Screening And Application Of Novel N-acetylglucosamine Deacetylase

Glucosamine(GlcN)has rich biological activity and is widely used in many fields such as medicine and food.The current production methods of GlcN include acid hydrolysis,microbial fermentation and biocatalysis.The traditional chitin acid hydrolysis method is the main way of GlcN industrial production,but this method has defects in the source of raw materials,environmental protection and product safety.N-acetyl-D-glucosamine(GlcNAc)was produced by microbial fermentation,and further deacetylation to obtain GlcN has gradually become the main way of large-scale biomanufacturing of GlcN in recent years.However,this method still needs to use hydrolysis to deacetylate GlcNAc to generate GlcN.The process consumes a lot of concentrated acid,which can easily cause equipment corrosion and seriously pollute the environment.In response to the above problems,this study aims to discover efficient N-acetylglucosamine deacetylase and explore the enzymatic deacetylation pathway of GlcNAc,thus replacing the current acid hydrolysis deacetylation method and providing a basis for the realization of full-chain green biomanufacturing of GlcN.First,six protein sequences(TkDac,TpDac,TsDac?TspDac,TcDac and CqCBDA)from different sources(Thermococcus kodakarensis,Thermococcus profundus,Thermococcus siculi,Thermococcus sp.2319x1,Thermococcus celericrescens and Cyclobacterium qasimii)with potential n-acetylglucosamine deacetylase activity were screened from the database by bioinformatics.The screened genes were expressed heterologously by E.coli expression system,in which TpDac,TcDac and CqCBDA were induced to express soluble recombinant protein.The three recombinant enzymes were purified by Ni affinity chromatography column and characterize their enzymatic properties.The results showed that the three recombinant deacetylases all have N-acetylglucosamine deacetylase activity.The optimal pH of TpDac,TcDac and CqCBDA were 8.0,8.8 and 7.6,respectively,and the optimal reaction temperatures were 80?,75? and 40?.The specific enzyme activities were 271.8 U/mg,179.7 U/mg and 9.6 U/mg.respectively with GlcNAc as the substrate under the optimal conditions.Secondly,the recombinant deacetylase TcDac with high enzyme activity and high expression was selected to explore the application potential of enzymatic deacetylation of GlcNAc.The results showed that the content of GlcN can reach 28.0 g/L when the catalytic reaction time is 1.5 h under the conditions of substrate GlcNAc concentration of 40 g/L and reaction temperature of 50?.and the conversion rate of GlcNAc is 95.4%.the content of GlcN can reach 44.4 g/L when the catalytic reaction time is 2.5 h under the conditions of substrate GlcNAc concentration of 60 g/L.and reaction temperature of 30?.and the conversion rate of GlcNAc is 88.9%.The lower the initial concentration of the substrate GlcNAc is,the higher the conversion rate will be,but the yield of GlcN will be low.the lower the temperature is,the less the loss of GlcN will be,but the reaction time will be long.The optimal application conditions of TcDac catalyzed GlcNAc deacetylation to produce GlcN obtained by the above experiment are:substrate concentration of 40?60 g/L,reaction temperature of 30?40 ?,pH of 7.5?8.0,reaction time of 1?2.5 h.In summary,this study screened three novel deacetylases with N-acetylglucosamine deacetylase activity.Among them,TcDac has high expression level,high specific enzyme activity,high catalytic conversion rate and good stability.It has good potential for large-scale enzymatic production of GlcN.The research results provided the basis for the related research of enzymatic green preparation of GlcN.