1). The mutations at this site decreased or lost the activity except the RgPAL-Q137E. The RgPAL-Q137E mutant had an extended optimum pH 7–9 with the activity of about 1.8-fold higher than that of the wild type at pH 7 ( Fig. 6). The optimum pH of an enzyme depends on the ionizable amino acids at active site which are involved in catalysis [10] and [11] or stabilization of transition state

by electrostatic interaction [17] and [18] Selleckchem U0126 and enzyme substrate binding [6]. The Glu137 might be involved in the stabilization of transition state by electrostatic interaction. According to the Friedel–Crafts-type mechanism, the carbocation intermediate is large energy barrier which must be surpassed [3]. The improvements of RgPAL-Q137E at pH 7 may be due to the negative charge of Glu137 which facilitates

stabilization of carbocation intermediate to reduce the energy barrier through electrostatic interaction. The kcat value provides an assessment of the specificity and is directly related to the free energy of activation of the transition state [8], [17] and [18]. Therefore, the calculation of mutational effects using kcat provides insights on the contribution of electrostatics. As shown in Table 1, the kcat mutational effect was 0.56 at pH 7, Alectinib cell line which represents a ΔΔG‡ of 1.56 kcal/mol, and the kcat mutational effect was found to be 0.93 at pH 9, which represents a ΔΔG‡ of 0.19 kcal/mol. Therefore, a negative charge at position 137contributes 1.32 kcal/mol (1.56 kcal/mol − 0.19 kcal/mol) of net free energy toward the electrostatic stabilization of the transition state. In addition, the negative charge of Glu137 is also likely to counteract the adverse effects of the positive charge of His136, which favors a protonated state at pH 7. The pKa value of the imidazole group of His is approximately 5–7, and the His136 tends to exhibit

the protonated state with a positive charge at pH 7, which is disadvantageous to the electrophilic attack MIO on the aromatic ring of the substrate by the MIO. RgPAL could be used to resolve dl-phenylalanine to produce Adenosine triphosphate optical pure d-phenylalanine, since the pH is one of the most important quality parameters for PAL catalytic reaction to resolve the dl-phenylalanine. In this study, the optimum pH of RgPAL was shifted toward the acidic side through site-directed mutagenesis based on the analysis of catalytic mechanism and structure. The RgPAL-Q137E mutant exhibited a wide pH range from 7 to 9. When this mutant was used to resolve the dl-phenylalanine, the conversion rate and eeD value increased by 29% and 48%, and the ultimate conversion rate and eeD value achieved 93% and 86%, respectively. However, the eeD value and conversion rate using RgPAL-Q137E need to be further improved, such research is currently carrying out in our lab. This work provides an effective strategy to shift the optimum pH for the enzyme application.