Zhu Longbao, Zhou Li, Cui Wenjing, Liu Zhongmei, Zhou Zhemin
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Biotechnol Rep (Amst). 2014 Jun 6;3:21-26. doi: 10.1016/j.btre.2014.06.001. eCollection 2014 Sep.
Phenylalanine ammonia-lyase (PAL) from JN-1 stereoselectively catalyzes the conversion of the l-phenylalanine into -cinnamic acid and ammonia, and was used in chiral resolution of dl-phenylalanine to produce the d-phenylalanine under acidic condition. However, the optimum pH of PAL is 9 and the PAL exhibits low catalytic efficiency at acidic side. Therefore, a mutant PAL with a lower optimum pH is expected. Based on catalytic mechanism and structure analysis, we constructed a mutant PAL-Q137E by site-directed mutagenesis, and found that this mutant had an extended optimum pH 7-9 with activity of 1.8-fold higher than that of the wild type at pH 7. As revealed by Friedel-Crafts-type mechanism of PAL, the improvement of the PAL-Q137E might be due to the negative charge of Glu137 which could stabilize the intermediate transition states through electrostatic interaction. The PAL-Q137E mutant was used to resolve the racemic dl-phenylalanine, and the conversion rate and the value of d-phenylalanine using PAL-Q137E at pH 7 were increased by 29% and 48%, and achieved 93% and 86%, respectively. This work provides an effective strategy to shift the optimum pH which is favorable to further applications of PAL.
来自JN-1的苯丙氨酸解氨酶（PAL）可立体选择性地催化L-苯丙氨酸转化为反式肉桂酸和氨，并用于在酸性条件下拆分外消旋苯丙氨酸以生产D-苯丙氨酸。然而，PAL的最适pH为9，在酸性条件下催化效率较低。因此，期望获得最适pH较低的突变型PAL。基于催化机制和结构分析，我们通过定点诱变构建了突变型PAL-Q137E，发现该突变体的最适pH范围扩展至7-9，且在pH 7时的活性比野生型高1.8倍。如PAL的傅克型机制所示，PAL-Q137E活性的提高可能是由于Glu137的负电荷通过静电相互作用稳定了中间过渡态。利用PAL-Q137E拆分外消旋苯丙氨酸，在pH 7时，D-苯丙氨酸的转化率和e值分别提高了29%和48%，分别达到93%和86%。这项工作提供了一种有效的策略来改变最适pH，有利于PAL的进一步应用。
