Hydrolyses and transglycosylations performed by purified alpha-D-glucosidase of the marine mollusc Aplysia fasciata

The purification and characterisation of the alpha-glucosidase from the marine mollusc Aplysia fasciata are reported. Overall substrate specificity of the pure enzyme for both hydrolytic and transglycosylation reactions was studied. Remarkable characteristics of this enzyme are indicated by the results of the interesting survey of transglycosylation reactions reported: pyridoxine glucosylation, synthesis of chromophoric (pNP) di- and trisaccharides, glucosylation of cellobiose and sucrose. For these last two acceptors both the yields of reactions and the concentrations of products are comparable to those obtained using glycosyl transferases; in addition, synthesis of pyridoxine and chromophoric glycosides were still possible using a 1:1 ratio maltose:acceptor which is a very interesting characteristic from a synthetic point of view (effortless purification, productivity of each reaction batch, etc.).     

The effect of maltose on dextran yield and molecular weight distribution

Dextran synthesis has been studied since the Second World War, when it was used as blood plasma expander. This polysaccharide composed of glucose units is linked by an α-1,6-glucosidic bond. Dextransucrase is a bacterial extra cellular enzyme, which promotes the dextran synthesis from sucrose. When, besides sucrose, another substrate (acceptor) is also present in the reactor, oligosaccharides are produced and part of the glucosyl moieties from glucose is consumed to form these acceptor products, decreasing the dextran yield. Although dextran enzymatic synthesis has been extensively studied, there are few published studies regarding its molecular weight distribution. In this work, the effect of maltose on yield and dextran molecular weight synthesized using dextransucrase from Leuconostoc mesenteroides B512F, was investigated. According to the obtained results, maltose is not able to control and reduce dextran molecular weight distribution and synthesis carried out with or without maltose presented the same molecular weight distribution profile.     

Enzymatic Synthesis of Polyol- and Masked Polyol- Glycosides using β-Glycosidase of Sulfolobus Solfataricus

The use of commercially available mesophilic glycosidases in the enzymatic synthesis of glycosides of different types is a well established method suffering from some drawbacks such as a poor yield. Substrates with three or four hydroxyl groups have been subjected to enzymatic glucosylation using crude homogenate of the thermophilic archaeon Sulfolobus solfataricus containing a β-glycosidase activity able to transfer glucose, galactose and fucose from different donors. The stereochemistry of this reaction was interpreted in terms of interaction with a possible “glucose” active site of the enzyme. In addition masked or protected derivatives of tetritols and some simple unsaturated alcohols were glycosylated yielding glycosides in yields very competitive with those obtained using mesophilic enzymes, examples of further chemical manipulation of these compounds were reported. When using a scarce amount of acceptor, a reasonable amount of products could be obtained by adding different aliquots of donor at time intervals.     

Studies on glucosyltransferase and endogenous glucosyl acceptor in Bacillus cereus AHU 1030 membranes

A glucosyltransferase, extracted from the membranes of Bacillus cereus AHU 1030 with Tris-HCl buffer containing 0.1% Triton X-100 at pH 9.5, was separated from an endogenous glucosyl acceptor by chromatography on DEAE-Sepharose CL-6B subsequent to chromatography on Sepharose 6B. Structural analysis data showed that the glucosyl acceptor was a glycerol phosphate polymer linked to beta-gentiobiosyl diglyceride. The enzyme catalyzed the transfer of glucosyl residues from UDP-glucose to C-2 of the glycerol residues of repeating units of the acceptor. On the other hand, a lipoteichoic acid which contained 0.3 D-alanine residue per phosphorus was isolated from the cells by phenol treatment at pH 4.6. Except for the presence of D-alanine, this lipoteichoic acid had the same structure as the glucosyl acceptor. The rate of glucosylation observed with the D-alanine-containing lipoteichoic acid as the substrate was less than 40% of that observed with the D-alanine-free lipoteichoic acid, indicating that the ester-linked D-alanine in the lipoteichoic acid interferes with the action of the glucosyltransferase. The enzyme also catalyzed glucosylation of poly(glycerol phosphate) which was synthesized in the reaction of a separate enzyme fraction with CDP-glycerol. Thus, it is likely that the glucosyltransferase functions in the synthesis of cell wall teichoic acid.     

Highly regioselective glucosylation of 2'-deoxynucleosides by using the crude β-glycosidase from bovine liver

An enzymatic regioselective approach for the glucosylation of a series of 2′-deoxynucleosides was described by using the crude β-glycosidase from bovine liver that is less expensive and can be simply prepared in a standard organic laboratory. With the glucosylation of 2′-deoxyuridine as a model reaction, the effects of several key factors on the enzymatic reaction were examined. The optimum enzyme dosage, buffer pH and temperature were 0.05 U/ml, 9.5 and 42 °C, respectively. The presence of alkali β-glycosidase as the main active component in the crude enzyme extract might account for the high glucosylation activity at pH 9.5. In addition, the desired 5′-O-glucosylated derivatives of 2′-deoxynucleosides were synthesized with the yields of 22-72% and exclusive 5′-regioselectivities (>99%).     

