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丙氨酸依赖型转氨酶高通量快速检测方法的建立

刘欢, 吴旭日, 陈依军

刘欢, 吴旭日, 陈依军. 丙氨酸依赖型转氨酶高通量快速检测方法的建立[J]. 中国药科大学学报, 2018, 49(4): 490-495. DOI: 10.11665/j.issn.1000-5048.20180416
引用本文: 刘欢, 吴旭日, 陈依军. 丙氨酸依赖型转氨酶高通量快速检测方法的建立[J]. 中国药科大学学报, 2018, 49(4): 490-495. DOI: 10.11665/j.issn.1000-5048.20180416
LIU Huan, WU Xuri, CHEN Yijun. Rapid and sensitive high-throughput determination method for alanine-dependent transaminase[J]. Journal of China Pharmaceutical University, 2018, 49(4): 490-495. DOI: 10.11665/j.issn.1000-5048.20180416
Citation: LIU Huan, WU Xuri, CHEN Yijun. Rapid and sensitive high-throughput determination method for alanine-dependent transaminase[J]. Journal of China Pharmaceutical University, 2018, 49(4): 490-495. DOI: 10.11665/j.issn.1000-5048.20180416

丙氨酸依赖型转氨酶高通量快速检测方法的建立

基金项目: 国家自然科学基金资助项目(No.21778076);江苏高校“青蓝工程”资助项目

Rapid and sensitive high-throughput determination method for alanine-dependent transaminase

  • 摘要: 本研究以来源自Vibrio fluvialis JS17的丙氨酸依赖型转氨酶VfTA为研究对象,偶联丙酮酸氧化酶和辣根过氧化物酶,建立了一种基于反应液颜色变化的转氨酶活性测定新方法,反应条件优化后,考察了VfTA活力单位数与反应液400 nm处吸收度的线性关系,并将此法用于商业化转氨酶ATA117活力的研究。结果显示:该法对转氨酶VfTA的活力检测限可达0.45 U/mL,对ATA117的活力检测限可达0.5 U/mL,并且可根据反应液颜色深浅初步判断酶活力情况。
    Abstract: A new method for determining transaminase activity based on the color change of the reaction solution was established, by using alanine-dependent transaminase VfTA from Vibrio fluvialis JS17 as the research object coupled with pyruvate oxidase and horseradish peroxidase. After the optimization of the conditions, the linear relationship between VfTA activity units and the absorbance at 400 nm was investigated. This method was also applied to determine the activity of commercial transaminase ATA117. The results showed that the detection limit of transaminase VfTA activity was up to 0. 45 U/mL and the detection limit of ATA117 activity was up to 0. 5 U/mL. The transaminase activity could be quickly judged according to the color depth of the reaction solution.
  • [1] Ward J,Wohlgemuth R.High-yield biocatalytic amination reactions in organic synthesis[J].Curr Org Chem,2010,14(17):1915.
    [2] Desai A. Sitagliptin manufacture: a compelling tale of green chemistry,process intensification,and industrial asyetric catalysis[J].Angew Chem Int Edit,2011,50(9):1974.
    [3] Ran N,Zhao L,Chen Z,et al.Recent applications of biocatalysis in developing green chemistry for chemical synthesis at the industrial scale[J].J Cheminformatics,2008,39(29):361-372.
    [4] Ghislieri D,Turner NJ.Biocatalytic approaches to the synthesis of enantiomerically pure chiral amines[J].Top Catal,2014,57(5):284-300.
    [5] Busto E,Gotor-Fernández V,Gotor V.Hydrolases in the stereoselective synthesis of N-heterocyclic amines and amino acid derivatives[J].Chem Rev,2011,111(7):3998-4035.
    [6] Abrahamson MJ, Wong JW, Boarius AS. The evolution of an amine dehydrogenase biocatalyst for the asyetric production of chiral amines[J].Adv Synth Catal,2013,355(9):1780-1786.
    [7] Malik MS,Park ES,Shin JS.ω-Transaminase-catalyzed kinetic resolution of chiral amines using L-threonine as an amino acceptor precursor[J].Green Chem,2012,14(8):2137-2140.
    [8] Green AP,Turner NJ,O′Reilly E.Chiral amine synthesis using ω-transaminases:an amine donor that displaces equilibria and enables high-throughput screening[J].Angew Chem Int Edit,2014,53(40):10714-10717.
    [9] Shin JS,Yun H,Jang JW,et al.Purification,characterization,and molecular cloning of a novel amine:pyruvate transaminase from Vibrio fluvialis JS17[J].Appl Microbiol Biotech,2003,61:463-471.
    [10] Rosche B,Breuer M,Hauer B,et al.Role of pyruvate in enhancing pyruvate decarboxylase stability towards benzaldehyde[J].J Biotechnol,2005,115(1):91-99.
    [11] Genz M, Vickers C, van den Bergh T, et al. Alteration of the donor/acceptor spectrum of the(S)-amine transaminase from Vibrio fluvialis[J].Int J Mol Sci,2015,16:26953-26963.
    [12] Yun H,Cho BK,Kim BG.et al.Kinetic resolution of(R,S)-sec-butylamine using Omega-transaminase from Vibrio fluvialis JS17 under reduced pressure[J].Biotechnol Bioeng,2004,36(3):773-780.
    [13] Sam M,Giyoung S,Minsu S,et al.High throughput screening methods for ω-transaminases[J].Biotechnol Bioeng,2013,18:1-7.
    [14] Katrin W,Margit W,Glieder A,et al.A novel multi- enzymatic high throughput assay for transaminase activity[J].Tetrahedron,2012,68:7586-7590.
    [15] Wu X,Fei M,Chen Y,et al.Enzymatic synthesis of L-norephedrine by coupling recombinant pyruvate decarboxylase and ω-transaminase[J].Appl Microbiol Biot,2014,98(17):7399-7408.
    [16] Fuchs CS,Simon RC,Riethorst W,et al.Synthesis of(R)- or(S)-valinol using ω-transaminases in aqueous and organic media[J].Bioorgan Med Chem,2014,22(20):5558-5562.
    [17] Weiß MS,Pavlidis IV,Vickers C,et al.Glycine oxidase based-throughput solid-phase assay for substrate profiling and directed evolution of(R)- and(S)-selective amine transaminases[J].Anal Chem,2014,86(23):11847-11853.
    [18] Scheidt T,Land H,Anderson M,et al.Fluorescence-based kinetic assay for high-throughput discovery and engineering of stereoselective ω-transaminases[J].Adv Synth Catal,2015,357(8):1721-1731.
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出版历程
  • 刊出日期:  2018-08-24

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