耐热β-半乳糖酶在乳酸克鲁维酵母及毕赤酵母中的表达研究

Study on expression of thermostable β-galactosidasein Kluyveromyces lactis and Pichia pastoris expression system

将嗜热脂肪芽孢杆菌来源的耐热β-半乳糖酶基因bgaB分别插入穿梭质粒pKLAC1、pPIC9k的α-因子信号肽下游,构建bgaB基因的乳酸克鲁维酵母真核表达载体pKLAC1-bgaB及毕赤酵母真核表达载体pPIC9k-bgaB。载体经酶切线性化后采用电击方法分别转化到乳酸克鲁维酵母K.lactis GG799及毕赤酵母GS115中,并通过同源区各自整合到宿主基因组中。重组酶进行镍柱纯化、Western Blot杂交鉴定及酶学性质分析。结果表明,耐热β-半乳糖苷酶在酵母表达系统中可以实现外源表达且热稳定性保持良好,但不能被酵母系统有效分泌。

Thermostable β-galactosidase gene bgaB from Geobacillus stearothermophilus were cloned into pKLAC1 and pPIC9k downstreams of the α-mating factor domain,resulting in construction of eukaryotic expression vectors of Kluyveromyces lactis and Pichia pastoris expression systems.Introduction of the linearized expression cassettes in K.lactis and P.pastoris cells was achieved by electrotransformation,and expression cassettes were inserted into the host genomes at the homology locus.Recombinant enzymes were purified by Ni2＋-NTA affinity chromatography,identified by western blot and the enzyme property was characterized.The results suggested that the recombinant thermostable β-galactosidases were capable of expression in yeast expression system and the stability was preserved well,but recombinants can not be secreted effectively.