粘红酵母酪氨酸解氨酶基因的克隆表达与酶学性质
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摘要
为研究粘红酵母酪氨酸解氨酶(Rg TAL)的酶学性质,利用生物信息学分析方法对Rg TAL的氨基酸序列和蛋白结构进行分析;利用大肠杆菌BL21(DE3)和p ET-32a表达系统获得重组Rg TAL;用高效液相色谱(HPLC)检测酶的催化活性。结果:克隆获得完整的Rg TAL编码基因,氨基酸同源性分析发现,与已有的Rg TAL序列的同源性只有74%,序列具有活性结构区域位点Ala217、Ser218、Gly219和底物特异性决定位点His143。重组的Rg TAL最适反应温度40℃,最适合反应p H值5.0。对Rg TAL的动力学分析表明,催化底物L-酪氨酸的活力不高,有待进一步的研究。本文测定了重组Rg TAL的酶学性质,为今后利用Rg TAL生产香豆酸的和为进一步改造Rg TAL奠定了基础。
Objective: To investigate the characteristic of tyrosine ammonia-lyase(TAL) from Rhodotorula glutinis.Methods: To explore the amino acid sequence and protein structure of Rg TAL by bioinformatics analysis; Rgtal was expressed in Escherichia coli BL21(DE3) and p ET-32 a system. High performance liquid chromatography(HPLC) analyses were carried out to measure the catalytic activity of Rg TAL. Results: In this report, a tal gene from Rhodotorula glutinis was cloned. Amino acid analysis showed that only 74% homology with existing Rg PAL. Rg TAL contained the active site residue for important for catalytic structure(Ala217, Ser218, Gly219) and for substrate selection(His143). The optimum temperature for the recombined Rg TAL activity was 40 ℃, and the optimum p H for the activity of this enzyme was 5.0.The kinetic analysis of Rg TAL showed that its catalytic efficiency was low and further study was needed. Conclusions:The characteristic of Rg TAL were measured, which lays a foundation for the p-coumaric bioproduction and rational enzyme modification research.
Objective: To investigate the characteristic of tyrosine ammonia-lyase(TAL) from Rhodotorula glutinis.Methods: To explore the amino acid sequence and protein structure of Rg TAL by bioinformatics analysis; Rgtal was expressed in Escherichia coli BL21(DE3) and p ET-32 a system. High performance liquid chromatography(HPLC) analyses were carried out to measure the catalytic activity of Rg TAL. Results: In this report, a tal gene from Rhodotorula glutinis was cloned. Amino acid analysis showed that only 74% homology with existing Rg PAL. Rg TAL contained the active site residue for important for catalytic structure(Ala217, Ser218, Gly219) and for substrate selection(His143). The optimum temperature for the recombined Rg TAL activity was 40 ℃, and the optimum p H for the activity of this enzyme was 5.0.The kinetic analysis of Rg TAL showed that its catalytic efficiency was low and further study was needed. Conclusions:The characteristic of Rg TAL were measured, which lays a foundation for the p-coumaric bioproduction and rational enzyme modification research.
引文
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[2]邓菊香,王祖元.苯丙氨酸的生产现状与发展前景[J].安徽化工,2007,33(4):1-3.
[3]VANNELLI T,X XUE Z,BREINIG S,et al.Functional expression in Escherichia coli of the tyrosine-inducible tyrosine ammonia-lyase enzyme from yeast Trichosporon cutaneum for production of p-hydroxycinnamic acid[J].Enzyme Microb Tech,2007,41(4):413-422.
[4]CHENG J C,DAI F,ZHOU B,et al.Antioxidant activity of hydroxycinnamic acid derivatives in human low density lipoprotein:Mechanism and structure-activity relationship[J].Food Chem,2007,104(1):132-139.
[5]NSANGOU M,FIFEN J J,DHAOUADI Z,et al.Hydrogen atom transfer in the reaction of hydroxycinnamic acids with(OH)-O-center dot and(HO2)-H-center dot radicals:DFT study[J].J Mol Struc-Theochem,2008(1/2/3),862:53-59.
[6]ABDEL-WAHAB M H,EL-MAHDY M A,ABD-ELLAH M F,et al.Influence of p-coumaric acid on doxorubicin-induced oxidative stress in rat’s heart[J].Pharmacol Res,2003,48(5):461-465.
[7]BODINI S F,MANFREDINI S,EPP M,et al.Quorum sensing inhibition activity of garlic extract and p-coumaric acid[J].Lett Appl Microbiol,2009,49(5):551-555.
[8]冉桂梅,何彬,杨凌,等.对香豆酸在大鼠体内的药动学研究[J].中国药学杂志,2005,40(24):1889-1891.
[9]VOGT T.Phenylpropanoid biosynthesis[J].Mol Plant,2010,3(1):2-20.
[10]NEISH A.Formation of m-and p-coumaric acids by enzymatic desamination of the corresponding isomers of tyrosine[J].Phytochem,1961,1(1):1-24.
[11]KYNDT J,MEYER T,CUSANOVICH M,et al.Characterization of a bacterial tyrosine ammonia-lyase,a biosynthetic enzyme for the photoactive yellow protein[J].FEBS Lett,2002,512(1/2/3):240-244.
[12]XUE Z X,MCCLUSKEY M,CANTERA K,et al.Improved production of p-hydroxycinnamic acid from tyrosine using a novel thermostable phenylalanine/tyrosine ammonia lyase enzyme[J].Enzyme Microb Tech,2007,42(1):58-64.
[13]KANG S Y,CHOI O,LEE J K,et al.Artificial biosynthesis of phenylpropanoic acids in a tyrosine overproducing Escherichia coli strain[J].Microb Cell Fact,2012,11(1):153.
[14]HUANG Q,LIN Y H,YAN Y J.Caffeic Acid production enhancement by engineering a phenylalanine overproucing Escherichia coli strain[J].Biotechnol Bioeng,2013,110(12):3188-3196.
[15]WU J J,LIU P R,FAN Y M,et al.Multivariate modular metabolic engineering of Escherichia coli to produce resveratrol from L-tyrosine[J].J Biotechnol,2013,167(4):404-411.
[16]龙燕,杨升.几种提取热带假丝酵母总RNA方法比较[J].实验技术与管理,2012,29(9):48-50.
[17]TROTT O,OLSON A J.Auto Dock Vina:improving the speed and accuracy of docking with a new scoring function,efficient optimization,and multithreading[J].J Comput Chem,2010,31(2):455-461.
[18]WATTS K T,MIJTS B N,LEE P C,et al.Discovery of a substrate selectivity switch in tyrosine ammonia-lyase,a member of the aromatic amino acid lyase family[J].Chem Biol,2006,13(12):1317-1326.
[19]ZHOU J W,DU G C,CHEN J.Novel fermentation processes for manufacturing plant natural products[J].Curr Opin Biotech,2014,25(1):17-23.