A concentration-dependent effect of methanol on Candida antarctica lipase B in aqueous phase

• A concentration-dependence effect of methanol on CALB.
• The conformational integrity of the CALB was essential for its activity.
• The use of DLS and CD for the analysis of interaction mechanism.

The influence of methanol on enzyme is of great importance in enzymatic synthesis. The influence of methanol on the activity of Candida antarctica lipase B (CALB) upon hydrolysis of tributyrin was investigated by enzyme kinetics analysis. Dynamic light scattering (DLS) and circular dichroism (CD) was used to measure the variation of hydrodynamic diameter and secondary structure change of CALB at different methanol concentration, respectively. A concentration-dependent influence of methanol on CALB was observed. The activity of CALB slightly increased with less than 1.2 mol l−1 of methanol, followed by sustained decrease at higher methanol concentrations. DLS result indicated that the negative effect of methanol on CALB was supposed to follow different mechanisms due to the significant variation of particle size of CALB at different methanol concentrations. Both reversible competitive inhibition based on enzymatic kinetics analysis and inactivation based on CD spectroscopy study were supposed to contribute to the negative effect of methanol on CALB with methanol concentration in the range of 1.2 mol l−1 to 6 mol l−1. And after arriving at percolation threshold concentration of 6 mol l−1, methanol was mainly performed as aggregate and precipitate agent for CALB, which resulted in low effective enzyme concentration in the solution.

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