毕赤酵母Kex2蛋白酶的同源表达及酶学性质 <sup>*</sup>

doi:10.13523/j.cb.20190105

中国生物工程杂志

2019, Vol. 39

Issue (1): 38-45 DOI: 10.13523/j.cb.20190105

研究报告

毕赤酵母Kex2蛋白酶的同源表达及酶学性质 ^*

王彤,徐岩,喻晓蔚(

)

1 江南大学工业生物技术教育部重点实验室　无锡　214122
2 宿迁市江南大学产业技术研究院　宿迁　223814

Homologous Expression and Characterization of Pichia pastoris Kex2

Tong WANG,Yan XU,Xiao-wei YU(

)

1 Key Laboratory of Industrial Biotechnology of Jiangnan University, Wuxi　214122, China
2 Suqian Industrial Technology Research Institute of Jiangnan University,Suqian　223814, China

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摘要：

Kex2蛋白酶是一种来源于酵母的前体加工蛋白酶。利用毕赤酵母(Pichia pastoris)同源表达来源于毕赤酵母的Kex2蛋白酶(PPKex2),研究其表达特性和酶学性质,同时与毕赤酵母表达的酿酒酵母(Saccharomyces cerevisiae)Kex2蛋白酶(SCKex2)进行比较。首先,分别从毕赤酵母和酿酒酵母基因组中获得Kex2基因,将其插入到表达载体pPIC9K中,并转化毕赤酵母菌株GS115。重组菌株经甲醇诱导表达后,结果表明PPKex2的发酵上清液比活是SCKex2的7倍。Kex2蛋白酶经Q-FF强阴离子交换柱纯化后进行酶学性质研究。酶学性质研究结果表明,PPKex2的最适反应pH是8.0~9.0,最适反应温度是37℃,与SCKex2性质相近。在稳定性方面,PPKex2在pH 7.0时最稳定,在碱性条件下的稳定性高于SCKex2,在酸性条件下的稳定性低于SCKex2,另外PPKex2的温度稳定性略低于SCKex2。酶促反应动力学研究表明,PPKex2的k_cat和k_cat/K_m值分别为SCKex2的4.8倍和3.3倍。首次报道了同源表达毕赤酵母Kex2蛋白酶的表达特性及酶学性质,为其今后的研究及应用奠定了基础。

关键词： Kex2; 毕赤酵母; 同源表达; 酶学性质

Abstract:

Kex2 protease is a precursor-processing protease from yeast. Kex2 protease from Pichia pastoris (PPKex2) was homologous expressed in Pichia pastoris. Then the expression and enzymatic characteristics of PPKex2 were studied and further compared with the Kex2 protease from Saccharomyces cerevisiae (SCKex2) expressed in Pichia pastoris. Firstly, these Kex2 genes were cloned from the genomes of Pichia pastoris and Saccharomyces cerevisiae, inserted into pPIC9K expression vector, and then transformed into Pichia pastoris GS115. The expression of PPKex2 and SCKex2 were induced by methanol and purified with anion exchange chromatography (Q-FF). Finally, the enzymatic characteristics of these Kex2 protease were characterized. The specific enzyme activity of PPKex2 in the supernatant was seven times higher than that of SCKex2. Enzymatic characterization showed that PPKex2 was similar to SCKex2 and had the optimal activity at pH 8.0-9.0 and 37℃. In terms of stability, PPKex2 was most stable at pH 7.0. PPKex2 was more stable than SCKex2 in alkaline pH and less stable than SCKex2 in acidic pH. Meanwhile, the thermostability of PPKex2 is lower compared with that of SCKex2. The kinetic analysis showed that the k_cat and k_cat/K_m of PPKex2 was 4.8- and 3.3-fold higher than that of SCKex2, respectively. The first time report the expression and enzymatic characteristics of PPKex2 homologous expressed in Pichia pastoris, which shows potential application in the future.

Key words: Kex2 Pichia pastoris Homologous expression Enzymatic characteristics

收稿日期: 2018-08-17 出版日期: 2019-02-28

ZTFLH:

Q814

基金资助: * 国家自然科学基金面上项目(31671799);江苏省“大人才高峰”计划资助项目(NY-010)

通讯作者: 喻晓蔚 E-mail: bioyuxw@aliyun.com

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引用本文:

王彤,徐岩,喻晓蔚. 毕赤酵母Kex2蛋白酶的同源表达及酶学性质 ^*[J]. 中国生物工程杂志, 2019, 39(1): 38-45.

Tong WANG,Yan XU,Xiao-wei YU. Homologous Expression and Characterization of Pichia pastoris Kex2. China Biotechnology, 2019, 39(1): 38-45.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190105 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I1/38

图1 PPKex2和SCKex2氨基酸序列对比图

图2 pPIC9K-PPKex2和pPIC9K-SCKex2的质粒图

图3 PPKex2和SCKex2菌株发酵液上清的SDS-PAGE图

图4 纯化的PPKex2和SCKex2的SDS-PAGE图

图5 PPKex2和SCKex2的最适反应pH和最适反应温度

图6 PPKex2和SCKex2的pH稳定性和温度稳定性

表1 PPKex2和SCKex2动力学参数

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