Three-phase partitioning and immobilization of Bacillus methylotrophicus Y37 cellulase on organo-bentonite and its kinetic and thermodynamic properties

Abstract

AbstractIn this study, for the first time Bacillus methylotrophicus Y37 cellulase was purified and recovered in a single step by three-phase partitioning (TPP). The optimal purification parameters for TPP were 40% ammonium sulfate saturation (m/v) with a 1.0:1.0 (v/v) ratio of crude extract:t-butanol, which gave 5.8-fold purification with 155% recovery of cellulase. Non-covalent immobilization of the partitioned cellulase was performed using bentonite as a support material. The activity observed in the 20th experiment was 100%. The optimal pH values and temperatures determined for the free enzyme and the immobilized enzyme were 5.0 and 6.0 and 45°C and 50°C, respectively. The Arrhenius activation energy (Ea) of the immobilized enzyme was lower than that of the free enzyme, whereas the Michaelis–Menten constant (Km) and maximum velocity (Vm) of the immobilized enzyme increased. The turnover number (kcat) and the catalytic performance (kcat/Km) demonstrated the improved catalytic properties of the immobilized enzyme compared to the free enzyme. Immobilization of cellulase is thermodynamically preferred.