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

关键词:  黑曲霉ASP-S21  生淀粉糖化酶  淀粉结合域  In-fusion TM PCR Cloning  融合表达  毕赤酵母GS115

Key words:  Aspergillus niger ASP-S21  raw starch-digesting glucoamylase  starch-binding domain (SBD)  In-fusionTM PCR Cloning  fusion expression  Pichia pastoris GS115

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-fusionTM PCR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pPIC9K-psg,实现融合酶基因psg在毕赤酵母GS115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60~70℃,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。

Abstract:

[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 of 60~70℃ 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.