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

微生物学报 Pub Date : 2017-01-04

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

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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.

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[蜡样芽孢杆菌磷脂酶C在乳酸克鲁维菌中的异源表达、纯化和特性研究]。

目的:构建重组乳酸克吕酵母菌生产蜡样芽孢杆菌磷脂酶C (PLC)的菌株。对重组酶进行了纯化和表征。方法克隆蜡样芽孢杆菌PLC编码基因bccp。将扩增片段插入pKLAC1中获得表达质粒。培养含有上述质粒的K. lactis表达PLC，通过HisTrapTM亲和层析纯化PLC并对其进行表征。结果:克隆出蜡样芽孢杆菌PLC，并在乳酸菌中表达。以对硝基苯基磷胆碱为底物时，酶活性为19251 U/mg。纯化后的PLC最适温度为80℃，最适pH为9.0。重组酶在40℃和7.0 ~ 8.0 pH范围内稳定。Cu2+和Co2+抑制其活性，而Zn2+、Mn2+、Ca2+和Mg2+则刺激其活性。结论:首次在乳酸菌中表达并鉴定了PLC基因。这些研究结果为重组PLC的研究提供了参考。

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微生物学报

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期刊介绍： Acta Microbiologica Sinica(AMS) is a peer-reviewed monthly (one volume per year)international journal,founded in 1953.It covers a wide range of topics in the areas of general and applied microbiology.The journal publishes original papers,reviews in microbiological science,and short communications describing unusual observations. Acta Microbiologica Sinica has been indexed in Index Copernicus (IC),Chemical Abstract (CA),Excerpt Medica Database (EMBASE),AJ of Viniti (Russia),Biological Abstracts (BA),Chinese Science Citation Database (CSCD),China National Knowledge Infrastructure(CNKI),Institute of Scientific and Technical Information of China(ISTIC),Chinese Journal Citation Report(CJCR),Chinese Biological Abstracts,Chinese Pharmaceutical Abstracts,Chinese Medical Abstracts and Chinese Science Abstracts.

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