Preparation And Characterization Of Lipase Immobilized On Reversibly Soluble-insoluble N-(2-carboxylbenzoyl) Chitosan

Lipases belong to the carboxylic ester hydrolases and catalyse the hydrolysisof triacylglycerols to glycerol and free fatty acids. However, their abilities to performhydrolysis, alcoholysis, esterification,transesterification, and interesterification givethem numerous applications throughout the biotechnological fields, includingoleochemical industry, dairy industry, food industry, cosmetic industry,pharmaceutical industry, and chemical industry.Enzyme immobilization on insoluble solid supports, besides facilitating therecovery and further reuse of catalyst, provides additional important advantagesover free enzyme such as increasing enzyme stability and improving enzymeproperties. Various methods available for immobilization of enzymes can bedivided into two general classes: physical methods, where there are only weakinteractions between support and enzyme, and chemical methods, where covalentbonds are formed between support and enzyme. Specifically, the immobilization ofenzymes includes following four methods: adsorption onto an insoluble solidmaterial, entrapment or encapsulation in a polymeric matrix, crosslinking with abifunctional reagent, and covalent binding to an insoluble solid carrier. But inbiochemical reactions, the enzymes immobilized on insoluble solid carrier usuallyexhibit lower activity than their free form due to diffusional limitation and sterichindrance in a heterogeneous reaction system. To overcome this problem, it hasbeen suggested to immobilize enzymes to a reversibly soluble-insoluble polymer.The immobilized enzymes could be utilized in a soluble form, after which theenzymes can be recovered in an insoluble state by changing the external conditions,such as pH or temperature Chitosan is a natural polysaccharide composed of randomly distributedb-1,4-linked D-glucosamine and N-acetyl-D-glucosamine and producedcommercially by deacetylation of chitin which is the second most abundantbiomass next to cellulose. The reactive amino group in chitosan can provide easyderivatization of this polysaccharide with some functional reagents for widerapplication. Chitosan has been widely used in industrial and biomedical fields andis considered to be a suitable support for enzyme immobilization because of itsreproducibility, biocompatibility, biodegradability and processability.In the present work, N-(2-carboxybenzoyl) chitosan (CBC) which exhibitedreversible solubility in aqueous solution with pH change was prepared byintroducing phthaloyl groups into chitosan N-terminal of the glucosamine units. Acommercial lipase was covalently immobilized on this reversibly soluble–insolublepolymer using glutaraldehyde as coupling agent to assess potential application ofCBC as the carrier material for the enzyme immobilization. The properties ofimmobilized lipase were systematically studied and compared with its freecounterpart. The obtained CBC exhibited reversible solubility in aqueous solution;it was soluble at pH above3.8and precipitated at pH below3.4. The porcinepancreatic lipase was covalently immobilized on CBC with glutaraldehyde as thecrosslinking agent. Under the optimal immobilization condition, the retentionactivity of the immobilized lipase was found to be69.8%. The maximum activityof lipase immobilized on CBC was observed at40℃, pH8.0, while the free lipasepresented maximum activity at37℃,pH7.5. The lipase immobilized on CBCexhibited improved thermal and storage stabilities and retained58.7%of its initialactivity after9times of repeated use.