里氏木霉纤维二糖水解酶Ⅱ在毕赤酵母中的高效表达

本工作采用巴氏毕赤酵母Pichiapastoris表达系统进行了里氏木霉Trichodermareesei纤维二糖水解酶Ⅱ(CellobiohydrolaseII)的表达。用RT-PCR的方法从经稻草粉诱导的里氏木霉培养物中分离出纤维二糖水解酶Ⅱ的基因,将其插入到巴氏毕赤酵母的表达载体pPICZαA中,并使之处于α-因子信号肽序列的下游,得到重组质粒pPICZαA-cbh2。通过电穿孔的方法用线性化的pPICZαA-cbh2转化巴氏毕赤酵母GS115菌株,经过大量筛选后得到可以高效表达纤维二糖水解酶的毕赤酵母菌株P.pastorisCBHⅡ1。在甲醇诱导的条件下培养P.pastorisCBHⅡ1,培养液中的CMC活性可达到3.82U/mL,SDS-PAGE分析结果表明纤维二糖水解酶在P.pastorisCBHⅡ1中的表达量远远高于里氏木霉。对表达产物进行了LC-MS分析,结果表明所表达的蛋白为里氏木霉的纤维二糖水解酶。

EFFECTIVE EXPRESSION OF TRICHODERMA REESEI CELLOBIOHYDROLASE Ⅱ IN PICHIA PASTORIS

The gene of cellobiohydrolase II (cbh2) from Trichoderma reesei was effectively expressed in Pichia pastoris. The cbh2 gene was isolated through RT-PCR method, and was ligated with the Pichia expressing vector pPICZαA, generating a recombinant plasmid pPICZαA-cbh2. Linearized pPICZαA-cbh2 was transformed into P. pastoris GS115 with the method of electroporation, and thereby the cbh2 gene was inserted into the Pichia genome downstream, the strong AOX1 promoter through homologous recombination. The most efficient recombinant Pichia strain, which was designated as P. pastoris CBHⅡ1, expressed large amount of CBHⅡ when cultivated in methanol containing medium. The amino acid sequence of the recombinant CBHⅡ was verified to be identical to the native CBHⅡ through LC-MS analysis and Matrixscience database searching. As indicated by CMC activity and SDS-PAGE analysis, the CBHⅡ produced by P. pastoris CBHⅡ1 was much more than T. reesei, while CMC activity of the cultivation supernatant of recombinant P. pastoris was lower. This work exhibits an alternative approach to improve the cellulolytic enzyme from filamentous fungi other than traditional mutation and screening.