重组Taxus chinensis 苯丙氨酸氨基变位酶性质表征及合成R-β-芳香丙氨酸

食品科学 ›› 0, Vol. ›› Issue (): 0-0.

• 生物工程 • 下一篇

重组Taxus chinensis 苯丙氨酸氨基变位酶性质表征及合成R-β-芳香丙氨酸

丰国强¹,徐文亮¹,宋平²,李婉珍¹,葛飞¹,陶玉贵¹,朱龙宝¹

1. 安徽工程大学
2. 安徽工程大学生物与化学工程学院

收稿日期:2019-12-12 修回日期:2020-11-25 出版日期:2021-03-25 发布日期:2021-03-22
通讯作者: 朱龙宝 E-mail:lbzhu2008@126.c0m
基金资助:
国家自然科学基金;安徽工程大学拔尖人才项目

Characterization of recombinant phenylalanine aminomutase from Taxus chinensis and synthesis of R-β-arylalanine

²,Ping SONG³, ², ²,

Received:2019-12-12 Revised:2020-11-25 Online:2021-03-25 Published:2021-03-22

摘要/Abstract

摘要： 摘要：为实现酶法合成高附加值的β-芳香丙氨酸，首先化学合成来源于紫衫（Taxus chinensis）的氨基变位酶(Tcpam)基因，构建了大肠杆菌重组质粒Pet-sumo-Tcpam，转入大肠杆菌中进行异源诱导表达，采用镍柱亲和层析制备电泳纯的重组酶TcPAM，用于催化合成β-芳香丙氨酸。实验结果表明：重组质粒Pet-sumo-Tcpam成功在大肠杆菌中实现高效表达，获得可溶性的重组TcPAM，经过亲和层析制备出电泳纯的重组TcPAM。质谱和圆二色谱检测分析结果表明，TcPAM能够催化α-芳香丙氨酸异构化为R构型的β-芳香丙氨酸。TcPAM在最适温度30 ℃、pH9的条件下，酶活达到4.11 U/mg，金属离子K+、Fe2+和 Ca2+对TcPAM的活性影响较小，而Cu2+和Zn2+有强烈抑制性，表面活性剂CTAB、SDS、Triton X-100和Tween 80对重组酶的活性影响较小，相对酶活保持在90%以上。进一步利用TcPAM催化α-芳香丙氨酸异构合成β-芳香丙氨酸，结果表明：苯环上携带不同基团的α-芳香丙氨酸为底物时，苯环上携带吸电子基团比供电子基团的底物转化率更高，底物的α-氨基容易转移至β位，其中4-MeO-β-苯丙氨酸的产率最高，达到45%，为建立酶法合成β-芳香丙氨酸奠定基础。

关键词: 关键词：紫衫, 苯丙氨酸氨基变位酶, β-芳香丙氨酸

Abstract: Abstract: In order to synthesize high value β-arylalanine, the gene of phenylalanine aminomutase form Taxus chinensis was cloned and expressed in E.coli. The expression vector Pet-sumo-Tcpam was successfully constructed and transferred into E.coli BL21 to express the recombinant enzyme (TcPAM). The electrophoretically pure TcPAM was prepared using affinity chromatography. The results of MS and CD showed that TcPAM could catalyze isomerization of α-arylalanine to R-β-arylalanine. The activity of TcPAM was 4.11 U/mg at optimum condition of 30℃ and pH9.Metal ions K+, Fe2+, Ca2+ and surfactants CTAB,SDS,Triton X-100,Tween 80 had little effect on the activity, and about 90% of activity remained, while Cu2+and Zn2+ had strong inhibitory effect on TcPAM, Furthermore, the α-arylalanines with different groups at benzene ring were used as substrates. The results suggested that the electron-withdrawing group at benzene ring of substrate promote the shift of α-amino group to β site to improve yield of β-aralyalaines, the yield of 4-MeO-β-phenylalanine reached 45 %.

Key words: Keywords: Taxus chinensis, Phenylalanine ainomutase, β-arylalanine

中图分类号:

Q814

丰国强徐文亮宋平李婉珍葛飞陶玉贵朱龙宝. 重组Taxus chinensis 苯丙氨酸氨基变位酶性质表征及合成R-β-芳香丙氨酸[J]. 食品科学, 0, (): 0-0.

Ping SONG. Characterization of recombinant phenylalanine aminomutase from Taxus chinensis and synthesis of R-β-arylalanine[J]. FOOD SCIENCE, 0, (): 0-0.

