Exo-?-sialidases:gene Mining,transglycosylation And Molecular Evolution

Human milk oligosaccharides(HMOs)are a group of complex unconjugated glycans that are highly abundant in and unique to human milk.Numerous reports have detailed the effects of HMOs promote the growth of desired bacteria in the infant's intestine and,in particular,members of the genus Bifidobacterium.Besides,feeding of breast milk to infants is linked to several other beneficial effects,like reduce microbial infections by serving as antiadhesive antimicrobials and directly modulate immune responses.The sialylated oligosaccharides are a particularly interesting subgroup of HMOs.They are present in large quantities in colostrum(1-3.3 g/L),especially the 6'-sialyllactose accounts for 0.25-1.3 g/L.SL and sialylated oligosaccharides are thought to have significant health benefits for the neonate,because of their roles in supporting resistance to pathogens,gut maturation,immune function,and cognitive development.While HMOs are abundant in human milk,only trace amounts are present in infant formula based on bovine milk.Therefore the production of HMOs is receiving increased attention,both for commercial use as well as for functional studies of individual HMOs.Some methods for the synthesis of these sialyllactose are reported:chemical methods,and enzymatic methods.In chemical methods,the structure of sialic acid makes the sialylation reaction particularly difficult to execute in an elimination-free and stereo-controlled manner.In addition,these procedures often involve many steps with protections and deprotections.Enzymatic approaches employing trans-sialidases,sialidases and sialyltransferases as synthetic tools have offered an alternative access to the family of sialylated glycostructures with excellent stereospecificity and varying regioselectivity depending on the type and source of enzyme.Sialyltransferases,the natural enzymes for specific synthesis of oligosaccharides and polysaccharides,can transfer sialic acid from cytidine monophosphate-sialic acid(CMP-sialic acid)to an acceptor,but this process requires the activated CMP-sialic acid and generally exhibit strict acceptor specificity.Among enzymatic methods,use of sialidases(neuraminidases;EC 3.2.1.18)were reported in the synthesis of sialic acid-containing substances.Despite the limitations of the low transglycosylation yield resulting from the decrease of the reaction products by self-hydrolysis,bacterial sialidases are attractive enzyme resources for enzymatic synthesis due to their substrate preferences,which allow the recognition of various sialic acid-containing structures,and are capable of synthesizing regioselective sialoglycoconjugates using cheaper glycosidesIn this work,seven exo-a-sialidases derived from gut bacteria,including 3 from Bacteroides fragilis NCTC9343,3 from Clostridium perfringens ATCC13124,and 1 from Bifidobacterium bifidum JCM1254,were subjected to gene cloning and heterogeneous expression in Escherichia coli.The recombinant enzymes were purified,characterized for substrate specificity.These enzymes have broad substrate specificities and can hydrolyze ?2-3,?2-6,and ?2-8 various bond-type sialic acid substrates.We have prepared oligosialic acid donors by polysialic acid hydrolysis,which is cheaper than other sialic acid donors.A sialidase,designated as BfN 3,from B.fragilis NCTC9343 was found to possess excellent transglycosylation activity for the synthesis of sialylated human milk oligosaccharide.The native BfN3 was a homodimer with a molecular weight of 113.6 kDa.The Km and kcat values for 4-methylumbelliferyl N-acetyl-a-D-neuraminic acid and sialic acid dimer were determined to be 0.06 mM and 283.18 s-1,and 0.75 mM and 329.64 s-1,respectively.The enzyme was able to transfer sialyl from sialic acid dimer or oligomer to lactose with high efficiency and strict ?2-6 regioselectivity.The influences of initial substrate concentration,pH,temperature,and reaction time on the transglycosylation were investigated in detail.Using 40 mM sialic acid dimer(or 40 mg/ml oligomer)and 1 M lactose(pH 6.5)at 50? for 10 min,BfN3 could specifically produce 6'-sialyllactose,a dominant sialylated human milk oligosaccharide,at a maximal conversion ratio above 20%.The receptor selectivity of BfN3 was studied and it was found that the enzyme receptor has a wide range of properties such as lactose,melibiose,galactose,N-acetylgalactosamine and glucose can be used as receptors.This enzyme provided a promising alternative to the current synthetic methods for obtaining important sialylated oligosaccharides.Sialidase follows the general mechanism of glycosidase reaction,and the reaction product can be re-hydrolyzed by the enzyme as a substrate.Even if the reaction conditions are optimized,the yield of the product can only be increased within a certain range.Therefore,we conducted a molecular transformation study of BfN3 in order to obtain high Efficiency mutant enzyme.Firstly,the BfN3 gene was randomly mutated by error-prone PCR and screened with methanol and lactose as transglycosyl acceptors.A total of 2457 mutants were screened.It was found that the mutant enzyme T1231 had the best effect,compared to the wild-type BfN3 transglycosylation.The ability(lactose receptor)increased by 41.5%.At the same time,rational design of was carried out based on the structural characteristics of the highly efficient synthesizing of ?2-6 sialic acid oligosaccharides,namely Trypanosoma cruzi TcTS and Trypanosoma brucei TbTS.The amino acid positions were:I205L,G207A,G271V,M294Y,G295Y,W300Y,P349A,R418Y,G419Y,V448G,and G509A.The results showed that among the 11 mutant enzymes,the G271V and G294Y mutant enzyme activities were lost,and the activity of the remaining 9 mutant enzymes was increased.Among them,V448G,G207A,W300Y,and P349A had the most significant effects,with sialic acid oligosaccharides.The transglycosylation activity of the donor and lactose receptors as substrates was increased by 42.9%,57.2%,57.2%,and 64.3%,respectively,compared to the wild-type enzyme.The availability of these mutant enzymes provides a useful tool for the synthesis of sialic acid-associated oligosaccharides and glycoside compounds.