Studies On The Fusion Expression,purification And Applications Of Synthetic Enzymes Related To Three Sugar Nucleotides

Glycans are biomolecules widely distributed in nature,which play important roles as structural or functional compounds in the process of life activities.The accessibility of glycans through extraction and purification,chemical synthesis,enzymatic synthesis or other means is the basis for their functional and application research.Enzymatic synthesis of glycans has attracted more and more attention because of its rapid and efficient reaction,high regional and stereoselectivity,mild reaction conditions,and so on.Leloir-type glycosyltransferases(GTs)are the main enzymes for glycans synthesis.Since Leloir-type GTs require sugar nucleotides(SNs)as the activated glycosyl donors,the availability of SNs is essential for the enzymatic synthesis of glycans.However,there are still many challenges to obtain SNs,including timeconsuming and laborious fermentation and chromatographic purification of multiple enzymes,high production cost and difficulty to scale-up.Seeking to overcome these challenges,we constructed the fusion proteins of enzymes involved in the biosynthesis of SNs with elastin-like polypeptide(ELP)as the purification tag and purified the recombinant protein through inverse transition cycling(ITC)in order to establish a new method to obtain these biocatalysts efficiently.Galactokinase from Streptococcus pneumoniae(SpGalk)and UDP-sugar pyrophosphorylase from Arabidopsis thaliana(AtUSP)were fused with ELP tag separately or jointly and the ITC purification conditions for these fusion enzymes were screened.After ELPmediated ITC purification,the results showed that 91 mg of ELP-SpGaIK,67 mg of ELPAtUSP and 97 mg of ELP-AtUSP-SpGalK could be purified from one liter of E.coli culture,respectively.The yield of ELP-AtUSP-SpGalK was lower than that of SpGalK-His6,but higher than that of AtUSP-His6.Subsequently,we determined the enzymatic properties of the fusion enzymes and found that the enzyme activity of SpGalK in ELP-AtUSP-SpGalK was increased compared with SpGalK-His6,while the enzyme activity of AtUSP was reduced.Based on the results of enzymatic properties,the gram-scale preparation of uridine diphosphate galactose(UDP-Gal)was easily realized via reaction catalyzed by ELP-AtUSP-SpGalK under the reaction conditions of Tris-HCl buffer(pH 8.0)and 40℃.Sialic acid aldolase from Pasteurella multocida P-1059(PmNanA)and CMP-sialic acid synthase from Neisseria meningitidis(NmCSS)were fused with ELP tag separately or jointly.The purification conditions and enzymatic properties were explored.After ELP-mediated ITC purification,the results showed that 101 mg of ELP-NmCSS,63 mg of ELP-PmNanA and 118 mg of ELP-NmCSS-PmNanA were purified from one liter of E.coli culture,respectively.The yield of ELP-NmCSS-PmNanA was lower than that of His6-PmNanA and His6-NmCSS.On the basis of the results of enzymatic properties and optimum reaction conditions,cytidine monophosphate-N-acetylneuraminic acid(CMP-Neu5Ac)was successfully synthesized catalyzed by ELP-NmCSS-PmNanA under the reaction conditions of Tris-HCl buffer(pH 8.5)and 40℃.The bifunctional enzyme FKP from Bacteroides fragilis was used to synthesize guanosine diphosphate-fucose(GDP-Fuc).FKP was fused with ELP to construct the recombinant expression vector.After recombinant protein expression and ELP-mediated ITC purification,the results showed that 97 mg ELP-FKP was obtained from one liter of E.coli culture,which was significantly higher than the recombinant FKP with His6 as the purification tag.Subsequently,we determined the optimal reaction conditions of ELP-FKP,and found that the optimal reaction conditions of ELP-FKP were Tris-HCl buffer(pH 8.0)and 40℃.Under the optimal reaction conditions,the enzyme activity of ELP-FKP was 171.2 U/μmol and GDP-Fuc was successfully synthesized by using ELP-FKP as the catalyst.In summary,the method to obtain biocatalysts for SNs synthesis with ELP as the purification tag and protein fusion strategy is feasible.Compared with the traditional single enzyme expression system,the biocatalysts for the synthesis of SNs can be obtained by one fermentation of ELP fusion protein,which greatly reduce the fermentation cost and improve the protein production efficiency.Furthermore,ELP-mediated inverse transition cycling(ITC)non-chromatographic purification technique to purify protein is cheaper,more environmentally friendly,simpler and faster compared with the traditional column chromatography.The method to obtain SNs biocatalysts also has the potential to be applied to the multi-enzyme reaction system of glycans synthesis and provides tool enzymes for glycans synthesis.