登录    |    注册

您好,欢迎来到中国测试科技资讯平台!

首页>《中国测试》期刊>本期导读>路邓葡萄球菌单宁酶基因的克隆、表达、纯化与改造

路邓葡萄球菌单宁酶基因的克隆、表达、纯化与改造

334    2018-09-27

¥0.50

全文售价

作者:刘鳐, 胡雪丽, 钟秋梅, 吕蝶, 吴明波

作者单位:成都医学院, 四川 成都 610500


关键词:路邓葡萄球菌;单宁酶;三明治结构;定点突变;没食子酸甲酯


摘要:

为提高路邓葡萄球菌(Staphylococcus lugdunensis)单宁酶(Sl-tan)的活性,该文利用化学合成方法获得Sl-tan基因,将该基因连接到重组表达质粒pET43.1-A,再转化到大肠杆菌感受态细胞BL21-DE3中进行表达,通过亲和层析柱纯化,以没食子酸甲酯为底物进行酶活性测定以及酶学性质分析,并基于生物信息学分析,结合定点突变技术对Sl-tan进行人工改造。结果显示,获得的重组单宁酶产量明显增高,最高可达42 mg/L发酵液;酶学性质研究显示该酶在pH 8.0,温度40 ℃时获得的活性最高(40 U/mg);Ala460突变为Pro460后的Sl-tan活性可提高82.5%。


Cloning, expression, purification and modification of tannase gene from Staphylococcus lugdunensis

LIU Yao, HU Xueli, ZHONG Qiumei, LÜ Die, WU Mingbo

Chengdu Medical College, Chengdu 610500, China

Abstract: In order to improve the activity of tannase (Sl-tan) from Staphylococcus lugdunensis, Sl-tan gene was chemically synthesized and constructed into protein expression vector pET43.1-A. The construct vector was expressed in E. coli BL21-DE3 cells and purified with affinity chromatography, and methyl gallate was used as the substrate to study the activity of tannase. Besides, artificial modification was carried out for Sl-tan based on bioinformatics analysis and site-directed mutagenesis technique. Results show that production of obtained recombinant tannase is obviously increased (reaching 42 mg/L fermentation liquor at most). Research on enzymatic properties show that activity of the enzyme is the highest (40 U/mg) when pH value is 8.0 and temperature is 40℃, and the activity of Sl-tan after Ala460 mutates into Pro460 can be improved for 82.5%.

Keywords: Staphylococcus lugdunensis;tannase;sandwich structure;site-directed mutagenesis;methyl gallate

2018, 44(9): 63-68  收稿日期: 2018-05-11;收到修改稿日期: 2018-06-09

基金项目: 四川省科技厅应用基础研究项目(2016GZ0364,2018JY0208);四川省教育厅科研项目(16ZA0287,18ZB0152);四川省卫计委项目(18PJ586,18PJ006);成都医学院科研创新团队项目(CYTD16-04);成都医学院校基金(15Z106)

作者简介: 刘鳐(1988-),女,四川绵阳市人,助理实验师,硕士,研究方向为微生物学

参考文献

[1] WHITE T. Tannins-their occurrence and significance[J]. J Sci Food Agric, 1957, 8(7):377-385
[2] BAXTER N J, LILLEY T H, HASLAM E, et al. Multiple interactions between polyphenols and a salivary proline-rich protein repeat result in complexation and precipitation[J]. Biochemistry, 1997, 36(18):5566-5577
[3] BELUR P D, MUGERAYA G, NIRMALA K R, et al. Production of novel cell-associated tannase from newly isolated Serratia ficaria DTC[J]. J Microbiol Biotechnol, 2010, 20(4):732-736
[4] BLTLER L G. Anti-nutritional effects of condensed and hydrolysable tannins[J]. Basic Life Sci, 1992, 59:693-698
[5] SHARMA S, BHAT T K, DAWRA R K. A spectrophotometric method for assay of tannase using rhodamine[J]. Anal Biochem, 2000, 279(1):85-89
[6] AGUILAR C N, RODRIGUEZ R, GUTIERREZ-SANCHEZ G, et al. Microbial tannases:advances and perspectives[J]. Appl Microbiol Biotechnol, 2007, 76(1):47-59
[7] HASLAM E, STANGROOM J E. The esterase and depsidase activities of tannase[J]. Biochem J, 1966, 99(1):28-31
[8] LOPES L, COSTA B, GOUVEIA M J, et al. Kinetic and thermodynamic parameters, and partial characterization of the crude extract of tannase produced by Saccharomyces cerevisiae CCMB 520[J]. Nat Prod Res, 2018, 32(9):1068-1075
[9] TSAI C L, CHIU Y M, HO T Y, et al. Gallic acid induces apoptosis in human gastric adenocarcinoma cells[J]. Anticancer Res, 2018, 38(4):2057-2067
[10] LI R, FU F G, LIU C, et al. Tannase immobilisation by amino-functionalised magnetic Fe3O4-chitosan nanoparticles and its application in tea infusion[J]. Int J Biol Macromol, 2018, Mar 16, 114:1134-1143
[11] 宁井铭, 方世辉, 夏涛, 等. 酶澄清绿茶饮料研究[J]. 食品与发酵工业, 2005, 31(9):122-124
[12] LEKHA P K, LONSANE B K. Production and application of tannin acyl hydrolase:state of the art[J]. Adv Appl Microbiol, 1997, 44:215-260
[13] RODRIGUEZ-DURAN L V, VALDIVIA-URDIALES B, CONTRERAS-ESQUIVEL J C, et al. Novel strategies for upstream and downstream processing of tannin acyl hydrolase[J]. Enzyme Res, 2011, 2011(1):823619
[14] WU C, ZHANG F, LI L, et al. Novel optimization strategy for tannase production through a modified solid-state fermentation system[J]. Biotechnol Biofuels, 2018, 11(1):92
[15] VARADHARAJAN V, VADIVEL S S, RAMASWAMY A, et al. Modeling and verification of process parameters for the production of tannase by Aspergillus oryzae under submerged fermentation using agro-wastes[J]. Biotechnol Appl Biochem, 2017, 64(1):100-109
[16] IWAMOTO K, TSURUTA H, NISHITAINI Y, et al. Identification and cloning of a gene encoding tannase (tannin acylhydrolase) from Lactobacillus plantarum ATCC 14917(T)[J]. Syst Appl Microbiol, 2008, 31(4):269-277
[17] WU M, PENG X, WEN H, et al. Expression, purification, crystallization and preliminary X-ray analysis of tannase from Lactobacillus plantarum[J]. Acta Crystallogr Sect F:Struct Biol Cryst Commun, 2013, 69(Pt4):456-459
[18] SUZUKI K, HORI A, KAWAMOTO K, et al. Crystal structure of a feruloyl esterase belonging to the tannase family:A disulfide bond near a catalytic triad[J]. Proteins, 2014, 82:2857-2867
[19] WU M, WANG Q, MCKINSTRY WJ, et al. Characterization of a tannin acyl hydrolase from Streptomyces sviceus with substrate preference for digalloyl ester bonds[J]. Appl Microbiol Biotechnol, 2015, 99(6):2663-72
[20] REN B, WU M, WANG Q, et al. Crystal structure of tannase from Lactobacillus plantarum[J]. J Mol Biol, 2013, 425(15):2737-2751