纤维二糖差向异构酶在毕赤酵母中的表达及酶学性质研究  

Expression of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus in Pichia pastoris and its enzymatic characterization

作　　者：韩亮[1] 杨瑞金[1,2] 赵伟[1] 沈秋云[1] 张文斌[1] 华霄[1] 

机构地区：[1]江南大学食品学院,江苏无锡214122 [2]江南大学食品科学与技术国家重点实验室,江苏无锡214122

出　　处：《工业微生物》2016年第1期16-21,共6页Industrial Microbiology

基　　金：十二五国家科技支撑计划项目(编号:2011BAD23B03);国家自然科学基金面上项目重点项目(编号:31230057)

摘　　要：首先将来源于Caldicellulosiruptor saccharolyticus的纤维二糖差向异构酶基因CsCEm进行密码子优化,然后进行全基因合成,再将其引入到载体pPIC9K中,构建重组质粒pPIC9K-Cs CEm并转化入毕赤酵母GS115,得到酵母工程菌株。经微孔板筛选、摇瓶筛选得到酶活最高的重组工程菌GS115-4-19。该菌株经甲醇诱导144 h后,摇瓶发酵液上清酶活达到0.42 U/m L。酶学性质研究结果表明：该酶的最适pH为7.5,且在pH 6.0~8.0范围内相对酶活都在80%以上;在pH 4~9的缓冲液中放置24 h后仍保持原酶活力的80%以上;最适温度为80℃,在60℃~80℃保温30 min后,相对酶活在80%以上。动力学研究结果表明该酶对底物乳糖的Km和Vmax分别为（120.27±9.96）mmol/L和（1.035±0.05）mmol/L/min。纤维二糖差向异构酶在毕赤酵母中的成功表达为生物酶法合成乳果糖提供了重要参考。In this study, the cellobiose 2-epimerase（ Cs CE） from Caldicellulosiruptor saccharolyticus codon was optimized,and then the optimized gene Cs CEm was synthesized. Based on the gene engineering technology,the yeast expression vector pPIC9K-CsCEm was constructed,and then transformed into chromosome of P. pastoris GS115 strain.Recombinant strain（ GS115-4-19） was screened by microplate and shake flask,and cultured in shake flask under the optimal conditions for enzyme production. The maximum activity of the culture supernatant was 0. 42 U / m L. Furthermore,the enzymatic properties of recombined Cs CE was characterized in detail. Results showed that recombinant Cs CE had the high relative enzyme activity at pH 6. 0 ~ 8. 0,especially at 7. 5. Over 80% of enzyme activity was remained even after storing in the buffer solution at pH 4. 0 ~ 9. 0 for 24 h. The optimal temperature was around 80 ℃. It had the relative activity more than 80% at 60 ℃ ~ 80 ℃. When lactose was used as substrate,Kmand Vmaxof Cs CE were 120. 27 ± 9. 96 mmol / L and 1. 035 ± 0. 05 mmol / L / min. Expression of Cs CE in Pichia pastoris provided reference for synthesis of lactulose by enzymatic method.

关 键 词：纤维二糖差向异构酶 毕赤酵母 性质 

分 类 号：TQ925[轻工技术与工程—发酵工程]