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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2017, Vol. 37 Issue (10): 42-52    DOI: 10.13523/j.cb.20171006
研究报告     
Rhodococcus ruber CGMCC3090腈水合酶纯化、酶学性质及结晶研究
王世伟1, 王敏2, 王卿惠1
1. 齐齐哈尔大学 生命科学与农林学院 齐齐哈尔 161000;
2. 工业微生物教育部重点实验室 天津科技大学生物工程学院 天津 300457
Purification,Crystallization and Characterization of a Nitrile Hydratase from Rhodococcus ruber CGMCC3090 Strain
WANG Shi-wei1, WANG Min2, WANG Qing-hui1
1. College of Life Science of Qiqihar University, Qiqihar 161006, China;
2. Key Laboratory of Fermentation Microbiology, Tianjin University of Science and Technology, Ministry of Education, Tianjin 300457, China
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摘要： Rhodococcus ruber CGMCC309菌株为酰胺酶及腈水解酶双重缺陷菌株，研究表明该菌能产宽泛底物特异性的腈水合酶。对该菌株产生的新型腈水合酶（NHase-3090）进行纯化和结晶，并研究了其酶学性质。采用疏水、离子交换及凝胶过滤3种层析方法，使该酶纯化倍数达到17.14，得率高达26.2%。电泳分析表明，全酶分子量为105 kDa，由α（24.3 kDa）和β（28.0kDa）2个亚基组成，并构成α2β2四聚体。酶的最适pH和温度分别为7.5和30℃。该酶明显受不同金属离子影响。动力学研究表明，Km为178.8 mM；Vmax为209.1 μmol/L·min·mg。研究发现3种金属离子Zn2+，CO2+和Cd2+有利于酶蛋白结晶。结晶最佳条件是：采用112-34#试剂（0.05mol/L水合硫酸镉、0.1mol/L HEPS和1.0mol/L三水醋酸钠），蛋白质浓度为15 mg/ml，结晶温度为16℃，pH为7.5，结晶时间为30 d。腈水合酶蛋白单晶经X射线衍射，分辨率达到了3.7Å。该腈水合酶的纯化和结晶为进一步深入研究其结构和功能奠定了基础。
关键词： 赤红球菌CGMCC3090;  纯化;  结晶;  酶学性质;  腈水合酶    
Abstract: Rhodococcus ruber CGMCC3090 was separated from soil polluted by nitriles at Tianjin suburb.The strain can transform nitriles into correspondent amides with broad substrate spectra and higher catalytic activity, stronger substrate tolerance, good regioselectivity especially to dinitrile substrates (e.g. adiponitrile).The new type of NHase was purified to homogenety after sonication, ammonium sulfate fractionation, hydrophobic, ion exchange and gel-filtration column chromatography. About 17.14-fold purification was achieved with 26.2% yield. The holoenzyme purified (Mr 105kDa;α2β2) consists of α subunit (Mr 24.3 kDa) and β subunit (Mr 28 kDa) respectively. The optimal temperature and pH was 30℃ and pH 7.5 respectively.The enzyme purified was inhibited or promoted by different metals in various degrees. The Km and Vmax values of purified NHase respectively were 178.8 mM and 209.1 μmole/min/mg protein using 3-cyanopyridine as substrate.The nitrile hydratase crystallization conditions were screened and optimized by using 6 crystallization reagents screened from 194 reagents from 4 set Kit(Hampton Research).A single crystal from the NHase was obtained at the optimum conditions(15 mg protein/ml,16℃,pH 7.5,30 d)by using 112-34# reagent (0.05mol/L cadmium sulfate hydrate,0.1mol/L HEPS and 1.0mol/L sodium acetate trihydrate). the Nitrile hydratase single crystal had a 3.7Å resolution by X ray diffraction analysis.It paved the way for in-depth study of the structure and function of the nitrile hydratase from Rhodococcus ruber CGMCC3090 strain.
Key words: Rhodococcus ruber    Nitrile hydratase    Purification    Characterization    Crystallization
收稿日期: 2017-06-27 出版日期: 2017-10-25
ZTFLH:  Q813.11  
基金资助: 2016年黑龙江省大学生创新创业训练计划（201610221026）、黑龙江省教育厅科学技术研究项目（12541855）资助项目
通讯作者: 王世伟,wsw888535@sohu.com     E-mail: wsw888535@sohu.com
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引用本文:

王世伟, 王敏, 王卿惠. Rhodococcus ruber CGMCC3090腈水合酶纯化、酶学性质及结晶研究[J]. 中国生物工程杂志, 2017, 37(10): 42-52.

WANG Shi-wei, WANG Min, WANG Qing-hui. Purification,Crystallization and Characterization of a Nitrile Hydratase from Rhodococcus ruber CGMCC3090 Strain. China Biotechnology, 2017, 37(10): 42-52.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20171006    或    https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I10/42

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