【摘要】 目的:提高毕赤酵母工程菌P.pastoris-CBHⅡ液体发酵产纤维二糖水解酶(CBHⅡ)的能力。方法:分别构建了用于胞外及胞内表达透明颤菌血红蛋白(VHb)基因的毕赤酵母重组表达质粒pPICZαA-vhb与pPICZαA(-s)-vhb。通过电转化将两种质粒转化巴氏毕赤酵母重组菌体P.pastoris-CBHⅡ菌株,经过筛选分别获得可正确表达具有生物活性的VHb蛋白的重组菌株P.pastoris-CBHⅡ-vhb(胞内表达VHb)及P.pastoris-CBHⅡ-vhb(-s)(胞外表达VHb)。结果:摇瓶发酵实验表明,血红蛋白在贫氧条件下可促进CBHⅡ的分泌表达,培养液上清的CMC酶活分别从出发菌株P.pastoris-CBHⅡ的1.81U/ml提高到2.04U/ml(胞内表达VHb)与1.93U/ml(胞外表达VHb)。VHb蛋白胞内表达菌株比胞外表达菌株效果更明显。更多还原

【Abstract】 Objective:In order to improve the extracellular secretion of CBHII from P.pastoris-CBHⅡon liquid-state fermentation.Methods:Recombinant vectors pPICZαA-vhb(for extracellular expression) and pPICZαA(-s)-vhb(for intracellular expression) were constructed.These two recombinant vectors were then transformed into the P.pastoris-CBHⅡ with the method of electroporation respectively,resulting in the recombinant strains P.pastoris-CBHⅡ-vhb(extracellular secretion) and P.pastoris-CBHⅡ-vhb(-s)(intracellular expression) which can correctly produce VHb protein with biological activity.The shake flask fermentation experiments showed that the presence of VHb can efficiently enhanced the recombinant strains’ secretive expression of CBHII on the condition of oxygen deficiency.Results:The CMC activities in the culture supernatant of two vhb harboring strains were 2.04U/ml(intracellular) and 1.93U/ml(extracellular),higher than the P.pastoris-CBHⅡ(1.81U/ml).These results indicated that intracellular expression of VHb was more beneficial to the expression of CBHⅡ than that of extracellular expression.更多还原