肺炎克雷伯氏菌漆酶基因的克隆及其在毕赤酵母中的高效表达  

Cloning of the Laccase Gene from Klebsiella pneumoniae and its Efficient Expression in Pichia pastoris

作　　者：王建玲[1] 桂爽 付禹 张妤彤 郑东 路福平[1] 刘逸寒[1] WANG Jianling;GUI Shuang;FU Yu;ZAHNG Yutong;ZHENG Dong;LU Fuping;LIU Yihan(Key Laboratory of Industrial Fermentation Microbiology,Ministry of Education,Tianjin Key Laboratory of IndustrialMicrobiology,National Engineering Laboratory for Industrial Enzymes,College of Biotechnology,Tianjin University ofScience & Technology,Tianjin 300457,China)

机构地区：[1]工业发酵微生物教育部重点实验室天津市工业微生物重点实验室工业酶国家工程实验室天津科技大学生物工程学院,天津300457

出　　处：《天津科技大学学报》2019年第1期12-17,共6页Journal of Tianjin University of Science & Technology

基　　金：天津市科技计划资助项目(16YFZCSY01040;17JCYBJC23700);大学生实验室创新基金资助项目(1604A207)

摘　　要：以肺炎克雷伯氏菌(Klebsiellapneumoniae)基因组为模板,通过PCR扩增得到其漆酶基因lac;基于毕赤酵母密码子偏爱性优化后,得到新型漆酶基因lacm;将其与大肠杆菌–毕赤酵母(E. coli-P. pastoris)穿梭表达载体pPIC9K连接,构建重组质粒pPIC9K-lacm;将该质粒转化毕赤酵母(Pichiapastoris)GS115中,实现该漆酶的胞外分泌表达,发酵液中重组漆酶(rLACM)活力达0.37 U/m L.经对rLACM酶学性质分析表明:rLACM的最适温度为70℃,最适pH为8.0,在30～70℃、pH 5.0～9.0活力稳定.The gene of lac was obtained from the genomic DNA of Klebsiella pneumoniae through PCR amplification.A novel laccase gene lacm was obtained based on the yeast codon bias optimization.It was inserted into an E.coli-P.pastoris shuttle vector pPIC9K to construct recombinant plasmid pPIC9K-lacm.The recombinant plasmid was then transformed into Pichia pastoris GS115.The recombinant laccase was successfully expressed in P.pastoris and secreted into the culture medium.In optimized fermentation conditions,the activity of the recombinant laccase(rLACM)in fermentation broth was up to 0.37 U/mL.The optimal enzyme activity of rLACM was observed at pH 8.0 and 70℃temperature.Stability studies showed that rLACM was stable when temperature was 30-70℃and pH 5.0-9.0.

关 键 词：漆酶 毕赤酵母 异源表达 酶学性质 

分 类 号：Q814.4[生物学—生物工程]