화학공학소재연구정보센터
Chinese Journal of Chemical Engineering, Vol.26, No.2, 380-385, 2018
Biosynthesis of 4-hydroxyphenylpyruvic acid from L-tyrosine using recombinant Escherichia coli cells expressing membrane bound L-amino acid deaminase
4-Hydroxyphenylpyruvic acid (4-HPPA), a kind of alpha-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 alpha-keto acid is thought to be both economic and efficient. Among the enzymes that convert amino acid to alpha-keto acid, membrane bound L-amino acid deaminase (mf-AAD), which is anchored to the outer side of the cytomembrane, becomes an ideal enzyme to prepare alpha-keto acid since there is no cofactors needed and H2O2 production during the reaction. In this study, the mf-AAD from Proteus vulgaris was used to prepare whole-cell catalysts to produce 4-HPPA from L-tyrosine. The secretory efficiency of mf-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 mf-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 mf-AAD expression. The reaction conditions for mf-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. (C) 2017 The Chemical Industry and Engineering Society of China, and Chemical Industry Press. All rights reserved.