Stabilization of β-glucosidase in Aspergillus phoenicis QM 329 pellets

Abstract

Glucose, derived from the enzymatic hydrolysis of cellulose, can be used as a carbon source to produce liquid fuel by fermentation. To get a higher hydrolysis rate in lignocellulose degradation, it is necessary to supplement the cellulase from Trichoderma with exogenous β-glucosidase. Aspergillus phoenicis QM329 produces β-glucosidase at a high level. It can grow in pellet form. This can be used as an immobilized enzyme preparation for enzymatic hydrolysis of cellobiose. Stabilization is necessary owing to the enzyme leakage from the dried pellet due to the cell wall damage which has followed during the drying. Stabilization was performed with glutaraldehyde which cross-links the enzyme molecules as well as various cell compartments. Different stabilization times and glutaraldehyde concentrations were used, and the pellets were evaluated in consecutive cellobiose hydrolysis series. Using 0.1, 0.25, and 0.5% (v/v) glutaraldehyde concentration and 24, 48, and 72 h stabilization time, it was found that both parameters increased the operational stability which is effected on the enzyme leakage and the number of functioning active sides. To examine the behavior of the self-immobilized pellets on a longer time scale, 60-day hydrolyses were performed by using stabilized pellets with 0.05, 1.025, and 2.0% (v/v) glutaraldehyde concentration and 24, 60, and 96 h stabilization time according to the experimental design. To decrease uncertainty, three series of experiments were run. By the analysis of the initial hydrolysis rate, the 1.2% (v/v) glutaraldehyde concentration was found to get optimal operational stability pellets and the stabilization time was not strongly influencing the stabilization. Finally, the operational stability of optimally treated pellets and the uncertainty of treatments with glutaraldehyde were examined, and they were found to be appropriate and reproducible.