| Carbohydrates are fundamental substrates that build up the living organism.They usually exist in forms such as monosaccharide,oligosaccharide and polysaccharide,and they can also be integrated with protein or lipid to form glycoconjugates.These glycoconjugates participate in the cell recognition,hormone regulation,fertilization,occurrence,development,differentiation,and other important life processes.The structures of carbohydrates are complicated,which heavily affected their functions.Therefore,the synthesis and structure study of carbohydrates are important for their functional studying and application.Glycosidases(EC 3.2.1),which also called glycoside hydrolases,are a sort of enzymes that specifically hydrolyze the glycosidic bonds.Meanwhile,many glycosidases can also catalyze in vitro transglycosylation reaction when suitable donor and receptor are offered.Glycosidase catalyzed glycoside synthesis occurs in mild reaction condition,which means it does not need the participation of organic solvent,so the food and drug safety can be guaranteed.And glycosidases are wide spread,each kind has its own hydrolytic substrate preferences.The chain type,glycosidic bond and configuration of transglycosylation products synthesized by glycosidases,are varied.Glycosidases occupies an important position in the synthesis of glycosides.?-L-Fucosidases(EC 3.2.1.51)are a kind of enzymes involved in the metabolism of fucosyl-oligosaccharides.According to the amino acid sequence and hydrolysis substrate specificity of enzymes,?-L-fucosidases can be divided into two glycoside hydrolase families,GH95 and GH29.?-L-Fucosidases of GH95 family are also called?a-1,2-L-Fucosidase,which specifically hydrolyze ?1,2 bonds by using reverting mechanism while they cannot hydrolyze artificial substrates such as p-nitrophenyl-?-L-fucoside(pNPF).Enzymes of GH29 family are further divided into GH29-A and GH29-B subfamilies according to the substrate specificity of enzymes.GH29-A enzymes have relatively broad substrates specificity,which can hydrolyze both fucosyl-oligosaccharides and pNPF.Enzymes of GH29-B family are called?-1,3/4-L-fucosidase,which specifically hydrolyze ?1,3 as well as ?1,4 bonds.Both GH29-A and GH29-B subfamily use retaining mechanism.As the hydrolysis mechanism,only GH29-A subfamily can directly be used as tool to synthesis fucosyl-oligosaccharides.Fucosyl residues exist in various oligosaccharides and glycoconjugates,including human blood sugar chain,N-glycoside,O-glycoside and human milk oligosaccharides.Fucosyl residues are also found on the surface of some tumor cells.Fucose has an important role to the function of Fucosyl-oligosaccharides.Research had proved that fucosylation rich oligosaccharide has special immunogenicity.They can effectively improve the antibody mediated cellular immune responses against of GloboH containing mice tumor cell line LLC1.In current,some researchers have committed to synthesize fucosyl-oligosaccharide using ?-L-fucosidase,but the linkage type of transglycosylation products are limited,and the synthetic efficiency is relatively low.In this work,five ?-L-fucosidases of Bacteroides fragilis NCTC 9343 were cloned,expressed and characterized.Transglycosylation activity screening of ?-L-Fucosidase turns out that two ?-L-fucosidase,BF29A2 and BF29A4,can catalytic the transglycosylation reactions using pNPF as donor.According to the analysis result of CAZY(http://www.cazy.org/GH29.html,B.fragilis NCTC 9343 has 13 ?-L-Fucosidases.Four of them are grouped into GH95 family,and nine of them are grouped into GH29 family.We analyzed the sequence similarity of GH95 enzymes of B.fragilis with GH95 enzymes that have been reported.Sequences of GH29 enzymes of NCTC 9343 were aligned with that of other GH29 enzymes,and a development of the evolutionary tree was constructed.According to the evolutionary tree,nine GH29 family enzymes of B.fragilis were grouped into GH29-A and GH29-B subfamilies.We finally successffully expressed four enzymes of GH29-A subfamily(BF29AI BF29A2 BF29A4,BF29A5)and one GH95 enzyme(BfAfc3)for further study.Each of the five enzymes was cloned and overexpressed in E.coli.The substrate specificity of these five enzymes was studied using fucosyl-oligosaccharides and pNPF as substrates.BfAfc3 could specifically hydrolyze al-2 bond.Study of the change of anomeric carbon of product in time course revealed that BfAfc3 uses reverting mechanism,which was consisted with the classification of BfAfc3 into GH95 family.All the four enzymes of GH29-A subfamily could hydrolyze pNPF.BF29A1 could only hydrolyze pNPF;BF29A2 could also hydrolyze ?1-2/3/4 bonds,while pNPF was its most suitable substrates;BF29A4 showed limited hydrolysis activity on different fucosyl-oligosaccharides,with preferences for ?1-3,and ?1-6 fucosyl linkage;BF29A5 showed the highest hydrolytic activity for al-3 fucosyl linkage.Transglycosylation activity screening of ?-L-Fucosidase turned out that only BF29A2 and BF29A4 can catalytic the transglycosylation reactions using pNPF as donor.The Km of BF29A2 for pNPF was 10.18 mM,the maximum reaction rate was 0.72 nM/min.The optimum pH of BF29A2 was 5.5-6.5,and BF29A2 was stabled at pH 5.0-11.5.The optimal reaction temperature of BF29A2 was 40?,and the enzyme was stable when temperature was lower than 40 ?.BF29A2 could be activated by Zn2+,Fe3+,and be inhibited by Ag+,Cu2+,Hg2+.N-Acetylgalactosamine(GalNAc)and pNPF were used as acceptor and donor respectively for the study of transglycosylation activity of BF29A2.After optimization of the acceptor and donor concentration,pH,temperature and reaction time,transglycosylation yield of BF29A2 reach to 53%.Mass spectrum(MS)and nuclear magnetic resonance(NMR)analysis showed that the transglycosylation product of BF29A2 was Fucal-3GalNAc.The Km of BF29A4 for pNPF was 0.86 mM,and the maximum reaction rate was 4.51 nmol/min.Compared to BF29A2,BF29A4 had higher affinity for pNPF,faster hydrolysis rate.The optimum pH of BF29A4 was 5.5-7.5,and it was stable at pH 5.0-11.The optimal temperature was 45?,and it was stable when temperature was lower than 45 ?.BF29A4 was not activated by any measured ion.But BF29A4 can be inhibited by Ca2+ and Ni2+,Ag+,Fe3+,Hg2+ and EDTA.We chose pNPF as donor and NAcetylglucosamine(GlcNAc)as acceptor to study the transglycosylation activity of BF29A4.After optimization of the acceptor and donor concentration,pH,temperature and reaction time,total transglycosylation yield of BF29A4 reached to 79%.What interesting was that the proportion of two transglycosylation products(named productl and product2)changed along with pH and temperature.Under acid condition,pH 4.5-6.5,the producing rate of productl was low,and the proportion to product2 was about p1:p2 = 0.3:1.In alkaline conditions,the proportion of productl rose gradually,and the proportion to product2 was about p1:p2 = 0.7:1.In addition,temperature showed distinct effect on the production of two products..The proportion of two kinds of roducts was about p1,p2 = 0.1:1 at 20?,while the ratio was p1,p2 = 1.7:1 at 50?Therefore,lower temperature was conducive to product2 synthesis,and higher temperature contributed to productl synthesis,.By ion chromatography analysis,productl and product2 were deduced to be Fuc?1-3GlcNAc and Fucal-6GlcNAc respectively. |
PDF Full Text Request