奇异变形杆菌工程化L-氨基酸脱氨酶催化生成苯丙酮酸
Production of phenylpyruvic acid by engineered L-amino acid deaminase from Proteus mirabilis.
作者信息
Liu Jia, Liu Jianmin, Yang Bin, Gao Cong, Song Wei, Hu Guipeng, Liu Liming, Wu Jing
机构信息
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China.
International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, 214122, China.
出版信息
Biotechnol Lett. 2022 Jun;44(5-6):635-642. doi: 10.1007/s10529-022-03245-y. Epub 2022 Apr 16.
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 M2 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.
目的
本研究旨在开发一种高效的酶促策略，用于从L-苯丙酮酸（L-Phe）工业生产苯丙酮酸（PPA）。
结果
奇异变形杆菌的L-氨基酸脱氨酶在大肠杆菌BL21（DE3）中表达，并通过构象动力学工程进行改造以消除产物抑制。基于结构分析，确定了两个残基（E145/L341），以减少产物与酶之间的相互作用并增加蛋白质的灵活性，从而促进产物释放。与野生型相比，突变体M2的产物抑制降低了3.84倍，催化效率提高了1.35倍。最后，在5-L生物反应器中获得了81.2 g/L的PPA产量，转化率为99.6%。
结论
工程化催化剂可显著降低产物抑制，促进PPA的有效工业合成。
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