Production Of N-acetyl-D-glucosamine From Mycelial Waste By A Recombinant Chitinase

N-acetyl-β-D-glucosamine（GlcNAc）is a kind of monosaccharide with many bioactivities and functions,which has been extensively used in medicine,food and cosmetic industries.Enzymatic production of GlcNAc by hydrolyzing chitin-containing materials is a very potential method for its mild condition and environmentally friendly process.Mycelium of Aspergillus niger,a waste of fermentation industry,is a cheap and safe material that is rich in chitin（about25%）.So it is desirable to find an efficient method for enzymatic production of GlcNAc using mycelium waste as the material.In this article,a chitinase from A.niger（AnChi）was recombinantly expressed,purified and characterized.Furthermore,the industrial application potential of AnChi has been explored.The results obtained in this thesis include:（1）The gene of AnChi was obtained from the data base of NCBI.AnChi was predicted as a glycoside hydrolase family 18（GH18）chitinase by sequence analysis and homology modeling.AnChi was predicted to be a true chitinase but with weaker substrate binding affinity than chitinase A from Serratia marcesens（SmChi A）.（2）The whole catalytic domain of AnChi was successfully expressed in Escherichia coli BL21（DE3）.And AnChi was purified by immobilized metal ion affinity chromatography.40mg of AnChi was obtained from 1 L culture medium.（3）Characterization of enzymatic properties showed that AnChi had an optimum temperature 40℃and an optimum pH 6.0 respectively.Kinetic parameters shown that AnChi had higher affinity and catalytic efficiency towards 4-MU-β-（GlcNAc）₂ than ethylene glycol chitin.AnChi had higher hydrolytic activity towards mycelium waste than classical SmChiA and commercial chitinase.（4）AnChi was successfully expressed in Bacillus subtilis KO7 using a constitutive promoter P_ylb.The culture medium was isolated by ion-exchange chromatography.And AnChi is identified by SDS-PAGE and enzyme activity.（5）The mycelium waste is hydrolyzed by culture medium.The concentration of product came to 3.8 mmol/L and got a high yield of 78%.The product was identified by HPLC as（GlcNAc）₂.When adding OfHex1 to the reaction system,the product was transformed into GlcNAc.（6）The reuse of An Chi was explored.The mycelium waste was used to adsorb AnChi in culture medium.However the rate of adsorption was only 25%which was not enough for reusing.