| Steviol glycosides,known as natural low-calorie or no-calorie sweeteners with 50～400 times sweetness than sucrose,have been approved as safety food addictives by China,United States,European Food Safety Authority(EFSA),and etc.In the meantime,they also have important medicinal values in antihypertension,antihyperglycemia,antidiarrhea,antiinflammatory and antitumor.As the ideal substitutes of sucrose,they would have great promising prospects in the market.But the defective attribute of bitterness reflects their applications in the developing natural food additives market.Enzymatic glucosylation modifications have been applied to improve the sensory quality using glycosyltransferases(EC 2.4)with nucleotide sugars as the donor substrates,as well as glycosidases(EC 3.2.1)with oligosaccharides as the donor sbustrates.About 57 different naturally occurring steviol glycosides have been found in stevia leaves,among them,stevioside is the most abundant followed by rebaudioside A(RA),and they occupy 80%above of the total steviol glycosides.Stevioside and RA are the two main steviol-type substrates used in the enzymatic modifications,and RA possesses higher sweetness and lower bitterness than stevioside.However,the lingering bitterness of RA still restricts its commercial applications.With the organoleptic superiority,the glucosylated products of RA has been regarded as promising candidates for food additives.Rebaudioside M(RM),as the sweetest steviol glycoside with the best sensory quality,has been used in food and beverage by PureCircle and Coca-Cola.However,RM occupied only 0.4～0.5%of the total steviol glycosides in stevia leaves,and the multi-step purification and low yield made difficulties in the large-scale preparation and limited its applications in industrial.This study is focus on enzymatic glucosylation modification of steviol glycosides,which including the following four aspects:1.Two-step enzymatic conversion process of RA into RA1GAs the bitterness of rebaudioside Q3(RQ3,the mono-α-1,4-glucosylated RA derivative with glycosylation occurred on the C-13 site)and the mono-a-1,6glucosylated RA derivative decreased,we hypothesized that the sensory quality would be better by improving the yield of mono-glucosylated products.In this paper,a new two-step enzymatic conversion process was established for the modification of RA via transglycosylation followed by hydrolyzation.In the first step of transglycosylation,using β-cyclodextrin(CD)as the donor substrate and RA as the acceptor substrate,three commercial cyclodextrin glucanotransferases(CGTases)were screened for the transglycosylation activity and the conversion of RA was 77.2,71.1 and 4.7%,respectively.CGTase Toruzyme 3.0 L with the highest RA conversion was subsequently selected and used in the transglycosylation reactions,and the reaction conditions including initial substrate concentration,donor variety,temperature and reaction time were detailedly investigated.The maximum conversion of 87.8%of RA into glucosylated RA derivatives was achieved by incubating 2 U/mL enzyme,82.5 mg/mLβ-CD and 82.5 mg/mL RA at 60℃ for 5 h.Multiple glucosylated products were formed in this reaction,among them,six main derivatives of P1-P6 were analyzed by LC-MS.P1 was the monoglucosylated derivative with a glucosyl unit added to the C-13 site,P2 was the mono-glucosylated derivative with a glucosyl unit added to the C-19 site,P3 was the di-glucosylated derivative with a glucosyl unit added to the C-13 site and a glucosyl unit added to the C-19 site,P4 was the di-glucosylated derivative with two glucosyl units added to the C-19 site,and P5 was the tri-glucosylated derivative with two glucosyl units added to the C-13 site and a glucosyl unit added to the C-19 site.Considering that P6 was a mixture of tri-and tetra-glucosylated products,the glycosylation positions could not be identified by MS analysis.Two mono-glucosylated products of P1 and P2 were formed with a total yield of 18.6%.In the second step of hydrolyzation,four commercial enzymes including three a-amylases and a glucoamylase were screened for shortening the newly synthesized a-glucans of the glucosylated RA