β-甘露聚糖酶基因和木聚糖酶基因在毕赤酵母中的共表达

Coexpression of β-mannanase Gene and Xylanase Gene in Pichia pastoris

摘要:

为实现β-甘露聚糖酶基因和木聚糖酶基因在毕赤酵母中的共表达,作者将经SalⅠ线性化的pPIC9K-xynⅡ电转化至工程菌GS115/Anman5A中,经G418浓度梯度筛选后获得能同时高产β-甘露聚糖酶和木聚糖酶的双重重组子GS115/Anman5A-xynⅡ。SDS-PAGE显示目的蛋白的相对分子质量分别约为52 000,24 100。摇瓶发酵筛选出产酶活性最强的转化株,命名为GSMX2,随后对该菌株的表达条件进行初步优化,优化后的表达条件为:诱导时间120 h,培养基起始pH值为7.0,甘油添加量为1.5%,甲醇添加量为1.5%。在此培养条件下,产β-甘露聚糖酶和木聚糖酶的活性分别达到37.1 U/mL和193.6 U/mL。

Abstract:

β-mannanase and xylanase are extensively used in food industry.Synergistic action between the two enzymes is obersved to effectively improve digestibility of the hemicelluloses of food material.In order to coexpress β-mannanase gene and xylanase gene in Pichia pastoris,pPIC9K-xynⅡ was linearized with Sal I and transformed into GS115/Anman5A by electroporation.GS115/Anman5A-xynⅡ with high production of β-mannanase and xylanase was screened by using G418.SDS-PAGE demonstrated two protein bands with apparent molecular weight of about 52 000 and 24 100.One GS115/Anman5A-xyn Ⅱ transformant producing the highest activities of two enzymes in shake flasks was selected and numbered as GSMX2.Subsequently the expression conditions for enzymes production by using GSMX2 were optimized,the optimum expression conditions listede as follows:initial pH 7.0,glycerol concentration 1.5%,methanol concentration 1.5%,induction for 120 h.Under these conditions,the activities of β-mannanase and xylanase were achieved at 37.1 U/mL and 193.6 U/mL,respectively.