Reaction mechanism of isomaltooligosaccharides synthesis by α-glucosidase fromAspergillus carbonarious

Summary An -glucosidase fromAspergillus carbonarious CCRC 30414 was employed for investigating the enzymatic synthesis of isomaltooligosaccharides from maltose. The enzyme transferred a glucose unit from the nonreducing end of maltose and other -linked glucosyl oligosaccharides to glucose and other glucosyl oligosaccharides which function as accepting co-substrates. The transfer of a glucose unit occurs most frequently to the 6-OH position of the nonreducing end of acceptor, but transfer to 4-OH position also occurs. Treatment of 30 % (w/v) maltose with the enzyme under optimum conditions afforded more than 50% isomaltooligosaccharides.     

Modification of flavonoid using lipase in non-conventional media: effect of the water content

The enzymatic acylation of a flavonoid (naringin) was investigated in this work. This atypic substrate for a lipase was esterified very selectively by the immobilized Candida antarctica lipase: a single product was synthesized and was assumed to be the 6-O-palmitate naringin ester acylated on the glucose moiety. As lipase-catalyzed esterification reactions in organic media are greatly influenced by the water content, the effect of the initial hydration level of the reaction medium components was pointed out for naringin palmitate synthesis. 2-Methyl 2-butanol (solvent) and naringin (acyl acceptor) provided high amounts of water and when dried increased the conversion yield by 63% and the specific activity by 60%. On the contrary, the enzyme must not be dried because water is essential for the three-dimensional structure of the protein and, if absent, results in a 67% loss of activity. As water was produced in parallel to ester synthesis, the equilibrium of the reaction might be shifted by its removal. When the reaction was carried out with 100 g l(-1) molecular sieves 4A added after 24 h of reaction, a conversion yield of 43% was reached after 55 h reaction.     

Sucrose phosphorylase: a powerful transglucosylation catalyst for synthesis of α-D-glucosides as industrial fine chemicals

Abstract

Sucrose phosphorylase is a bacterial transglucosidase that catalyzes conversion of sucrose and phosphate into α-D-glucose-1-phosphate and D-fructose. The enzyme utilizes a glycoside hydrolase-like double displacement mechanism that involves a catalytically competent β-glucosyl enzyme intermediate. In addition to reaction with phosphate, glucosylated sucrose phosphorylase can undergo hydrolysis to yield α-D-glucose or it can decompose via glucosyl transfer to a hydroxy group in suitable acceptor molecules, giving new α-D-glucosidic products. The glucosyl acceptor specificity of sucrose phosphorylase is reviewed, focusing on applications of the enzyme in glucoside synthesis. Polyhydroxylated compounds such as sugars and sugar alcohols are often glucosylated efficiently. Aryl alcohols and different carboxylic acids also serve as acceptors for enzymatic transglucosylation. The natural osmolyte 2-O-(α-D-glucopyranosyl)-sn-glycerol (GG) was prepared by regioselective glucosylation of glycerol from sucrose using the phosphorylase from Leuconostoc mesenteroides. An industrial process for production of GG as active ingredient of cosmetic formulations has been recently developed. General advantages of sucrose phosphorylase as a transglucosylation catalyst lie in the use of sucrose as a high-energy glucosyl donor and the usually weak hydrolase activity of the enzyme towards substrate and product.     

Naringinase-catalyzed hydrolysis of naringin adsorbed on macroporous resin

Naringenin, a natural plant flavonoid found in citrus fruits, has been reported to exhibit a wide range of pharmacological functions, including anticancer, antioxidant, antiatherogenic, antithrombotic, and vasodilator activities. Naringenin can be produced from the naringinase (NGase)-catalyzed enzymatic hydrolysis of naringin. However, the poor solubility of naringin in aqueous systems considerably limits the efficiency of naringenin biocatalysis. In this work, a novel substrate adsorption system was proposed for naringin adsorption to increase the efficiency of naringin hydrolysis and naringenin production. Three Amberlite macroporous resins, namely, XAD-4, XAD-7HP and XAD-16, were investigated for their naringin adsorption capacities and effects on NGase hydrolysis. Results indicated that the physical properties of the resins played a critical role in naringin adsorption and naringenin enzymatic synthesis. Naringin hydrolysis was carried out using free and adsorbed substrates. The substrate adsorption strategy could increase the catalytic efficiency at a high naringin concentration. In addition, the reaction conditions for enzymatic naringenin synthesis were optimized, and naringenin was prepared at a liter scale with a high substrate concentration. These results suggested that substrate adsorption is a promising strategy to increase the enzymatic hydrolysis efficiency of naringenin in aqueous systems.     

