The CD spectra were measured on MOS-450/AF-CD-STP-A (Bio-Logic, France) at a protein concentration of 0.1 mg/mL in 50 mM Tris/HCl buffer (pH 8.6) using a 1 cm path-length

quartz cuvette. To minimize the signal baseline drift, the spectropolarimeter and xenon lamp were warmed up at least 30 min prior to each experiment. The enzyme data in the 190–240 nm bands were collected, and which the spectrum obtained for a buffer blank was subtracted from these data. The assay to determine the kinetic parameters were performed using different concentrations of l-phenylalanine (1–20 mM) (Sigma–Aldrich, Germany). The reactions were initiated by the addition of an appropriate quantity of RgPAL to each reaction system. The reaction was conducted at 40 °C and check details stopped by addition of 0.5 mL of methanol. The formation of

trans-cinnamic acid was BIBW2992 nmr measured by HPLC (Hitachi, Japan) at 290 nm, the mobile phase contained 50% methanol. The obtained experimental dependences of the initial catalytic rates on the substrate concentrations were fitted to Michaelis–Menten equation through nonlinear regression analysis using Origin (7.5 versions). One enzyme activity unit was defined as the amount of enzyme that produced 1 μmol trans-cinnamic acid per minute at 40 °C. The effects of the pH were determined at 40 °C using a series of buffers with various pH values (pH 5.0–7.0, 50 mM sodium acetate buffer; pH 7.0–9.0, 50 mM Tris–HCl buffer; pH 9.0–12.0, 50 mM sodium carbonate buffer). The chiral resolutions of dl-phenylalanine using RgPAL and RgPAL-Q137E were performed at pH 7 and pH 9, respectively. The experiments were carried out in

500 mL batch conical flasks with lid in a rotating shaker and contained 300 mL of dl-phenylalanine (100 mM) and 250 μg of pure enzyme at 40 °C. The conversion rate of l-phenylalanine and the eeD value of d-phenylalanine were calculated by the following equations: conversion rate=[(Lphe,in−Lphe,out)Lphe,in]×100%eeD=[(Dphe−Lphe,out)(Dphe+Lphe,out)]×100%;where the eeD is the enantiomeric excess of d-phenylalanine; the Lphe, in is the initial concentration of l-phenylalanine; the Lphe,out is the residual concentration of l-phenylalanine selleck chemical after resolution; the Dphe is the concentration of d-phenylalanine. The d-phenylalanine and l-phenylalanine are detected through HPLC (Hitachi, Japan) at 205 nm according to the method described by Fukuhara [7]. The mobile phase contained 20% methanol and a complex of optically active L-Pro-Cu(II) (1.5 mM L-Pro and 0.75 mM CuSO4). The “mutational effect” was determined by dividing the kcat value of the mutant enzyme by that of the wild type, and the free energy (ΔΔG‡) was calculated from the following equation: ΔΔG‡ = −RTln (mutational effect) as described by Olucha (2011, 2012) [17] and [18].