The Glycosylation Of Phenolic Compounds By ?-galactosidase

Phenolic compounds are a class of chemicals containing a hydroxyl group directly bonded to the aromatic hydrocarbon group.They are widely distributed in nature,such as in fruits,vegetables,legumes,cereals,tea and medicinal herbs.Phenols have gained wide attention thanks to their positive physiological and pharmacological functions such as anti-tumor,antioxidant,anti-bacterial and anti-inflammatory.They can also be used in the prevention and treatment of cardiovascular or cerebrovascular diseases.Despite the importance of phenolic compounds,their use is limited because they are poorly soluble in water and easily degraded by light irradiation in aqueous solution.Glycosylation of phenolic compounds is considered to be a very useful method to increase their solubility and stability in water.Glycosylation can also improve the biological and pharmacological functions by decreasing the toxicity and side effects.The enzymatic method for the glycosylation can accomplish through one simple step with stereoseletivity and regioselectivity in an environment-friendly way.?-Galactosidase(EC3.2.1.23)are among the most important glycosidases for galactosylation.However,these enzymes are poor in glycosylation of phenolic compounds,with limited acceptor selectivity and low product yields.In this work,the?-galactosidase from L.bulgaricus L3 was subject to molecular evolution to yield mutants with improved translgycosylation activity and broadened acceptor specificity toward phenolic compounds.Using the mutants,a series of novel phenolic galactosides were produced.Thus,this work provided a new tool for enzymatic glycosylation of phenolic compound,as well as novel phenolic glycosides with potential applications in pharmaceutical industry.Homology modeling of the ?-galactosidase BgaL3 from L.bulgaricus L3 showed that the W980 residue located at the entrance of the active center and might play a key role in the initial substrate selection of the enzyme.This residue was subjected to site-saturation mutagenesis.The resulting 19 mutants displayed reduced hydrolysis activity toward oNPGal and decreased self-transglycosylation activity toward lactose as compared to the wild-type enzyme.When using lactose as the donor and hydroquinone as the acceptor,however,a W980F mutant exhibited increased transglycosylation yield toward hydroquinone.In addition,the glycosyl donor specificity of W980F was broadened.The enzyme could hydrolyze pNP-?-D-fucoside and even catalyze fucose transfer to generate pNP-p-D-Fuc-(1?3)-?-D-Fuc.The transglycosylation activity of W980F toward various phenolic compounds was tested.These compounds included phenol,catechol,hydroquinone,phloroglucinol,pyrogallol,gallic acid,chlorogenic acid,rutin,quercetin,caffeic acid phenethyl ester,clove acid,and caffeic acid.The W980F showed increased glycosylation yields by 7.6%to 53.1%for phenol,catechol,hydroquinone,and phloroglucinol as compared to BgaL3.It also showed broadened acceptor specificity and could glycosylate pyrogallol(32.3%product yield)and caffeic acid(7.0%product yield).Both BgaL3 and W980F had no transglycosylation activity toward gallic acid,chlorogenic acid,rutin,quercetin,and clove acid.The transglycosylation reactions of W980F was performed by incubation of the enzyme with 200 mM lactose and 100 mM acceptor including phenol,catechol,hydroquinone,pyrogallol or caffeic acid at 45 ? for 45 min.The glycoside products were purified by Bio-Gel P2 and HPLC and their structures were determined by MS and NMR analysis.These products were identified to be phenol-O-?-galactoside,hydroquinone-O-?-galactoside,catechol-O-?-galactoside,pyrogallol-l-O-?-galactoside,pyrogallol-2-O-?-galactoside,caffeic acid-3-O-?-galactoside,caffeic acid-4-O-?-galactoside.In addition to site mutagenesis,the ?-galactosidase from L.bulgaricus L3 was simultaneously subjected to random mutagenesis.A mutant 252 was screened to catalyze glycosyl transfer to resveratrol,based on blue-white clone screening with X-gal and subsequent detection of transglycosylation activity using TLC.The sequence of the mutant 252 was analyzed and there were six amino acid mutations including Q40R,K85R,F177S,D433G,and V511A when compared to BgaL3.Single site mutagenesis of these amino acids showed that the mutants F177S,D433G,V511 A,and E707G could glycosylate resveratrol while the mutants Q40R and K85R did not display the transglycosylation activity toward resveratrol.The transglycosylation reaction of 252 was performed by incubation of the enzyme with 200 mM lactose and 44?M resveratrol at 45 ? for 30min.The glycoside product was separated by ethyl acetate extraction,followed by TLC and HPLC purification.The MS analysis of the purifed product revealed a peak signal at m/z[M+Na]+ 413.1,suggesting monogalactosylated products of resveratrol(Mr 390).From 1H NMR spectrum,there was a mixture of two isomers with ?-glycosidic bond.Their structures were presumed to be resveratrol-3-O-?-galactoside and resveratrol-4'-O-?-galactoside.Product purification and confirmation is still going on.