[蜡样芽孢杆菌磷脂酶C在乳酸克鲁维酵母中的异源表达、纯化及特性分析]

[Heterologous expression, purification and characterization of phospholipase C from Bacillus cereus in Kluyveromyces lactis].

作者信息

Xiao Chao, Zhang Liang, Li Yanyan, Xin Yu, Chen Guoan, Yang Shengrong

出版信息

Wei Sheng Wu Xue Bao. 2017 Jan 4;57(1):87-96.

Abstract

OBJECTIVE

In this study, we constructed recombinant Kluyveromyces lactis strains to produce phospholipase C (PLC) of Bacillus cereus. The recombinant enzymes were purified and characterized.

METHODS

We cloned the PLC encoding gene bcplc of Bacillus cereus. And the amplified fragments were inserted into pKLAC1 to obtain expression plasmids. K. lactis harboring the above plasmids was cultivated to express PLC that was purified by HisTrapTM affinity chromatography and characterized.

RESULTS

PLC of B. cereus was cloned and expressed in K. lactis. The recombinant enzyme had shown activity of 19251 U/mg when using p-nitrophenyl phosphorycholine as substrate. Purified PLC exhibited optimum temperature at 80 °C and optimal pH at 9.0. The recombinant enzyme was stable below 40 °C and pH between 7.0 and 8.0. Cu2+ and Co2+ inhibited its activity whereas Zn2+, Mn2+, Ca2+ and Mg2+ stimulated its activity.

CONCLUSION

It is the first time to express and characterize the PLC gene in K. lactis. These research results provide reference for the study of recombinant PLC.

摘要

目的

在本研究中，我们构建重组乳酸克鲁维酵母菌株以生产蜡样芽孢杆菌的磷脂酶C（PLC）。对重组酶进行了纯化和特性鉴定。

方法

我们克隆了蜡样芽孢杆菌的PLC编码基因bcplc。将扩增片段插入pKLAC1以获得表达质粒。培养携带上述质粒的乳酸克鲁维酵母以表达PLC，该PLC通过HisTrapTM亲和层析进行纯化并进行特性鉴定。

结果

蜡样芽孢杆菌的PLC在乳酸克鲁维酵母中被克隆并表达。当使用对硝基苯基磷酸胆碱作为底物时，重组酶显示出19251 U/mg的活性。纯化的PLC在80°C时表现出最佳温度，在pH 9.0时表现出最佳pH值。重组酶在40°C以下以及pH值在7.0至8.0之间时稳定。Cu2+和Co2+抑制其活性，而Zn2+、Mn2+、Ca2+和Mg2+刺激其活性。