作　　者： ; ; ; ;

机构地区： 华南理工大学生物科学与工程学院

出　　处： 《生物技术通报》 2013年第2期118-123,共6页

摘　　要： CBD是一类存在各种水解酶(木聚糖酶,纤维素酶等)中的能够结合并识别纤维素的结合域,能有效地增加酶在底物周围的浓度,增强酶水解底物的能力。尝试将来源于哈茨木霉内切葡聚糖酶Ⅱ(THEGⅡ)的CBD及连接肽基因融合到S7-xyn的N-末端区域中,以期能获得高酶活力的融合米黑根毛霉脂肪酶应用于工业生产,研究通过将融合基因克隆到pPICZαA载体中,构建分泌性表达质粒pPICZαA-CBD-RML,载体经线性化后在毕赤酵母GS115中融合表达。通过与没有融合CBD的RML比较,两者具有相似的最适温度和温度稳定性曲线,而融合蛋白CBD-RML的分泌量提高了17%。研究结果表明,来源于THEGⅡ的CBD可以作为异源蛋白在毕赤酵母中表达的分泌增强子。 Rhizomucor miehei lipase （ RML ） , one of the important enzymes applied in industries, has a broad prospect of application. CBD, which is capable of tightly binding cellulose, has been found in diverse hydrolytic enzymes （ cellulose, xylanase ） . The presence of a CBD is shown to increase the effective concentration of enzyme on substrates, thereby assisting the enhancement of hydrolytic enzyme activity. Here the CBD from Trichoderma harzianum endoglueanase II （ THEGII ） was connected to the N-terminal of RML through an endogenous linker peptide. Then the fusion genes were cloned into the secreted expression vector pPICZαA to construct recombinant plasmid pPICZαA-CBD-RML. The plasmid was linearized and transformed into Pichia pastoris GS115 to obtain Pichia pastoris recombinant strain. Compared with that of the mature RML without CBD-fusion, secretion of CBD-RML by this fusion construct was about 17% enhanced in addition to similar temperature activity and stability between RML and CBD-RML. Therefore, the CBD from THEGII can be used as a secretion enhancer for secretory production of heterologous protein in Pichia pastoris.

关 键 词： 毕赤酵母 米黑根毛霉脂肪酶 分泌表达

领　　域： [生物学]