Discovery Of N-Acetylglucosamine(GlcNAc) Monomer Specific Deacetylase CmCBDA And Its Function In The Biocatalytic Synthesis Of Glucosamine And Sialosides

Glucosamine(2-amino-2-deoxy-D-glucose,GlcN)is an important aminohexose.It is widely used as a kind of food supplement because it can improve the body immune system,reduce inflammation,protect the liver and treat arthritis.So far,the method of producing GlcN in the industry is mainly strong acid hydrolysis.For a long time,researchers have been trying to find a green and friendly biocatalytic method which can replace chemical method to produce GlcN.However,new deacetylases which can specifically convert GlcNAc into GlcN is a bottleneck in this chain.If we can find a new N-Acetylglucosamine deacetylase,this highly effective enzymatic production of GlcN provides a novel environmentally benign approach avoiding any harsh chemicals and allows the production of GlcN from both crustacean waste products and non-animal sources.Enzymatic synthesis of high value pharmaceutical and food raw materials and intermediates is the development trend of modern biochemistry.So,based on the purpose above,N-Acetylglucosamine deacetylase CmCBDA was successfully cloned and expressed from C marinum by genetic engineering technology.The recombinant CmCBDA only has activity to GlcNAc.The substrate specificity of CmCBDA was studied and the enzymatic properties of CmCBDA were explored.The optimized enzyme was applied to the production of GlcN by one-pot enzymatic method.In addition,CmCBDA also has acylation function.This discovery makes the production of sialosides by fully biocatalytic method possible in laboratory condition.Sialic acids in vivo are closely related to cell recognition and virus infection which is very important for the body.Specific research contents and results are as follows:1.Cloning,Recombinant Expression,Purification,Sequence Analysis and Activity Test of CBDA and Site-Directed Mutagenesis of CmCBDAThe target gene CBDAs were cloned from the genomes of C.marinum,E.faecalis and E.coli.They are named CmCBDA,EfCBDA and EcCBDA.The recombinant expression vectors were expressed inE.coli BL21.They were purified and the purity is quite high which is consistent with theoretical values.Bioinformatics technology is used to predict the proteomic properties.All three proteins are deacetylases of glycosylesterase family and have YdjC protein property.Theyhave no signal peptide and are non-secretory proteins.They have no transmembrane region and were not transmembrane proteins.A new method based on OPA reagentfor identifying the catalytic activity of deacetylases was established.With this method,product GlcN produced by deacetylases can be detected and quantified by high-throughput plater-reader.Besides OPA method,TLC,HPLC and NMR method were used to confirm that recombinant CmCBDA can catalyze GlcNAc.The conversion was almost 100%.The catalytic sites of CmCBDA were searched by sequence alignment.21 Asp and 230 His were the potential catalytic sites of CmCBDA and were mutated into neutral amino acids Asn and Ser by one-step PCR site-directed mutagenesis.Asp21 and his230 were identified as the catalytic sites of CmCBDA by enzyme activity verification.2.Substrate Preparation and Substrate Specificity of CmCBDAGlcNAc analogs(N-propionyl glucosamine,N-butyryl glucosamine,N-hexyl glucosamine,N-benzoyl glucosamine,N-azido-acetylglucosamine and so on),Fmoc-GlcN and Alloc-GlcN were synthesized by chemical method.NMR was used to confirm their structure and purity.CmCBDA is aGlcNAc deacetylase.It cannot catalyze monosaccharides such as GalNAc,ManNAc,GlcNAc-6-P,amino-protected glucosamine and acetylated amino acids.But it can catalyze some GlcNAc analogs;CmCBDA cannot catalyze chitosan,chitin or Nglycan.It means CmCBDA isn't a typical chitin deacetylase or N-glycan deacetylase.CmCBDA has the substrate specificity.It is a GlcNAc deacetylase.3.Biochemical Characteristics Determination of Recombinant CmCBDA and Its Application to Produce GlcN by One-Pot Enzyme CascadesThe biochemical characteristics of CmCBDA were determined.The optimum reaction temperature of CmCBDA was 42? and 37? is still very active.It was not a heatresistant enzyme.The activity had no change after incubation at 37? for 6 h if the reaction contains Mn2+.The optimum pH was 9.0,and8.0-10.0 is the optimum pH range.The catalytic reaction did not need metal ions.But Mn2+ could significantly promote itsactivity.Ni2+and Ca2+had no obvious effect on its activity.Zn2+completely inhibited its activity.Most denaturants and detergents could not