α-Chymotrypsin shows higher activity in water as well as organic solvents after three phase partitioning

Abstract

Alpha-Chymotrypsin was found to show a 119% increase in activity after three phase partitioning. The k_cat/K_m of the partitioned enzyme (TPP-C) for hydrolysis of Bz-Tyr-OEt in aqueous medium at 25°C was found to be 48.3×10⁴ mM⁻¹ min⁻¹ as compared to the corresponding value of 17.7×10⁴ mM⁻¹ min⁻¹ for the untreated control (C). The λ_max of the fluorescence emission spectrum of TPP-C showed 178% increase in the quantum yield when compared to C. TPP-C showed a 2.94 and 3.58 fold increase (as compared to C) in initial rates for formation of the ester Ac-Phe-OEt (from Ac-Phe and ethanol) in low water containing toluene and n-octane, respectively. It was found that TPP-C also showed the phenomenon of pH memory. At 5% (v v⁻¹) water (in t-amyl alcohol), while no esterification was observed with C, TPP-C still showed significant level of esterification activity.

Bz-Tyr-OEt, Benzoyl tyrosine ethyl ester; Ac-Phe, N-acetyl phenylalanine; Ac-Phe-OEt, N-acetyl phenylalanine ethyl ester; TPP, Three phase partitioning; C, Untreated α-chymotrypsin; TPP-C, α-Chymotrypsin subjected to TPP; k_cat, Catalytic efficiency; K_m, Michaelis constant

• α-chymotrypsin
• nonaqueous enzymology
• solvent effects
• three phase partitioning