[Cloning, heterologous expression and characterization of a thermostable esterase from Bacillus sp. HJ14 for diethyl-phthalate degradation].

微生物学报 Pub Date : 2016-12-04

Zheng Peng, Junmei Ding, Yunjuan Yang, Junjun Li, Yuelin Mu, Zunxi Huang

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引用次数: 0

Abstract

Objective: A thermostable esterase EstZ1 from Bacillus sp. HJ14 able to degrade diethyl-phthalate (DEP) was heterologously expressed in Escherichia coli BL21(DE3) and characterized.

Methods: Full-length EstZ1 was obtained based on specific amplification and genome sequencing, and amino acid sequence of EstZ1 was analyzed. EstZ1 was expressed in Escherichia coli BL21(DE3) using the pEASY-E2 expression system. EstZ1 was purified to electrophoretic homogeneity by Ni2+-NTA metal chelating affinity chromatography, and the enzyme was characterized. The degradation products from DEP were detected by high-pressure liquid chromatography and electrospray ionization mass spectrometry.

Results: The 903 bp full-length EstZ1 encoded 300 amino acid residues (EstZ1:33.84 kDa). EstZ1 showed the highest identity of 98% with hormone-sensitive lipase (HSL)-like family in NCBI databases. The optimal temperature and pH was 50℃ and 9.0, respectively, with p-NP butyrate as the best substrate. Meanwhile, it was stable between 40 and 70℃, pH 7.0 to 9.5. Most of metal ions, chemical agents had little impact. DEP could partially be degraded by EstZ1 to its corresponding monoalkyl and alcohol.

Conclusion: Our findings may serve as reference for phthalate esters degradation.

本刊更多论文

[Bacillus sp. HJ14耐热酯酶的克隆、异源表达及降解邻苯二甲酸二乙酯的鉴定]。

目的:从芽孢杆菌HJ14中分离出一种能降解邻苯二甲酸二乙酯(DEP)的耐热酯酶EstZ1，并在大肠杆菌BL21(DE3)中异种表达。方法:通过特异性扩增和基因组测序获得全长EstZ1，分析其氨基酸序列。EstZ1在大肠杆菌BL21(DE3)中通过pEASY-E2表达系统表达。采用Ni2+-NTA金属螯合亲和层析纯化EstZ1，使其达到电泳均匀性，并对酶进行了表征。采用高压液相色谱法和电喷雾质谱法对DEP的降解产物进行了检测。结果:全长903 bp的EstZ1编码300个氨基酸残基(EstZ1:33.84 kDa)。在NCBI数据库中，EstZ1与激素敏感脂肪酶(HSL)样家族的同源性最高，达98%。以丁酸p-NP为底物，最佳温度为50℃，pH为9.0℃。在40 ~ 70℃，pH 7.0 ~ 9.5范围内稳定。对大多数金属离子，化学药剂影响不大。EstZ1可将DEP部分降解为相应的单烷基和醇。结论:本研究结果可为邻苯二甲酸酯的降解提供参考。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

微生物学报

自引率

0.00%

发文量

7960

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.