米曲霉葡聚糖酶基因(AoEGLAI)在毕赤酵母中的表达研究及其酶学特性分析  

Expression and characterization of recombinant endo-β-1,4-glucanase from Aspergillus oryzae in Pichia pastoris

作　　者：何永梅[1] 王波[2] 李大伟[1] 田永生[2] 彭日荷[2] 姚泉洪[2] 

机构地区：[1]上海海洋大学食品学院,上海201306 [2]上海市农业科学院生物技术研究所,上海201106

出　　处：《上海农业学报》2016年第3期18-23,共6页Acta Agriculturae Shanghai

基　　金：上海市农业委员会重点项目(No.2011-1-8,2013D-8)

摘　　要：采用PTDS方法人工合成米曲霉葡聚糖酶基因(AoEGLAI),通过电击将其转化到毕赤酵母中表达,并采用DNS法对重组酶的酶学性质进行分析。结果表明:该酶耐酸但不耐热,最适反应温度为50℃,最适pH为3.0,在30—45℃和pH4—8时内切葡聚糖酶可保持90%以上酶活力,在底物CMC存在时其热稳定性有一定的提高。10 mmol/L的金属离子Cu^(2+)、Ca^(2+)、Zn^+、Gr^(3+)、Fe^(2+)和Fe^(3+)对酶有激活作用,Mn^(2+)对酶则起抑制作用。该酶对胰蛋白酶有一定的抗性,对胃蛋白酶抗性相对较差。The AoEGLAI gene was chemically synthesized by PTDS（PCR-based-two-steps DNA synthesis）strategy in this study.Then,the AoEGLAI gene was transformed into Pichia pastoris by electroporation and the enzymatic properties of recombinase were analyzed by DNS method.The results showed that the enzyme is acid proof but not heat resistant,the optimum temperature is 50 ℃,and the optimum pH is 3.The enzyme activity could be maintained 90%at 30—45 ℃ and pH 4—8.In the presence of substrate CMC,the enzyme＇s thermal stability improved.In addition,Cu（2＋）,Ca（2＋）,Zn（2＋）,Gr（3＋）,Fe（2＋） and Fe（3＋） of 10 mmol/L had active effects on the enzyme whereas Mn（2＋） had inhibitory effect on it.This enzyme had certain resistance to trypsin,and relatively poor resistance to pepsin.

关 键 词：米曲霉 内切葡聚糖酶 毕赤酵母 酶学性质 

分 类 号：Q78[生物学—分子生物学]