In vitro degradation of chitosan by a commercial enzyme preparation: effect of molecular weight and degree of deacetylation

Abstract

A commercially available almond emulsin β-glucosidase preparation has been reported to have chitobiose activity, and can hydrolyze chitin substrates due to a chitinase present in the enzyme preparation. This β-glucosidase preparation was used to investigate hydrolytic activity on five chitosan samples with different molecular weight and degree of deacetylation. The degree of deacetylation and molecular weight of the chitosan samples were determined using a circular dichroism and a viscometric method, respectively. The hydrolytic activity of this β-glucosidase preparation on chitosan was monitored viscometrically as the most convenient means of screening. Solutions of chitosan in pH 5.0 acetate buffer were prepared using the different viscosity grades of chitosan. The specific viscosity, measured after addition of β-glucosidase to the above solutions, decreased dramatically over time in comparison to that of the respective control mixture without enzyme. Eadie–Hofstee plots established that hydrolysis of chitosan by this enzyme preparation obeyed Michaelis–Menten kinetics. Apparent Michaelis–Menten parameters and initial degradation rates were calculated and compared to determine the influences of the degree of deacetylation and molecular weight on the hydrolysis. The results show that higher molecular weight and higher degree of deacetylation chitosans possessed a lower affinity for the enzyme and a slower degradation rate. Faster degradation rates, then, are expected with lower molecular weight and low degree of deacetylation chitosans. Hydrolysis of these chitosan samples confirms the existence of a chitinase in the almond emulsin β-glucosidase preparation, and further studies are warranted.