Production Of N-actyle D-glucosamine From Chitosan By A Combination Of Chitosanase CSN And Two Glucosidases

N-Acetyl-glucosamine?GlcNAc?usually polymerizes linearly with?1,4?-?-linkages a nd is the monomeric unit of the polymer chitin.It is the common monosaccharide der ivative of glucose.GlcNAc is not only the important part of the cell wall of bacterial,fungus and plants,but also a basic component of connective tissue of human.GlcNA c belongs to a large class of amino sugars that serve a number of functions and are l ocated throughout the human system.GlcNAc is the functional material with high pot ential in various fields,such as osteoarthritis prevention and healing,wound healing a nd anti-inflammatory.Thus it is wildly applied in the field of medical and healthcare,food and cosmetics.The producing of GlcNAc needs the chitin as the feed-stock.Chitin is the second most abundant organic compound on our planet after cellulose.It is the polymer whi ch is formed with GlcNAc.It is widely distributed worldwide,such as presents in ara chnids,most fungal cell walls,insect exoskeletons,the shells of crustaceans and parts of invertebrates.The water breeding of prawns and crabs covers a large propotion in the water breeding industry in the world.The annual output of prawns and crabs is o ver 2 million tons in our country,which provide a huge amount of shrimp and crabs shells as the material for producing GlcNAc.GlcNAc has historically been commercially prepared by several companies via a p rocess based on the acid hydrolysis of crude chitin or the physical methods.But the method that hydrolyzes chitin using strong acid and alkali,such as HCl and NaOH,c ausing several problems,including huge cost and low yield?below 65%?,and environ ment pollution.Thus the Enzymatic methods to produce GlcNAc attract wildly attentio n,which have several advantages including that the reaction conditions is mild,the pr ocess is environmentally friendly and the production has higher purity which is becom ing the hot area of research.Combining different enzymes hydrolyzing the chitin can improve the hydrolysis efficiency.In this study,the exo-?-N-acetylhexosaminidase Nag Z from Bacillus subtilis 168,exo-?-D-glucosaminidase(GlmA_TK)from Thermococcus kodakarensis KOD1 were expressed in Pichia pastoris Then purified NagZ was used t o hydrolyze the substrate of chitosan alone or coupled with CSN.More details are as follows:?1?The expression of exo-?-N-acetylhexosaminidase NagZ:the gene sequence of na gZ which coding the exo-?-N-acetylhexosaminidase from Bacillus subtilis 168 was fou nd in NCBI.After coden optimization according to the codon bias of Pichia pastoris,we got the target gene nagZ by overlapping PCR.Then we constructed the recombin ant vector of pHBM905A-nagZ and expressed in P.pastoris GS115.The activity of rec ombinant NagZ in fermentation supernatants can reach 0.56U/mL.The study of charac terization showed the the optimize temperature for NagZ is 60?and the optimize pH is 6.5.The enzyme is stable in the pH ranges from 4.5 to 10,the residual activity i s over 80%.After 24h incubation at 55?,the activity remains 85%.?2?The expression of exo-?-D-glucosaminidase:we expressed 3 kinds of exo-?-D-gl ucosaminidase in P.pastoris GS115 as before.The GlmA_TK from Thermococcus kodakar ensis KOD1,the CSX from Aspergillus fumigatus and GlmA_PH from Pseudozyma hubei ensis SY62.?3?The hydrolysis of substrates by NagZ and CSN:we use the NagZ to digest su bstrate chitin or chitosan.After the reaction in different temperatures,treatment time a nd substrates,the products was detected with HPLC.The results indicated that the bes t hydrolysis temperature is 37?and generate GlcNAc by hydrolysis colloidal chitin a nd chitosan.Coupling NagZ with CSN to hydrolysis colloidal chitosan can digest the chitosan.The conditions of reaction still need to be optimized to generate GlcNAc.The results of this work shows that we expressed the NagZ in Pichia pastoris su ccessfully and use it to hydrolyze chitin and chitosan.The coupling of NagZ and CS N can efficiently degrade chitosan,but the ratio of enzymes and treatment time need to be optimized to generate GlcNAc.This work provides a promising method for prod ucing GlcNAc from chitin.