derivatives.When three α-amylases were used for hydrolysis,yields of the mono-glucosylated products(P1 and P2)increased to 31.2%,28.5%and 20.8%,respectively.When the glucoamylase was used for hydrolysis,only a monoglucosylated product P2(denoted as RA1G)was produced,and reaction conditions including enzyme concentration,temperature and reaction time were optimized.The maximum yield of 53.3%of RA1G was achieved under the optimal conditions of incubating the reaction solution from the first step of transglycosylation(under the optimum conditions)with 1.5 U/mL glucoamylase at 60℃ for 3 h.By MS and NMR analysis,RA1G was identified as 13-[(2-O-β-D-glucopyranosyl-3-O-β-Dglucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-[(4-O-α-Dglucopyranosyl-β-D-glucopyranosyl)ester]which was a novel compound with monoα-1,4-glycosylation occurred on the C-19 site.Sensory evaluation showed that the sweetness of RA1G was almost the same as that of RA,whereas the bitterness decreased about 54%,which meant that RA1G has an improvement in the sensory quality in comparison with RA.In the present study,a new two-step enzymatic conversion process was constructed using CGTase and glucoamylase to modify the steviol glycoside of RA,and a novel compound of RA1G with mono-α-1,4glucosylation occurred on the C-19 site was formed as the single glucosylated product in the final reaction mixture,and its sensory quality was improved significantly.2.CGTW3:Screening and identification,gene cloning,expression,characterization,enzymatic properties and modification of RACGTases(EC 2.4.1.19)are a class of important glycosidases in glycoside hydrolase(GH)13 family that widely exist in microorganisms,plants and animals,and play important roles in the hydrolysis of starch and synthesis of α-,β-and γ-CD.In this work,four CGTase producing strains(W1～W4)were selected,and the transglycosylated activities were screened using β-CD as the donor substrate and RA as the acceptor substrate.CGTases produced by the strains of W1 and W3 were selected and the conversions of RA were 37%and 57%,respectively.The strain of W3 was subsequently identified as Bacillus cereus W3 based on the morphological and physiological characteristics and phylogenetic analysis of 16S rDNA sequence.Five cyclodextrin glucanotransferase genes(GenBank:KM200046.1,MN013353.1,KF269705.1,X68326.1 and EU644086.2)were searched from CAZy database and the conserved domains obtained by multi-sequences alignment were selected for the degenerate primers.A DNA fragment of～1700 bp was obtained by PCR amplification using the genomic DNA of B.cereus W3 as template,and the cyclodextrin glucanotransferase gene(GenBank:AF047363.1)of 3025 bp(gene of 703～2848 bp encoding an ORF of 2145bp)from Paenibacillus macerans with 97%homology was obtained by BLASTN analysis.This gene was used for primers design and template for PCR amplification,and a 3665 bp DNA fragment was obtained in which 1363-3508 bp encoding an ORF of 2145 bp with a 687 amino-acid protein(CGTW3)and a predicted 27 amino-acid signal peptide.The cgtW3 gene was then expressed in E.coli BL21(DE3).SDS-PAGE showed that the molecular weight of the single subunit was～75 kDa which was in accord with the predicted molecular mass of 75.1 kDa,and the native form of the enzyme turned out to be a homodimer since its natural molecular mass was nearly 138 kDa as determined by native gradient PAGE.The cyclization activity of CGTW3 was detected using starch as the substrate and the yields of α,β and γ-CD(52,17 and 6%,respectively)were analyzed by LC-MS.The enzyme was subsequently identified as α-CGTase.CGTW3 was stable at pH between 5.0 and 9.0 and temperature below 45℃,and was highly active at pH between 4.5 and 8.0 and temperature between 35 and 45℃.Ag+,Fe2+ and Fe3+ completely inactivated the enzyme activity,Co2+,NH4+,Cu2+ and Hg2+ partial inhibited the enzyme activity,whereas Mn2+,Mg2+,Ca2+ and Ni2+exhibited a promotion effect on enzyme activity.The transglycosylation activity was detected using RA as the acceptor substrate,and the influences of temperature,donor variety,enzyme concentration,reaction time and substrate weight ratio were investigated in detail.The