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 ...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.展开更多
基金Supported by the National Natural Science Foundation of China(31470793,31670804)China Postdoctoral Science Foundation(2016M592003)+1 种基金the Natural Science Foundation of Zhejiang Province(LZ13B060002)the General Scientific Research Project of Zhejiang Provincial Education Department(Y201432760)
文摘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.