completely inactivate the enzyme.Urea,mercaptoethanol,imidazole,surfactant NP40 and Triton X-100 had little effect on the activity of CmCBDA.DTT and protease inhibitor PMSF could strongly reduce its activity.Benzyl-phosphoglucosamine,GlcN derivative was a strong inhibitor of CmCBDA.Its Ki is 568.9 nM.IC50 is 2.2 uM.The Km value of CmCBDA to GlcNAc was 47.9 mM;the secondary structure of the enzyme remained unchanged by circular dichroism detection.In the laboratory condition,GlcN preparationmethod from GlcNAc was compared by enzyme method and chemical method.The products obtained by acid hydrolysis had color and by-products,which need to be purified for many times.And the yield is only 71%.The GlcN obtained by enzyme hydrolysis had high puritywithout by-products,and the yield is 96%.The final product is GlcN which is not the same as GlcNCl from acid hydrolysis.In the laboratory condition,Chitin and mushroom extract as substrates,CmCBDA and chitinase ZgBHex were used in one-pot,GlcN could be produced by whole biocatalytic method.Each gram of chitin can produce 17 mg of GlcN,and each gram of mushroom extract can produce 7 mg of GlcN.The whole pathway of GlcN production by biocatalytic method was successfully found.CmCBDA could be successfully immobilized on Amino C6 acrylate resin,and the immobilized CmCBDA could be recycled at 37? for six days,and the activity remained about 40%.4.Discovery of New Acylation Mechanismof CmCBDA and Its Application to Produce Sialosides by One-Pot Enzyme CascadesOrganic carboxylic acids such as propionic acid,butyric acid and azido acetic acid were used as acyl donors and GlcN as the acceptor.When the donorquantity is 5 equivlents of that of receptor,CmCBDA can acylate GlcN to generate different GlcNAc analogs(such as N-propionyl glucosamine,N-butyryl glucosamine,N-azido-acetylglucosamine).From the NMR quantity result,the acylation conversion was more than 50%.CmCBDA is a reversible enzyme and has acylation activity in specific condition.In addition,unlike the traditional chemical acylation,which requires the activation of donor acyl groups,CmCBDA does not need to activate the acyl groups in advance.Therefore,the acylation reaction catalyzed by CmCBDA is not only environmentally friendly,but also does not need the activation step.We found a new acylation mechanism which is different from the traditional chemical acylation.Other organic compounds containing carboxyl groups,such as biotin and y-aminobutyric acid,were used as acyl donors.CmCBDA could also catalyze the acylation of these organic compounds with GlcN to obtain corresponding products.The conversion from LC-MS result was 50%and 21%,respectively.In specific conditions,CmCBDA enzyme can take carboxylic acid as acyl donor and GlcN as acyl acceptor to directly catalyze acylation reaction to produce GlcN derivatives.Using GlcNAc as acyl acceptor,carboxylic acid(such as propionic acid,butyric acid)as acyl donor,CmCBDA can first plays the role of deacetylation,then acts its acylation function to obtain GlcNAc analogs(such as N-propionyl glucosamine,N-butyryl glucosamine).The conversion was between 31%-41%.The results can be applied to the production of sialosides and other glycosides with cheap chitin and organic acid as the starting substrate by one pot multi-enzyme cascades.The recent report shows that the starting substrate GlcNAc anologs was chemically synthetized to produce sialosides by one-pot four enzyme cascades.As CmCBDA could produce GlcNAc anologs instead of chemical method.Sialosides preparation by biocatalytic method by one-pot five-enzyme cascades could come true.The conversion and stability of the enzymes involved were studied at first.CmCBDA,PhGn2E and EcAldolase are the first three enzymes which are reversible.The conversion were53%?32%and 35%,respectively.The enzyme activity was0.071.6.59and 0.197,respectively.NmCTT and PdST6 are last two enzymes of the one-pot reaction,which had 100%and 97%conversion,respectively.The enzyme activity was209and 1.77,respectively.The last two enzymes are irreversible,which can drive the all reaction forward.Temperature stability showed that 20%or more activity remainedat 37? after20 h for these five enzymes,which is advantage for the production of sialosides in one-pot five-enzyme cascade.With the optimized conditions,GlcN and three carboxylic acids(acetic acid,propionic acid and hydroxyacetic acid)were used as the starting substrate,X-gal as the glycosyl acceptor,by one-pot five enzyme cascades.The yield of X-Gal-Neu5Ac is 98%,the yield of X-Gal-Neu5Prop is 94%and the yield of X-Gal-Neu5Gc is 29%.