The Study On Enzymatic Hydrolysis Of Chitosan To Prepare Chitooligosaccharide In Ionic Liquid System

Chitosan, is an important biomass resource, the content is only after cellulose,Chitooligosaccharide is a kind of water-soluble amino-sugar compound ofpolymerization degree between2to20, which is prepared by chitosan hydrolysis.Compared with chitosan, the chitooligosaccharide has received extensive attentionsbecause of its unique biological and physicochemical properties. Its water-solubility isbetter than that of chitosan, then it is more conducive for living organisms to digestand absorb, and has good physical and chemical properties.Enzymatic degradation process to prepare chitooligosaccharide is of many merits,such as mild reaction conditions, strong specificity, low environmental pollution,controllable molecular weight of the degradation products. Compared with the specificenzyme, the non-specific enzymes such as cellulase and amylase, were moreinexpensive and also showed good activity and stability on the degradation of chitosan,which can be indeed a very suitable alternative. However, the chitosan has stronghydrogen bonds, which can reduce the solubility of chitosan in the generic smallmolecule acidic solvents such as acetic acid, it increases the burden of processing andpreparation costs of the enzymatic preparation of chitooligosaccharide, and reducesproduction efficiency. Furthermore, the small molecule acid solvents in the preparationprocess will have a significant negative impact on the environment. Therefore, lookingfor a new green system that not only has good solubility for chitosan but also has goodcompatibility with the enzyme is the primary aspect to promote the study of chitosanhydrolysis. Ionic liquids, as new green solvents, have good stability, good solubilityand can recycle well, which is attracting widespread interest.This paper attempted to synthesize25kinds of ionic liquids, the structure of theionic liquids were verified by1H-NMR,13C-NMR and FT-IR. It was found that theionic liquid [Gly]BF₄could effectively dissolve chitosan to form a homogeneoussystem, and the FT-IR, SEM characterization of regenerated chitosan showed the ionic liquid could also promote the degradation of chitosan, meanwhile, the stability of theenzyme experiments confirmed that cellulase and amylase could still maintain a80%enzyme activity in the2wt%[Gly]BF₄after24h, which showed good biocompatibility.Cellulase as enzymatic catalyst, a typical homogeneous system was prepared bydissolving1.0g chitosan in99.0g2wt%[Gly]BF₄ionic liquid solution, the systematicinvestigation of reaction conditions on the enzymatic hydrolysis were carried out. Theoptimum conditions were as follows: degree of deacetylation of chitosan88.53%,pH5.0,50℃,2.5h and0.1g cellulase, then the average molecular weight ofchitooligosaccharide was about1900.Amylase as enzymatic catalyst, a typical homogeneous system was prepared bydissolving1.0g chitosan in99.0g2wt%[Gly]BF₄ionic liquid solution, the systematicinvestigation of reaction conditions on the enzymatic hydrolysis were carried out. Theoptimum conditions were as follows: degree of deacetylation of chitosan88.53%,pH5.0,50℃,2.5h and0.12g amylase, then the average molecular weight ofchitooligosaccharide was about2200.The degradation results of enzymatic hydrolysis of chitosan in the system of ionicliquid were much better than that in the system of acetic acid. And the ionic liquidcould be effectively reused, which was reused after5times, the average molecularweight of chitooligosaccharide could also reach2000.The moisture absorption and antibacterial antimicrobial activity ofchitooligosaccharide were investigated, results showed that, the smaller molecularweight and the higher the concentration were, the better its moisture absorption andantibacterial properties were.In this paper, the ionic liquid [Gly]BF₄was synthesized and successfully appliedto the chitosan enzymatic reaction, this innovative way will provide effective supportand inspiration for the development and utilization of the chitosan.