Mechanism-based site-directed mutagenesis to shift the optimum pH of the phenylalanine ammonia-lyase from Rhodotorula glutinis JN-1

Phenylalanine ammonia-lyase ( Rg PAL) from Rhodotorula glutinis JN-1 stereoselectively catalyzes the conversion of the l -phenylalanine into trans -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 Rg PAL is 9 and the Rg PAL exhibits low catalytic efficiency at acidic side. Therefore, a mutant Rg PAL with a lower optimum pH is expected. Based on catalytic mechanism and structure analysis, we constructed a mutant Rg 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 Rg PAL, the improvement of the Rg PAL-Q137E might be due to the negative charge of Glu137 which could stabilize the intermediate transition states through electrostatic interaction. The Rg PAL-Q137E mutant was used to resolve the racemic dl -phenylalanine, and the conversion rate and the ee D value of d -phenylalanine using Rg 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 Rg PAL.