Effects of organic solvents on the activity of free and immobilised laccase from Rhus vernicifera.

Rhus laccase (RL) was covalently immobilised onto chitosan, and the effects of immobilisation on pH optimum, enzyme activity, thermostability, and re-use evaluated, using either N,N-dimethyl-p-phenylenediamine or 2,6-dimethoxyphenol as substrate. Immobilisation greatly enhanced enzyme thermostability, resulted in negligible loss of activity, and showed excellent re-use potential, with >80% relative activity retained after 15 cycles in aqueous solvent. Immobilised Rhus laccase (I-RL) was more catalytically active in both hydrophobic and hydrophilic organic solvents than free RL. With water-immiscible organic solvents, both free RL and I-RL required a minimum water content to achieve activity. With water-miscible organic solvents, in general a water content of ∼20-50% (v/v) was required to achieve activity using free RL, whereas with I-RL less water was generally required to achieve enzyme activity, and therefore considerably higher relative activity was exhibited at lower water contents. Kinetic investigations showed that the rate of substrate disappearance generally followed a pseudo-first-order law, and for evaluated water-immiscible organic solvents rate constants generally increased with decrease of hydrophobicity, however, in water-miscible organic solvents no such relationship was observed. Some discussion of the potential interactions between organic solvent molecules and enzyme active centres was provided to explain obtained results.