Study On The Production And Expression Of Chitosanase

Chitosan oligosaccharide has good biological activity,has better water solubility and low molecular weight than chitosan.It is used in anti-oxidation,medical materials,food additives,agricultural products and even cancer treatment.It has a very broad application prospect.There are three methods for the preparation of oligochitosan:physical,chemical,and biological.Among them,enzymatic hydrolysis has great development potential as a promising method,but enzymatic hydrolysis also has its own shortcomings,such as enzyme activity and stability.Sex size,production cost,etc.Solving these problems is of great help to the development of the production process of chitosan oligosaccharides.The research purpose of this subject is to use gene editing methods to express chitosanase on yeast,and to study the enzymatic properties of the chitosanase produced,in order to optimize the production of chitosan oligosaccharides in the future.In this study,we first obtained a chitosanase gene sequence from Aspergillus fumigatus,and used Saccharomyces cerevisiae to insert the chitosanase gene into sites in Saccharomyces cerevisiae that have little effect on yeast biological activity.Compared with the traditional editing method,the editing method of GTR-CRISPR has the advantages of simplified operation steps and more flexible and efficient insertion sites.It provides a more reliable tool for expressing proteins in Saccharomyces cerevisiae.The chitosanase derived from Aspergillus fumigatus was successfully expressed in Saccharomyces cerevisiae.The size of recombinant chitosanase is about 60 kDa,which is quite different from the theoretical protein size(27 kDa).It was found through experiments on the reaction conditions of the enzyme.The optimal reaction temperature is 55℃,and the optimal pH=5.5.Secondly,a high-efficiency expression system was constructed using Pichia pastoris GS115.After a fed-batch fermentation method for highefficiency expression,the reaction was carried out under the reaction conditions of pH=5.5 and 55℃,and the highest enzyme activity obtained was 105 U/mL.The final protein concentration in the fermenter is 200 mg/L,and the final wet weight of the bacteria is 269 g/L.Experiments have verified that the optimal enzymolysis conditions for the chitosanase expressed by Pichia pastoris are pH=6,60℃,and the stability is relatively good.The research of this subject has obtained chitosanase products with higher reaction temperature and better stability.The types of enzymatic hydrolysis products are relatively clear,and the high-density fermentation of Pichia pastoris expression system produces chitosanase in large quantities,which is the mass preparation of oligochitosan products provides directions.