β-甘露聚糖酶AuMan5Aloop/H321G在乳酸克鲁维酵母中的高效表达  

High-Level Expression of A β-Mannanase AuMan5Aloop/H321G in Kluyveromyces lactis GG799

作　　者：唐诗涵[1] 李雪晴[1] 袁风娇[1] 李剑芳[1] 邬敏辰 TANG Shihan;LI Xueqing;YUAN Fengjiao;LI Jianfang;WU Minchen(School of Food Science and Technology,Jiangnan University,Wuxi 214122,China;Wuxi Medical School,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学食品学院,江苏无锡214122 [2]江南大学无锡医学院,江苏无锡214122

出　　处：《食品与生物技术学报》2019年第7期26-33,共8页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目（31271811）;江苏省普通高校研究生科研创新计划项目（SJLX16-0472）

摘　　要：为制备食品级β-甘露聚糖酶,将来源于宇佐美曲霉(Aspergillus usamii)的糖苷水解酶5家族β-甘露聚糖酶突变体基因(AuMan5Aloop/H321G)克隆至表达质粒pKLAC1信号肽α-MF下游,构建重组表达质粒pKLAC1-AuMan5Aloop/H321G,并电转化乳酸克鲁维酵母(Kluyveromyces lactis)GG799。采用半乳糖诱导表达和酶活性分析获得了高水平表达的重组β-甘露聚糖酶突变酶(reAuMan5Aloop/H321G)的转化子GG799/AuMan5Aloop/H321G,其产酶活力为54.9 U/mL。SDS-PAGE分析显示,reAuMan5Aloop/H321G的表观相对分子质量约为55 000。经YPG培养基初始pH、摇瓶装液量以及诱导剂的种类、初始质量浓度和添加方式的初步优化,GG799/AuMan5Aloop/H321G所产酶活力较工艺优化前提高了254.6%,从而实现了reAuMan5Aloop/H321G在乳酸克鲁维酵母中的高效表达。To prepare the food-grade β-mannanase,a β-mannanase mutant gene Auman5Aloop/H321G was expressed in Kluyveromyces lactis GG799. Auman5Aloop/H321G,gene constructed based on a glycoside hydrolase family 5 β-mannanase gene(Auman5 A) from Aspergillus usamii,was inserted into the signal peptide α-MF downstream of the expression plasmid pKLAC1,forming a recombinant plasmid p KLAC1-Auman5 Aloop/H321 G. Then,pKLAC1-Auman5Aloop/H321G was electroporated into Kluyveromyces lacti GG799. After induction by galactose and enzyme activity analysis,a transformant GG799/Auman5 Aloop/H321Gexpressing the highest recombinant β-mannanase(reAuMan5Aloop/H321G) activity of 54.9 U/mL was selected for subsequent studies. The apparent molecular weight of reAuMan5Aloop/H321Gwas estimated to be about 55 000 by SDS-PAGE analysis.Furthermore,the β-mannanase activity produced by GG799/Auman5Aloop/H321Gwas increased by 254.6% after primary optimization of the initial pH and loading volume of YPG medium as well as the species,initial concentration and supplementary mode of inducer. As a result,the high-level expression of reAuMan5Aloop/H321Gin K. lactis was actualized at the optimized fermentation process.

关 键 词：Β-甘露聚糖酶 乳酸克鲁维酵母 表达 诱导剂 发酵工艺 

分 类 号：Q786[生物学—分子生物学]