious protein band was 65 kDa. The optimal fermentature was 45℃ and pH value was 4.3. The activity of restructed laccase remain unchanged basically after it staied in 45℃ for 3 hours, the stability of avticity was good in the scope from 4.0 to 10.0 of pH.The enzyme activity of secretion expression achieve to 1.108 U/mL.