Enhanced activity,enantioselectivity and stability of papain in asymmetric hydrolysis of d,l-p-hydroxyphenylglycine methyl ester with ionic liquid

Papain-mediated asymmetric hydrolysis of D,L-p-hydroxyphenylglycine methyl ester (D,L-HPGME) was examined in the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM·BF₄) and different solvents. The activity of the enzyme varied widely with change in BMIM·BF₄ concentration, with 12.5% (v/v) being the optimum BMIM·BF₄ concentration for the reaction. Papain displayed much higher hydrolytic activity and enantioselectivity in phosphate buffer solution of 12.5% (v/v) BMIM·BF₄ (pH 7.0) than in other media examined. Comparative studies on the kinetics and activation energy (E_a) of this reaction performed in different media showed a higher V_max, a lower K_m and a lower E_a for the reaction taking place in phosphate buffer solution of 12.5% (v/v) BMIM·BF₄ than in other media tested. The stability of papain at 45°C was considerably enhanced in BMIM·BF₄ as compared with aqueous buffer, 2-propanol and acetonitrile. A half-life time of 169 h was observed with BMIM·BF₄ in the presence of substrate, which was 9.2–16.8-fold higher than those with the other solvents. These results suggested that BMIM·BF₄ is an excellent reaction medium for this reaction.