Last Step in the Conversion of Trehalose to Glycogen: A MYCOBACTERIAL ENZYME THAT TRANSFERS MALTOSE FROM MALTOSE 1-PHOSPHATE TO GLYCOGEN

We show that Mycobacterium smegmatis has an enzyme catalyzing transfer of maltose from [¹⁴C]maltose 1-phosphate to glycogen. This enzyme was purified 90-fold from crude extracts and characterized. Maltose transfer required addition of an acceptor. Liver, oyster, or mycobacterial glycogens were the best acceptors, whereas amylopectin had good activity, but amylose was a poor acceptor. Maltosaccharides inhibited the transfer of maltose from [¹⁴C]maltose-1-P to glycogen because they were also acceptors of maltose, and they caused production of larger sized radioactive maltosaccharides. When maltotetraose was the acceptor, over 90% of the ¹⁴C-labeled product was maltohexaose, and no radioactivity was in maltopentaose, demonstrating that maltose was transferred intact. Stoichiometry showed that 0.89 μmol of inorganic phosphate was produced for each micromole of maltose transferred to glycogen, and 56% of the added maltose-1-P was transferred to glycogen. This enzyme has been named α1,4-glucan:maltose-1-P maltosyltransferase (GMPMT). Transfer of maltose to glycogen was inhibited by micromolar amounts of inorganic phosphate or arsenate but was only slightly inhibited by millimolar concentrations of glucose-1-P, glucose-6-P, or inorganic pyrophosphate. GMPMT was compared with glycogen phosphorylase (GP). GMPMT catalyzed transfer of [¹⁴C]maltose-1-P, but not [¹⁴C]glucose-1-P, to glycogen, whereas GP transferred radioactivity from glucose-1-P but not maltose-1-P. GMPMT and GP were both inhibited by 1,4-dideoxy-1,4-imino-d-arabinitol, but only GP was inhibited by isofagomine. Because mycobacteria that contain trehalose synthase accumulate large amounts of glycogen when grown in high concentrations of trehalose, we propose that trehalose synthase, maltokinase, and GMPMT represent a new pathway of glycogen synthesis using trehalose as the source of glucose.     

Enzymatic synthesis of α-glucosyl-timosaponin BII catalyzed by the extremely thermophilic enzyme: Toruzyme 3.0L

Timosaponin BII (BII), a steroidal saponin showing potential anti-dementia activity, was converted into its glucosylation derivatives by Toruzyme 3.0L. Nine products with different degrees of glucosylation were purified and their structures were elucidated on the basis of ¹³C NMR, HR-ESI-MS, and FAB-MS spectra data. The active enzyme in Toruzyme 3.0L was purified to electrophoretic homogeneity by tracking BII-glycosylase activity and was identified as Cyclodextrin-glycosyltransferase (CGTase, EC 2.4.1.19) by ESI-Q-TOF MS/MS. In this work, we found that the active enzyme catalyzed the synthesis of alpha-(1→4)-linked glucosyl-BII when dextrin instead of an expensive activated sugar was used as the donor and showed a high thermal tolerance with the most favorable enzymatic activity at 100 °C. In addition, we also found that the α-amylases and CGTase, that is, GH13 family enzymes, all exhibited similar activities, which were able to catalyze glucosylation in steroidal saponins. But other kinds of amylases, such as γ-amylase (GH15 family), had no such activity under the same reaction conditions.     

Surface display of transglucosidase on Escherichia coli by using the ice nucleation protein of Xanthomonas campestris and its application in glucosylation of hydroquinone

A surface anchoring motif using the ice nucleation protein (INP) of Xanthomonas campestris pv. campestris BCRC 12,846 for display of transglucosidase has been developed. The transglucosidase gene from Xanthomonas campestris pv. campestris BCRC 12,608 was fused to the truncated ina gene. This truncated INP consisting of N- and C-terminal domains (INPNC) was able to direct the expressed transglucosidase fusion protein to the cell surface of E. coli with apparent high enzymatic activity. The localization of the truncated INPNC-transglucosidase fusion protein was examined by Western blot analysis and immunofluorescence labeling, and by whole-cell enzyme activity in the glucosylation of hydroquinone. The glucosylation reaction was carried out at 40 degrees C for 1 h, which gave 23 g/L of alpha-arbutin, and the molar conversion based on the amount of hydroquinone reached 83%. The use of whole-cells of the wild type strain resulted in an alpha-arbutin concentration of 4 g/L and a molar conversion of 16% only under the same conditions. The results suggested that E. coli displaying transglucosidase using truncated INPNC as an anchoring motif can be employed as a whole-cell biocatalyst in glucosylation.     

