Kinetics and stability of Chitosan-immobilized laccase

Abstract

Laccase, from Coriolus versicolor, was immobilized to the biopolymers of chitosan with varying average molecular weights (Mw= 130,214,432, and 505 kDa) using the carbodiimide active esther method. The effect of the biopolymer molecular weight on the chitosan-immobilized laccase were analyzed for the activity of soluble chitosan-immobilized laccase conjugates for the oxidation of the substrate 2,2'-azino-bis3-ethylbenzthiazoleine-6-sulfonic acid (ABTS). Although the chitosan Iaccase conjugates of different molecular weights maintained from 20 - 40% residual activity compared to free laccase, the immobilized laccase was capable of being recovered and reused multiple times. During repeated cycles ofprecipitation and dissolution of conjugates prepared with laccase immobilized to chitosan of Mw 214 kDa up to 60% of residual activity was retained after 15 cycles. Activities of the chitosan-laccase conjugates retained similar responsiveness to changes in pH (1.0 to 8.0) and temperature (20-80°C) compared to free laccase. Results of kinetic parameters (Km, Vmax, and Kcat) for analysis comparing the effect of Mw and laccase density on conjugation show two different relationships. Although the affinity of the conjugates, Km, for ABTS showed no apparent trend based on Mw of chitosan, laccase conjugated to chitosan (Mw 132 kDa) resulted in Vmax and Kcat (0.37 umole mL-1 min-1 and 2.1x10+6) to be approximately half the values for free laccase (0.60 umole mL-1 min-1 and 4.4x10+6 respectively) and decreased as Mw increased from 132 to 505 kDa. Increasing the amount of chitosan conjugated to laccase (2.2 to 8.8 mg chitosan/mL) decreased Km values from 349 to 82 uM compared to 65 uM for free chitosan. However, Vmax decreased (268 to 0.63 umole mL-1 min-1)as the amount of chitosan conjugated to laccase increased compared to Vmax for free chitosan (0.46 umole mL-1 min-1). Kcat for conjugated laccase remained 100 times less than that for free laccase (2.0x10+5 and 1.3x10+7, respectively). These results are indicative that the properties of chitosan affect the laccases catalytic sites and global structure of laccase conjugated to chitosan. Keywords: Biopolymer, Chitosan, Bioconjugate, Laccase