Jia Liu1,2, Jianmin Liu3, Bin Yang1,2, Cong Gao1,2, Wei Song4, Guipeng Hu4, Liming Liu1,2, Jing Wu5. 1. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China. 2. International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, 214122, China. 3. Shandong Huishilai Biotechnology Co., Ltd, Jinan, 250098, China. 4. School of Life Sciences and Health Engineering, Jiangnan University, Wuxi, 214122, China. 5. School of Life Sciences and Health Engineering, Jiangnan University, Wuxi, 214122, China. wujing@jiangnan.edu.cn.
Abstract
OBJECTIVES: This study aimed to develop an efficient enzymatic strategy for the industrial production of phenylpyruvate (PPA) from L-phenylpyruvic acid (L-Phe). RESULTS: L-amino acid deaminase from Proteus mirabilis was expressed in Escherichia coli BL21 (DE3) and modified to release product inhibition by employing conformational dynamics engineering. Based on structural analysis, two residues (E145/L341) were identified for reducing interactions between the product and enzyme and increasing flexibility of the protein, thereby facilitating the product release. The mutant M2E145A/E341A exhibited a 3.84-fold reduction in product inhibition and a 1.35-fold increase in catalytic efficiency in comparison to the wild type. Finally, 81.2 g/L PPA production with a conversion of 99.6% was obtained in a 5-L bioreactor. CONCLUSIONS: The engineered catalyst can significantly reduce product inhibition and facilitate the effective industrial synthesis of PPA.
OBJECTIVES: This study aimed to develop an efficient enzymatic strategy for the industrial production of phenylpyruvate (PPA) from L-phenylpyruvic acid (L-Phe). RESULTS: L-amino acid deaminase from Proteus mirabilis was expressed in Escherichia coli BL21 (DE3) and modified to release product inhibition by employing conformational dynamics engineering. Based on structural analysis, two residues (E145/L341) were identified for reducing interactions between the product and enzyme and increasing flexibility of the protein, thereby facilitating the product release. The mutant M2E145A/E341A exhibited a 3.84-fold reduction in product inhibition and a 1.35-fold increase in catalytic efficiency in comparison to the wild type. Finally, 81.2 g/L PPA production with a conversion of 99.6% was obtained in a 5-L bioreactor. CONCLUSIONS: The engineered catalyst can significantly reduce product inhibition and facilitate the effective industrial synthesis of PPA.
Authors: Usama M Hegazy; Mohamed I El-Khonezy; Abeer Shokeer; Somaia S Abdel-Ghany; Roqaya I Bassuny; Amal Z Barakat; Walaa H Salama; Rasha A M Azouz; Afaf S Fahmy Journal: J Biochem Date: 2019-02-01 Impact factor: 3.387