Preparation And Influence Parameters Of Cross-Linked Lipase Aggregates

As a new type of carrier-free immobilized enzyme, cross-linked enzyme aggregates(CLEAs) with characteristics of simple preparation, high activity per unit volume and good stability has been extensively used in many fields. The studies of CLEAs were focused on its preparation and characterization, and little attention to the particle size with the regulation and its influence on the performance of CLEAs. However, the particle size of CLEAs not only affects the mass transfer efficiency, but also related to the stability of CLEAs. Therefore, controlling of particle size on CLEAs is crucial to its research and application. Hence, this study explores and optimizes conditions on cross-linked enzyme aggregate’s preparation conditions with Candida lipase (Candida sp.1619), on this basis have been through different means to regulate the size of CLEAs and investigate the differences of them.(1) Use carrier-free immobilized enzyme technology, the CLEAs with ideal activity and stability was obtained with three times the volume of enzyme solution of acetone as the precipitant, glutaraldehyde(2.08%) as cross-linker, under the120min’ cross-linking time,20℃cross-linking temperature,4000rmin-1centrifugation speed.(2) With the modifying of surfactant Tween-20and BS A, the activity of CLEAs increased by7%and22%, respectively. Investigation on characteristic of immobilized enzyme showed that it was more stable than free lipase in thermo-stability and pH stability. For CLEAs, the optimum range of pH values was6.0～9.0, the activity retained more than60%with2h heat treatment under50℃. With the analysis of Kmå'ŒVmax to CLEAs and free lipase, the CLEAs’affinity to the substrate is weaker but the catalytic per unit time is better than the free enzyme.(3) With the analysis of influence factors to CLEAs’ particle size, it was showed that the cross-linking process is the key step. With the ultrasound in the process, the size of particles can be reduced significantly, but the morphology of particles was not obvious. With vortex or by adding appropriate amount of BSA, the particle size can be reduced from151-180nm to19-26nm effectively. Meanwhile, the higher concentration of cross-linking agent and its molecular weight, the bigger size of CLEAs will be obtain.