Expression And Application Of N-acetyl ?-D Glucosaminase

N-Acetyl-glucosamine(Glc NAc)is a monosaccharide derivative of glucose that usually polymerizes linearly with(1,4)-?-linkages.Glc NAc is the monomeric unit of the polymer chitin..Glc NAc is the constituet of the cell wall of bacterial,fungus and plants,and the basic component of connective tissue and glycoprotein in joints of human.Glc NAc has the several physiological functions,such as the prevention of joint damage,antibacterial and anti-inflammatory activity,and the promotion of wound healing,so it is widely used in medicine,health care products,food and cosmetics and other fields.Glc NAc is mainly produced by chitin as raw material,which is widely found in fungi,yeast and other cell walls,or insects and shrimp crabs and other exoskeletons.Chitin is the second most aboundant carbohydrate after cellulose,and every year more than tens millions tons of chitin from aquatic products goes unutilized.Therefore,chitin is a suitable biomass resource for the production of Glc NAc.The traditional method is to hydrolyse chitin through a strong acid,such as HCl,which may produce large quantities of chemical waste.This chemical method are not environmentally friendly.Therefore,the development of environmentally friendly enzymatic method of hydrolysis of chitin has become a research hotspot.Enzymatic degradation of chitin to prepare glucosamine has the advantages ofmild reaction condition,high purity and high yield of Glc NAc.The produced efficiency and yield can be improved by combining several chitinase,such as chitinase can hydrolyze chitin into chitosan,further hydrolyzed into Glc NAc by N-acetyl-?-D glucosamine(NAGases).Since NAGases is exo-enzyme,the activities were generally low,the efficiency of the preparation of Glc NAc by enzyme method is low.We hope to improve the expression and enzyme activity of NAGases and enhance the enzyme hydrolysis efficiency of colloidal chitin.In this study,the expression of N-acetyl-?-D-glucosamine Bs Nag Z was successfully expressed in Piahia pastoris and the expression of enzyme was improved by screening the strains with high copy integration,the purified Bs Nag Z and chitinase were coupled,and the preparation of N-acetyl-glucosamine monomer(Glc NAc)by hydrolyzed colloidal chitin substrate was studied.The details are as follows:The first is the high efficient expression of Bs Nag Z in P.pastoris:The NAGases gene nag Z from Bacillus subtilis 168 was optimized according to the codon preference of Pichia pastori,the synthesized Bsnag Z gene was cloned into the expressed vector p PICZ?A,and the heterologous expression was successfully carried out in P.pastoris X33 strain.Through the Zeocin resistance screening to a Bsnag Z 4 copy of the integrated strain,the fermentation of the enzyme activity reached 3.2 U/m L,compared with the single copy,the enzyme activity increased by 4.17 times times.Enzymatic properties of recombinant enzymes were analyzed:the optimum temperature was 60°C,and the optimum p H was 6.0.At the same time,the enzyme has good stability:range feom p H 4.5 to 10,all maintain the activity of more than 80%.After the enzyme is preproccess at 1 h,there is still 80%residual enzymatic activity.Secondly,in order to obtain high enzymatic activity of Nag Z,we performed homologous pairs in NCBI based on Bs Nag Z amino acid sequences to find Coagulans genes derived from Bacillus DMS1 nagz,named Bc Nag Z,and the nag Z derived from Bacillus Lichheniformis WX02',named Bl Nag Z.The two genes were cloned into p ET-26b vector and successfully expressed in Escherichia coli.After studied the enzymatic properties of recombinant proteins,the optimum p H of Bl Nag Z and Bc Nag Z are 6.0,while the optimum temperature of the Bc Nag Z is 75°C,which is higher than that of the Bl Nag Z60°C.Comparing the two kinds of proteins,Bl Nag Z has higher enzyme activity and better expression,then the Bl Nagh Z was selected to optimize the induction conditions.And after optimization,the enzyme activity of fermentation was 13.2 U/m L.Finally,the experimental analysis of substrate enzymatic hydrolysis by Nag Z:The recombinant Nag Z was applied to the enzymatic hydrolysis of colloidal chitin substrates.The results of enzymatic hydrolysis showed that the enzymatic hydrolysis effect was better under the condition of 50 ~oC,the enzyme solution of 150?L was added into 250?L 3%colloidal chitin substrate,and Glc NAc could be detected after hydrolysis of 1h,but the yield was a litter.However,it is still necessary to further optimize the enzymatic conditions and produce N-acetyl glucosamine monomer in large quantities.In addition,Bl Nag Z has higher enzyme activity than Bs Nag Z,so the hydrolysis efficiency of Bl Nag Z is faster then Bs Nag Z at the same enzyme amount.To sum up,for the first time,in this study,Bs Nag Z was first expressed in P.pastoris,and through Zeocin resistance screening to improve the number of copies of genes,so as to improve the expression of recombinant Bs Nag Z.At the same time,the Bs Nag Z was applied to the enzymatic hydrolysis colloidal chitin,and the Glc NAc was obtained.And through the coupling of chitinase,can effectively degrade colloidal chitin,increase the yield of Glc NAc.In order to obtain Nag Z with higher enzyme activity,we expressed the nag Z gene(Bc Nag Z)from Bacillus Coagulans DMS1 in Escherichia coli,as well as the nag Z gene(Bl Nag Z)from Bacillus Lichheniformis WX02.The fermentation expression of Bl Nag Z was optimized,and the enzyme solution was also carried out by enzymatic hydrolysis.The results showed that recombinant Nag Z can hydrolyze colloidal chitin to obtain Glc NAc.This research lays a foundation for the efficient production of Glc NAc by enzymatic method.