蜜蜂α-淀粉酶基因在酿酒酵母中的表达  

Expression of α-amylase gene from Apis mellifera L. in Saccharomyces cerevisiae

作　　者：李蓬茸 曾维斯 彭芳[1] 吴菁 李鑫 闫达中[1] LI Peng-rong;ZENG Wei-si;PENG Fang;WU Jing;LI Xin;YAN Da-zhong(School of Life Science and Technology,Wuhan Polytechnic University,Wuhan 430023,China)

机构地区：[1]武汉轻工大学生命科学与技术学院,武汉430023

出　　处：《武汉轻工大学学报》2022年第6期53-59,共7页Journal of Wuhan Polytechnic University

摘　　要：α-淀粉酶是一种可以水解淀粉中α-1,4-糖苷键的水解酶,它将大分子的淀粉水解为小分子的葡萄糖。对来自蜜蜂的α-淀粉酶进行密码子优化和全基因合成,将合成后的基因克隆至穿梭载体pYES2-α-factor,转化进宿主菌INVSc1。通过碘染法筛选鉴定阳性克隆子,确定重组菌株最佳诱导后产酶时间为22 h,利用DNS法确定该酶最适pH为9.0,最适温度为50℃,在最适条件下(28℃,诱导时间22 h)诱导,重组菌表达α-淀粉酶最大产酶浓度为2 809.1 U/mL。诱导表达收集其上清粗酶液后,经SDS-PAGE电泳,测定其分子量大约为60 kDa,比预期分子量稍大,可能与糖基化有关。α-amylase is a hydrolase that hydrolyses the α-1,4-glycosidic bond in starches, which hydrolyses large starches into small glucose molecules. In this study, the codon optimisation and full gene synthesis of α-amylase from Apis mellifera L. was carried out, and the synthesized gene was cloned into the expression vector pYES2-α-factor and transformed into the host INVSc1. The positive clone was identified by iodine staining. The optimal time for enzyme production after induction of recombinatant strains was determined to be 22 h. The maximum enzyme concentration of the recombinant α-amylase can be as high as 2809.1 U/mL when induced under the optimum conditions(after induction for 22 h at 28 ℃). After the induction of expression and collection of the supernatant crude enzyme solution, the molecular weight of interest protein was determined to be approximately 60 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),and the molecular weight was slightly larger than expected, which may be attributed to glycosylation.

关 键 词：蜜蜂 Α-淀粉酶 密码子优化 酿酒酵母 

分 类 号：TQ9251[轻工技术与工程—发酵工程]