maximal RA conversion of 91.7%was obtained under the optimal conditions of incubating 82.5 mg/mL soluble starch and 27.5 mg/mL RA with 1.2 U/mL CGTW3 at 40℃ for 24 h.Under these optimal conditions,nine main products of Pla-P9a with mono-to ennea-glucosylation occured on the C-19 site were formed with the total yield of 87.3%,and three products of P1b-P3b(P1b was the monoglucosylated derivative with a glucosyl unit added to the C-13 site,P2b was the di-glucosylated derivative with a glucosyl unit added to the C-19 site and a glucosyl unit added to the C-13 site,P3 was the tri-glucosylated derivative with two glucosyl units added to the C-19 site and a glucosyl unit added to the C-13 site)were formed with the total yield of 2.9%,which indicated that glycosylation preferred to occur on the C-19 site of RA.By NMR analysis,the structure of P1a was identified as 13-[(2-Oβ-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16en-19-oic acid-[(4-O-α-D-glucopyranosyl-β-D-glucopyranosyl)ester]which was in accord with the structure of RA1G,and the structure of P1b was identified as 13-[(2O-β-D-glucopyranosyl-(4-O-α-D-glucopyranosyl-3-O-β-D-glucopyranosyl)-β-Dglucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-β-D-glucopyranosyl ester]which was in accord with the structure of RQ3.The Km and kcat values of CGTW3 for RA1G was 4.27 mM and 561.8 s-1 while this enzyme could not hydrolyze RQ3,which indicated that CGTW3 possessed C-19 site-selectivity in the hydrolysis reactions.The glucoamylase was used for shortening the newly synthesized α-glucans of the glucosylated RA derivatives.The maximum yield of 72.2%of RA1G was achieved by incubating the reaction solution from the transglycosylation reactions catalyzed by CGTW3(under the optimum conditions)with 1.5 U/mL glucoamylase at 60℃ for 1 h.By sequence alignment and active site analysis,the putative catalytic residues of CGTW3 were the acid/base catalyst Glu259 and nucleophilic catalyst Asp230.Molecular docking analysis showed that the optimal binding model with the lowest binding energy was the C-19 site of RA entering the catalytic cavity of CGTW3,which meant that the C-19 site was easier to be glucosylated than that of the C-13 site.3.EXG1:gene cloning,expression,hydrolysis and transglycosylationExo-β-1,3-glucanases(EXG,EC 3.2.1.58)are a class of glucosidases in GH5 family that catalyze the hydrolysis of β-1,3 linkage from the nonreducing end of glucans and release D-glucoses.The hydrolysis capability of β-1,3 glycosidic linkage of EXG1 from Aspergillus oryzae has already been reported.In this paper,a DNA fragment of 1266 bp that containing two exons and one intron was amplified by PCR using the genomic DNA of A.oryzae 112822(preserved in our laboratory)as template,and the gene of exg1(1218 bp)with removed intron by homologous recombination was obtained and consistent with the reported sequence(GenBank:BAB92972.1).The exg1 gene was then successfully expressed in E.coli and SDS-PAGE showed that the molecular weight of the purified enzyme was～46 kDa which was in accord with the predicted molecular mass of 46.2 kDa.The hydrolysis capability of EXG1 towards β1,3 and β-1,6 glucosidic linkage was examined using 10 μM laminaribiose(3-O-β-Dglucopyranosyl-β-D-glucopyranosyl)and gentiobiose(6-O-β-D-glucopyranosyl-β-Dglucopyranosyl)as substrates.TLC analysis showed that the laminaribiose was almost completely hydrolyzed while the gentiobiose remained a lot at 3.5 h,which meant that EXG1 possessed a higher capability of hydrolyzing β-1,3 linkage than that of β-1,6 linkage.The transglycosylation activity was analyzed using laminarin as the donor substrate and RA as the acceptor substrate.The influences of enzyme concentration,reaction temperature,reaction time and pH were investigated in detail,and the maximal conversion of 43.7%was obtained under the optimal conditions of incubating 20 mg/mL laminarin and 20 mg/mL RA with 0.2 U/mL enzymes at 35℃ for 5 h.The main product was the mono-glucosylated derivative which occupied about 72%of the total products.By MS and NMR analysis,the mono-glucosylated product was identified as 