参考文献

[1]He L Y, Jagtap P G, Kingston D G I, et al.A Common Pharmacophore for Taxol and the Epothilones Based on the Biological Activity of a Taxane Molecule Lacking a C-13 Side Chain[J].Biochemistry, 2000, 39(14):3972-3978
[2]Wu B, Wiktor Szymański, Heberling M M, et al.Aminomutases: mechanistic diversity,biotechnological applications and future perspectives[J].Trends in Biotechnology, 2011, 29(7):352-362
[3]Prabhakaran P C, Woo N T, Yorgey P S, et al.Biosynthesis of blasticidin S from L-alpha-arginine. Stereochemistry in the arginine-2,3-aminomutase reaction.[J].Journal of the American Chemical Society, 1988, 110(17):5785-5791
[4]Yin X, O' Hare T, Gould S J, et al.Identification and cloning of genes encoding viomycin biosynthesis from Streptomyces vinaceus and evidence for involvement of a rare oxygenase[J].Gene, 2003, 312(30):215-224
[5]Kudo F, Miyanaga A, Eguchi T.Biosynthesis of natural products containing β-amino acids[J].Natural Product Reports, 2014, 31(8):1056-1073
[6]Walker K D, Klettke K, Akiyama T, et al.Cloning,Heterologous Expression,and Characterization of a Phenylalanine Aminomutase Involved in Taxol Biosynthesis[J].Journal of Biological Chemistry, 2004, 279(52):53947-53954
[7]Steele C L, Chen Y, Dougherty B A, et al.Purification,cloning,and functional expression of phenylalanine aminomutase: The first committed step in Taxol side-chain biosynthesis[J].Archives of Biochemistry & Biophysics, 2005, 438(1):0-10
[8]Mutatu W, Klettke K L, Foster C, et al.Unusual Mechanism for an Aminomutase Rearrangement: Retention of Configuration at the Migration Termini[J].Biochemistry, 2007, 46(34):9785-9794
[9]Wohlgemuth R.Asymmetric biocatalysis with microbial enzymes and cells[J].Current Opinion in Microbiology, 2010, 13(3):283-292
[10]Grayson J I, Roos J, Osswald S.Development of a Commercial Process for (S)-β-Phenylalanine[J].Organic Process Research & Development, 2011, 15(5):1201-1206
[11]Forro E, Fueloep F.Recent lipase-catalyzed hydrolytic approaches to pharmacologically important beta-and gamma-amino acids[J].Current Medicinal Chemistry, 2012, 19(36):6178-6187
[12]Ratnayake N D, Theisen C, Walter T, et al.Whole-Cell Biocatalytic Production of Variously Substituted β-Aryl- and β-Heteroaryl-β-Amino Acids[J].Journal of Biotechnology, 2016, 217(10):12-21
[13]Mathew S, Nadarajan S P, Sundaramoorthy U, et al.Biotransformation of β-keto nitriles to chiral (S)-β-amino acids using nitrilase and ω-transaminase[J].Biotechnology Letters, 2016, 39(4):535-543
[14]Mathew S, Bea H, Nadarajan S P, et al.Production of chiral β-amino acids using ω-transaminase from Burkholderia graminis[J].Journal of Biotechnology, 2015, 20(196-197):1-8
[15]Mathew S, Jeong S S, Chung T, et al.Asymmetric synthesis of aromatic β-amino acids using ω-transaminase: Optimizing the lipase concentration to obtainthermodynamically unstable β-keto acids[J].Biotechnology Journal, 2016, 11(1):185-190
[16]朱龙宝, 陶玉贵, 葛飞, 等.苯丙氨酸变位酶的制备、表征及催化合成β-芳香丙氨酸[J].高等学校化学学报, 2017, 38(02):206-211
[17]Parmeggiani F, Weise N J, Ahmed S T, et al.Synthetic and therapeutic applications of ammonia-lyases and aminomutases[J].Chemical Reviews, 2018, 118(1):73-118
[18]Huang S X, Lohman J R, Huang T, et al.A new member of the 4-methylideneimidazole-5-one-containing aminomutase family from the enediyne kedarcidin biosynthetic pathway[J].Proceedings of the National Academy of Sciences, 2013, 110(20):8069-8074
[19]Varga A, Gergely Bánóczi, Nagy B, et al.Influence of the aromatic moiety in α- and β-arylalanines on their biotransformation with phenylalanine 2,3-aminomutase from Pantoea agglomerans[J].Rsc Advances, 2016, 6(61):56412-56420
[20]Ratnayake N D, Wanninayake U, Geiger J H, et al.Stereochemistry and Mechanism of a Microbial Phenylalanine Aminomutase[J].Journal of the American Chemical Society, 2011, 133(22):8531-8533
[21]Chesters C, Wilding M, Goodall M, et al.Thermal Bifunctionality of Bacterial Phenylalanine Aminomutase and Ammonia Lyase Enzymes[J].Angewandte Chemie, 2012, 51(18):4344-4348
[22]Jennewein S, Croteau R.Taxol: biosynthesis,molecular genetics,and biotechnological applications[J].Applied Microbiology and Biotechnology, 2001, 57(1-2):13-19
[23]Chirpich T P, Zappia V, Costilow R N, et al.Lysine 2,3-aminomutasePurification and properties of a pyridoxal phosphate and S-adenosylmethionine-activated enzyme[J].Journal of Biological Chemistry, 1970, 245(7):1778-1789
[24] Takeshi Y, Mina O, Kaori Y, et al.Assignment of the CD Cotton Effect to the Chiral Center in Pseurotins, and the Stereochemical Revision of Pseurotin A2.[J].Marine Drugs, 2016, 14(4):74.
[25]朱龙宝, 杨瑾, 葛飞, 等.成团泛菌苯丙氨酸氨基变位酶的性质表征及用于合成β-苯丙氨酸[J].精细化工, 2019, 36(03):449-454

重组Taxus chinensis 苯丙氨酸氨基变位酶性质表征及合成R-β-芳香丙氨酸

Characterization of recombinant phenylalanine aminomutase from Taxus chinensis and synthesis of R-β-arylalanine

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 1

编辑推荐

Metrics

本文评价