Characterization and thermal inactivation kinetics of highly thermostable ramie leaf β-amylase.

Abstract

We characterized ramie leaf β-amylase, and determined its thermostability and kinetic parameters. The enzyme was purified 53-fold using ammonium sulfate fractionation (40-60% saturation), anion exchange chromatography on DEAE-cellulose and gel permeation chromatography on Superdex-200. The purified enzyme was identified as β-amylase with molecular mass of 42kD. The enzyme displayed Km and kcat values for soluble potato starch of 1.1mg/mL and 7.8s-1, respectively. The enzyme had a temperature optimum of 65°C, and its activity at 70°C was 92% of that at the optimal temperature after a 15-min incubation. Furthermore, enzyme activity was stable during treatment at 55°C for 60min but was inactivated rapidly at >75°C. This thermal behavior indicates that ramie leaf β-amylase has excellent intermediate temperature-stable enzyme properties for the baking and bio-industries. Inactivation of the enzyme followed first-order kinetics in the range of 55-80°C. The enthalpy change of thermal inactivation (ΔH‡), ΔG‡, and ΔS‡ were 237.2kJ/mol, 107.7kJ/mol, and 0.39kJ/molK at 333K, respectively. The D-value at 65°C (=110min) and the z-value (=9.4°C) are given for food processing.