Properties And Reaction Mechanism Of Cross-linked Cellulase Aggregates

Cross-linked enzyme aggregates(CLEAs) exhibit high active recovery, steady structure and stronger environment adaptability compared to the immobilized enzymes prepared by traditional methods. In this study, cross-linked cellulase aggregates were prepared by cellulase Trichoderma viride. The preparation process and properties of cross-linked cellulase aggregates (CLEAs-C) were investigated. The reaction mechanism of CLEAs-C was studied by analyzing the structure and characteristics. The main results listed as followed:CLEAs-C were prepared by ammonium sulfate used as the precipitant and glutaraldehyde used as and cross-linker. The results showed the optimum parameters of preparing CLEAs-C were: ammonium sulfate saturation of 95%, pH value of 6.0, the amount of cellulases of 50mg/mL, glutaraldehyde concentration of 3%, immobilization temperature of 30℃, immobilization time of 10h. Under the optimum parameters, the maximal relatively active recovery of CLEAs-C was about 80%.The enzymatic properties of CLEAs-C were studied in this paper. Compared with free cellulase, the optimal temperature of CLEAs-C was improved from 50℃to 60℃, the optimal pH value and ion concentration were 3.0 and 0.2mol/L respectively, which was the same as those of free cellulases. The stability of temperature and pH of CLEAs-C were superior to free cellulase, CLEAs-C had above 80% store activity recovery after 28 days compared with initial activity. Under the optimal conditions of enzymic preparation and hydrolysis, the highest activity recovery of CLEAs-C was increased by 250% compared with free cellulases.The characters and stutruct structure of CLEAs-C were analyzed using FTIR, and Atomic Force Microscope. The results showed the rigid structures of secondary structure were increased, exhibiting a better stability. It was speculated that there were some interspace in CLEAs-C, which make made the active sites of cellulases extend and allow the substrate molecule entering to contact with active sites, improving the enzymatic hydrolysis could be happened.The partical size of CLEAs-C prepared by different stirring rate was analyzed by Malvern Laser Particle Sizer, and the reaction mechanism of CLEAs-C was studied in terms of relatively active recovery of CLEAs-C. The results showed the activity of CLEAs-C increased firstly and reached the maximum, and then decreased with particle size. When the particle size of CLEAs-C was small, it meant less cellulases were cross-linked or less steric space inside the CLEAs-C, resulting into lower catalysis capacity. However, in the case of large particle size, the specific surface area of aggregates was small, decreasing the possibility of contacting between cellulase and substrate. Therefore, when CLEAs-C had proper particle sizes, proper specific surface area, proper steric space inside and proper degree of aggregation, CLEAs-C inhibited high catalysis capacity.