生淀粉结合域SBD及糖化酶基因在毕赤酵母中的融合表达  

Fusion Expression of Raw Starch Binding Domain with Glucoamylase in Pichia pastoris

作　　者：朱婧[1] 刘海余 王青艳[1] 米慧芝[1] 朱绮霞[1] 廖思明[1] 秦艳[1] 申乃坤[1] 黄日波[1] 

机构地区：[1]广西科学院国家非粮生物质能源工程技术研究中心,广西南宁530007

出　　处：《广西科学》2016年第1期7-11,共5页Guangxi Sciences

基　　金：国家自然科学基金项目(No.31160023);广西科学研究与技术开发计划项目(桂科合14123001-19);广西自然科学基金项目(No.2013GXNSFBA019102);八桂学者建设工程专项经费;2014年留学人员科技活动项目资助

摘　　要：【目的】研究生淀粉结合域SBD及糖化酶基因在毕赤酵母中的融合表达，提高酶的表达量和水解生淀粉的能力。【方法】利用In-fusionTMPCR克隆技术将淀粉结合域SBD无缝隙插入到黑曲霉糖化酶基因glu的5’端构建融合表达质粒pPICgK-psg，实现融合酶基因psg在毕赤酵母GSll5中的高效表达，并进行酶学性质研究。【结果】融合酶的最适作用条件及热稳定性均与原始酶无明显差别，但反应温度及pH值的范围更为宽泛，在反应温度6O～70℃，pH值为4．0～7．0时均较稳定；融合酶PSG降解生淀粉的能力较原始酶PG提高29．6％，比原始菌株提高86．5％。【结论】淀粉结合域SBD的融合提高了糖化酶水解生淀粉的能力。【Objective】In order to improve the expression of glucoamylase fromAspergillus niger ASP-S21 and the hydrolysis ability of raw starch,the fusion expression of starch binding domain(SBD)with glu was conducted in Pichia pastoris GS115.【Methods】The fusion expression plasmid was constructed by inserting the SBD fragment to 5′-end of glucoamylase gene glu with In-fusionTM PCR Cloning technology.The recombinant enzyme(PSG)was over expressed in Pichia pastoris GS115 and its enzymatic properties were characterized.【Results】The PSG showed strong raw cassava starch hydrolyzing activity,which was 29.6% higher than the wildtype PG,and 86.5% higher than parental strain.The reaction temperature and pH of the acfusion enzyme were more broad than that of the wildtype.It was stable in temperature range of60~70℃ and pH range from 4.0to 7.0.【Conclusion】The fusion of SBD with glucoamylase enables the fusion enzymes to hydrolyze raw starch more effectively.

关 键 词：黑曲霉ASP-S2 1 生淀粉糖化酶 淀粉结合域 In-fusion TM PCR Cloning 融合表达 毕赤酵母GSl15 

分 类 号：TS231[轻工技术与工程—粮食、油脂及植物蛋白工程] Q786[轻工技术与工程—食品科学与工程]