Biosynthesis of 4-hydroxyphenylpyruvic acid from L-tyrosine using recombinant Escherichia coli cells expressing membrane bound L-amino acid deaminase  

作　　者：Huanru Ding Weirui Zhao Changjiang Lu Jun Huang Sheng Hu Shanjing Yao Lehe Mei Jinbo Wang Jiaqi Mei 

机构地区：[1]School of Biotechnology and Chemical Engineering,Ningbo Institute of Technology,Zhejiang University,Ningbo 315100,China [2]Department of Chemical and Biolagical Engineering,Zhejiang University,Hangzhou 310027,China [3]School of Biological and Chemical Engineering,Zhejiang University of Science and Technology,Harlgzhou 310023,China [4]Department of Chemical Engineering,The University of utah,Salt Lake City 84102,America

出　　处：《Chinese Journal of Chemical Engineering》2018年第2期380-385,共6页中国化学工程学报（英文版）

基　　金：Supported by the National Natural Science Foundation of China(31470793,31670804);China Postdoctoral Science Foundation(2016M592003);the Natural Science Foundation of Zhejiang Province(LZ13B060002);the General Scientific Research Project of Zhejiang Provincial Education Department(Y201432760)

摘　　要：4-Hydroxyphenylpyruvic acid （4-HPPA）, a kind of α-keto acid, is an intermediate in the metabolism of tyrosine and has a wide range of application in food, pharmaceutical and chemical industry. Using amino acids as raw material to prod uce the corresponding α-keto acid is thought to be both economic and efficient. Among the enzymes that convert amino acid to α-keto acid, membrane bound L-amino acid deaminase （mL-AAD）, which is anchored to the outer side of the cytomembrane, becomes an ideal enzyme to prepare α-keto acid since there is no cofactors needed and H2O2 production during the reaction. In this study, the mL-AAD from Proteus vulgaris was used to prepare whole-cell catalysts to produce 4-HPPA from L-tyrosine. The secretory efficiency of mL-AAD conducted by its own twin-arginine signal peptide （twin-arginine translocation pathway, Tat） and integrated pelB （the general secretory pathway, Sec）-Tat signal peptide was determined and compared firstly, using two pET systems （pET28a and pET20b）. It was found that the Tat pathway （pET28a-mlaad） resulted in higher cell-associated mL-AAD activity and cell biomass, and was more beneficial to prepare biocatalyst. In addition, expression hosts BI21 （DE3） and 0.05 mmol. L- 1 IPTG were found to be suitable for mL-AAD expression. The reaction conditions for mL-AAD were optimized and 72.72 mmol,L 1 4-HPPA was obtained from 100 mmol.L 1 tyrosine in 10 h under the optimized conditions. This bioprocess, which is more eco-friendly and economical than the traditional chemical synthesis ways, has great potential for industrial application.4-Hydroxyphenylpyruvic acid(4-HPPA), a kind of α-keto acid, is an intermediate in the metabolism of tyrosine and has a wide range of application in food, pharmaceutical and chemical industry. Using amino acids as raw material to produce the corresponding α-keto acid is thought to be both economic and efficient. Among the enzymes that convert amino acid to α-keto acid, membrane bound L-amino acid deaminase(mL-AAD), which is anchored to the outer side of the cytomembrane, becomes an ideal enzyme to prepare α-keto acid since there is no cofactors needed and H_2 O_2 production during the reaction. In this study, the mL-AAD from Proteus vulgaris was used to prepare whole-cell catalysts to produce 4-HPPA from L-tyrosine. The secretory efficiency of mL-AAD conducted by its own twin-arginine signal peptide(twin-arginine translocation pathway, Tat) and integrated pelB(the general secretory pathway, Sec)-Tat signal peptide was determined and compared firstly, using two pET systems(pET28 a and pET20 b). It was found that the Tat pathway(pET28 a-mlaad) resulted in higher cell-associated mL-AAD activity and cell biomass, and was more beneficial to prepare biocatalyst. In addition, expression hosts B121(DE3) and 0.05 mmol ·L^(-1) IPTG were found to be suitable for mL-AAD expression. The reaction conditions for mL-AAD were optimized and 72.72 mmol·L^(-1) 4-HPPA was obtained from 100 mmol·L^(-1) tyrosine in 10 h under the optimized conditions. This bioprocess, which is more eco-friendly and economical than the traditional chemical synthesis ways, has great potential for industrial application.

关 键 词：4-Hydroxyphenylpyruvic acidc-tyrosine Membrane-bound L-amino acid deaminases Biocatalysis Molecular biology Biological engineering 

分 类 号：TQ245.4[化学工程—有机化工]