[Characterization and molecular modification of β-glucosidase from Citrobacter koser GXW-1].

微生物学报 Pub Date : 2017-03-04
Minhua Jiang, Houmin Lin, Jinyang Yin, Zilong Wang, Hao Pang, Ribo Huang, Liqin Du
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引用次数: 0

Abstract

Objective: The aim of this study was to characterize β-glucosidase from Citrobacter koser GXW-1 isolated from soil and to improve the enzyme by molecular modification.

Mehods: A bacterial strain with β-glucosidase activity was screened from the soil around Wuming sugar mill in Guangxi by esculin-ferric ammonium citrate selecting plate. The 16S rDNA of the strain was obtained and analyzed. By searching GenBank database, the genes encoding β-glucosidase from the same genus Citrobacter were found. These sequences were aligned. Then, a gene encoding β-glucosidase was amplified by PCR. The recombinant plasmid pQE-cbgl was constructed. The recombinant protein was purified with Ni-NTA. The enzyme properties of the recombinant protein CBGL were studied in detail. At last, the wild enzyme CBGL was reformed by error-prone PCR and site-directed random mutagenesis.

Results: C. koser GXW-1 with β-glucosidase activity was isolated from the soil. A gene encoding β-glucosidase was cloned from the wild strain GXW-1. The properties of CBGL were identified. Its optimal pH and temperature were 6.0 and 45℃. Its Km and Vmax value were (11.280±1.073) mmol/L and (0.1704±0.0073) μmol/(mg·min), respectively. Its Ki values was (66.84±3.40) mmol/L. CBGL can hydrolyze α-pNPG, stevioside, daidzin and genistin. CBGL was modified by error-prone PCR and site directed random mutagenesis. A positive mutant W147F was obtained successfully. Its Vmax was 2.54 times that of the wild enzyme CBGL.

Conclusion: CBGL not only can hydrolyze β-glycosidic bond, but also can hydrolyze the α-glycosidic bond in α-pNPG. Furthermore, CBGL can hydrolyze stevioside, daidzin and genistin. These characteristics indicate that the β-glucosidase CBGL has important applications in theoretical research and in industry.

[Citrobacter koser GXW-1 β-葡萄糖苷酶的表征及分子修饰]。
目的:研究从土壤中分离的科瑟柠檬酸杆菌GXW-1 β-葡萄糖苷酶,并对其进行分子修饰。方法:采用皮素-柠檬酸铁铵筛选板从广西武明糖厂周围土壤中筛选一株具有β-葡萄糖苷酶活性的细菌。获得菌株的16S rDNA并进行分析。通过对GenBank数据库的检索,找到了来自同一属Citrobacter的β-葡萄糖苷酶编码基因。这些序列是对齐的。然后,用PCR扩增β-葡萄糖苷酶编码基因。构建重组质粒pQE-cbgl。重组蛋白用Ni-NTA纯化。详细研究了重组蛋白CBGL的酶学性质。最后,利用易出错PCR和定点随机诱变技术对野生CBGL酶进行重组。结果:从土壤中分离到具有β-葡萄糖苷酶活性的葡萄球菌GXW-1。从野生菌株GXW-1中克隆了一个β-葡萄糖苷酶编码基因。鉴定了CBGL的性质。其最适pH为6.0,最适温度为45℃。其Km和Vmax分别为(11.280±1.073)mmol/L和(0.1704±0.0073)μmol/(mg·min)。Ki值为(66.84±3.40)mmol/L。CBGL可水解α-pNPG、甜菊苷、大豆苷和龙胆素。采用易出错PCR和定点随机诱变技术对CBGL进行了修饰。成功地获得了阳性突变体W147F。其Vmax为野生酶CBGL的2.54倍。结论:CBGL不仅能水解α-pNPG中的β-糖苷键,还能水解α-糖苷键。此外,CBGL还能水解甜菊苷、大豆苷元和龙胆素。这些特性表明,β-葡萄糖苷酶CBGL在理论研究和工业上具有重要的应用价值。
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
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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.
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