毕赤酵母高效表达整合有CBD 的米黑根毛霉脂肪酶

生物技术通报 ›› 2013, Vol. 0 ›› Issue (2): 118-123.

毕赤酵母高效表达整合有CBD 的米黑根毛霉脂肪酶

薛冲冲张俊辉林影韩双艳

（华南理工大学生物科学与工程学院，广州 510006）

收稿日期:2012-08-20 修回日期:2013-02-27 出版日期:2013-02-26 发布日期:2013-02-27
作者简介:薛冲冲，男，硕士研究生，研究方向：CBM 整合木聚糖酶、脂肪酶；E-mail ：x16c06@163.com
基金资助:
国家“863”计划项目（2011AA100905）

High Expression of Rhizomucor miehei Lipase in Pichia pastoris by Fusion to CBD

Xue Chongchong Zhang Junhui Lin Ying Han Shuangyan

（School of Bioscience and Bioengineering，South China University of Technology，Guangzhou 510006）

Received:2012-08-20 Revised:2013-02-27 Published:2013-02-26 Online:2013-02-27

摘要/Abstract

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。

关键词: 毕赤酵母, 米黑根毛霉脂肪酶, CBD, 分泌表达

Abstract: 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 endoglucanase 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.

Key words: Pichia pastoris, RML, CBD, Secreted expression

薛冲冲, 张俊辉, 林影, 韩双艳. 毕赤酵母高效表达整合有CBD 的米黑根毛霉脂肪酶[J]. 生物技术通报, 2013, 0(2): 118-123.

Xue Chongchong, Zhang Junhui, Lin Ying, Han Shuangyan . High Expression of Rhizomucor miehei Lipase in Pichia pastoris by Fusion to CBD[J]. Biotechnology Bulletin, 2013, 0(2): 118-123.

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