13-[(2-O-β-D-glucopyranosyl-(6-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl)-βD-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-[(β-D-glucopyranosyl)ester]which was a novel compound with mono-β-1,6-glycosylation occurred on the C-13 site(denoted as RAβ-1,6Glc).Under the optimized conditions,the conversion of RC,RM,St and Ru was 31.6,49.0,27.5 and 17.5%,respectively.The main glucosylated product of RC,RM,St and Ru was the mono-glucosylated derivative with glucosylation selectively occurred on the C-13 site,respectively.By sequence alignment and active site analysis,the putative catalytic residues of EXG1 were the acid/base catalyst Glu195 and nucleophilic catalyst Glu298.Molecular docking analysis showed that the binding energy of the C-13 site of RA,RC,St and Ru entering the catalytic center of EXG1(3.1,-2,-1 and-0.3 kcal/mol)was lower than that of the C-19 site(1.6,1.4,0.8 and 0.5 kcal/mol),which meant that it was easier for the C-13 site to be glucosylated than the C-19 site.4.Site-directed mutagenesis of UGT76G1 to improve the synthesis yield of RM with RD as the acceptor substrateIt has been reported that UDP-glycosyltransferase 76G1(UGT76G1)is a chameleon enzyme in glycosyltransferase(GT)1 family from Stevia rebaudiana that could catalyze kinds of reactions with UDP-glucose as the donor substrate,such as synthesis of RA from stevioside by β-1,3 glucosylation occurred on the C-13 site,synthesis of rebaudioside I from RA by β-1,3 glucosylation occurred on the C-19 site,synthesis of rebaudioside D(RD)from rebaudioside E(RE)by β-1,3 glucosylation occurred on the C-13 site,and synthesis of RM from RD by β-1,3 glucosylation occurred on the C-19 site.In this paper,the synthesized gene of ugt76g1(1374 bp)was successfully expressed in Saccharomyces cerevisiae W303.SDS-PAGE showed that the molecular weight of the purified enzyme was～53 kDa which was in accord with the predicted molecular mass of 52.9 kDa.The glycosylation reaction was performed by incubating 1 mM steviol glycoside(RE or RD),2 mM UDP-glucose and 3 mM MgCl2 at 37℃ for 12 h.Using RE as the acceptor substrate,UGT76G1 could synthesize RD with the yield of～100%,whereas using RD as the acceptor substrate,UGT76G1 could also synthesize RM,but the yield was only 36.7%.In order to improve the efficiency in synthesizing RM,the ligand molecule of RE was docked into the enzyme model to find out the positive subsites.Three positive subsite mutants(Q23N,T55S and S56A)of UGT76G1 using site-directed mutagenesis were carried out by changing polarity or steric hindrance,and mutant UGT76G1 T55S could improve the activity significantly with the yield of 64.6%.Considering that the structure of RD has one more 3-O-β-D-glucosyl than RE,we conducted a site-directed mutagenesis study of amino acid substitutions with relatively small polar or small side chain groups at Thr55.After that,site-specific mutations at Thr55(T55G,T55A and T55C)were performed,and the mutant UGT76G1 T55G could improve the yield of RM from 36.7%to 97.3%.The optimal temperature of UGT76G1 WT was 37℃ and it was stable below 50℃.The optimal temperature of UGT76G1 T55G was 37℃ and it was stable below 45℃.UGT76G1 WT was highly active at pH 7.5 and stable at pH 7.0 to 8.5.UGT76G1 T55G was highly active at pH 8.5 and stable at pH 7.0 to 7.5.The alignment models of UGT76G1 WT and T55G showed the amino acid in 55th site mutated from threonine to glycine could reduce the steric hindrance and broaden the catalytic cavity,which made the enterence of RD and the leavement of RM easier.These phenomena might eventually lead to the increasement of the activity.The new enzyme tool obtained by site-directed mutagenesis would achieve the synthesis of RM with high-potency in vitro.This study provided new processes for the enzymatic transglycosylation of steviol glycosides,supplied new enzyme tools for glycosides synthesis,and offered new idea to improve the transglycosylation activity of an enzyme.The novel compounds produced in this study would contribute to the further understanding of structuresweetness relationship of steviol glycosides and have great potential applications in the future. |
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