Cellulase immobilization on magnetite using a polymer spacer

Abstract

Cellulase was immobilized on magnetite particles with a polymer spacer. Several methods of immobilization were investigated for optimizing enzyme activity of the immobilized cellulase (IMC). The optimum method involved the steps which used 3-aminopropyltrimethoxysilane and polyethyleneglycol (PEG). The IMC prepared with the PEG hydrolyzed a highly crystalline cellulose with attachment efficiency ranging from 26 % to 196 % of free cellulase activity on a protein basis. The IMC's prepared with smaller amount of protein tended to show higher efficiency because of less degree of steric crowding. Presence of PEG spacer in IMC enhanced the activity by 3.30 times compared to IMC prepared in absence of PEG. The pH-activity and temperature-activity profiles of IMC were determined with the optimum activity attachment protocol. The optimum pH was shifted from 4.0 to 5.5 upon the immobilization, whereas the optimum temperature was not affected. Thermal denaturation of IMC was a two-stage process; the first stage being rapid with a 1.8 day half-life and the second stage with a 11.3 day half-life. Relative activities of Avicelase, CMCase, and cellobiase of IMC were different from those of free cellulase. It reflected a change occuring in composition of cellulase components during the immobilization reaction. Diffusional limitation was observed in production of reducing sugars from carboxymethyl cellulose with IMC. In contrast, no diffusional limitation was observed in production of glucose from Avicel with IMC. A fluidized-bed reactor was proved useful for continuous production of glucose from particulate cellulose with the IMC. While inhibitory products were continuously washed out of the reactor, the cellulose and IMC particles remained inside.