Increase in the glucosylated form of erythrocyte Cu-Zn-superoxide dismutase in diabetes and close association of the nonenzymatic glucosylation with the enzyme activity

Human erythrocytes contain glucosylated and nonglucosylated Cu-Zn-superoxide dismutases which can be separated by boronate affinity chromatography. The percentage of the glucosylated form is significantly increased in the erythrocytes of patients with diabetes as compared to normal erythrocytes. The nonglucosylated form of Cu-Zn-superoxide dismutase, which was washed through the boronate column, was glucosylated in vitro upon exposure to radioactive or non-radioactive D-glucose. Incorporation of D-glucose into the protein was observed, and with the increase in glucosylation, the enzymatic activity decreased, indicating that the glucosylation of the enzyme led to a low active form. This is the first demonstration that superoxide dismutase is glucosylated in erythrocytes and that the glucosylation leads to the inactivation of the enzyme.     

Production of glucooligosaccharides by an acceptor reaction using two types of glucansucrase from Streptococcus sobrinus

Glucooligosaccharides (GOS) were produced by using an acceptor reaction with two types of glucansucrase (GTF-S and GTF-I) from Streptococcus sobrinus. Acceptor reactions of GTF-S with maltose acceptor, gave a great number of GOS ranging from DP(degree of polymerization) 2 to DP15. At the both acceptor reactions with GTF-S or GTF-I, as the sucrose/maltose ratio was decreased, the amount of dextran and DP of oligosaccharides was decreased. A maximum GOS yield of 69% was achieved at the acceptor reaction with GTF-I and when the molar ratio of sucrose/maltose is 2:1, in which GOS of DP6~DP9 were major oligosaccharides and 17% of dextran. The polymeric size of GOS could be controlled by varying the ratio of sucrose to the acceptor (maltose in this work).     

Regioselective O-glucosylation by sucrose phosphorylase: a promising route for functional diversification of a range of 1,2-propanediols

1,2-Propanediol and 3-aryloxy/alkyloxy derivatives thereof are bulk commodities produced directly from glycerol. Glycosylation is a promising route for their functional diversification into useful fine chemicals. Regioselective glucosylation of the secondary hydroxyl in different 1,2-propanediols was achieved by a sucrose phosphorylase-catalyzed transfer reaction where sucrose is the substrate and 2-O-α-d-glucopyranosyl products are exclusively obtained. Systematic investigation for optimization of the biocatalytic synthesis included prevention of sucrose hydrolysis, which occurs in the process as a side reaction of the phosphorylase. In addition to ‘nonproductive’ depletion of donor substrate, the hydrolysis also resulted in formation of maltose and kojibiose (up to 45%) due to secondary enzymatic glucosylation of the glucose thus produced. Using 3-ethoxy-1,2-propanediol as the acceptor substrate (1.0 M), the desired transfer product was obtained in about 65% yield when employing a moderate (1.5-fold) excess of sucrose donor. Loss of the glucosyl substrate to ‘glucobiose’ by-products was minimal (<7.5%) under these conditions. The reactivity of other acceptors decreased in the order, 3-methoxy-1,2-propanediol > 1,2-propanediol > 3-allyloxy-1,2-propanediol > 3-(o-methoxyphenoxy)-1,2-propanediol > 3-tert-butoxy-1,2-propanediol. Glucosylated 1,2-propanediols were not detectably hydrolyzed by sucrose phosphorylase so that their synthesis by transglucosylation occurred simply under quasi-equilibrium reaction conditions.     

Functional characteristics of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. BL-31 highly specific for intermolecular transglycosylation of bioflavonoids

The functional characteristics of a beta-cyclodextrin glucanotransferase (CGTase) excreted from alkalophilic Bacillus sp. BL-31 that is highly specific for the intermolecular transglycosylation of bioflavonoids were investigated. The new beta-CGTase showed high specificities for glycosyl acceptor bioflavonoids, including naringin, rutin, and hesperidin, and especially naringin. The transglycosylation of naringin into glycosyl naringin was then carried out under the conditions of 80 units of CGTase per gram of maltodextrin, 5 g/l of naringin, 25 g/l of maltodextrin, and 1 mM Mn2+ ion at 40 degrees C for 6 h, resulting in a high conversion yield of 92.1%.     

Determining the optimum model parameters for oligosaccharide production efficiency using response surface integrated particle swarm optimization method: an experimental validation study

Abstract

Glucansucrases (GTFs) catalyzes the synthesis of α-glucans from sucrose and oligosaccharides in the presence of an acceptor sugar by transferring glucosyl units to the acceptor molecule with different linkages. The acceptor reactions can be affected by several parameters and this study aimed to determine the optimal reaction parameters for the production of glucansucrase-based oligosaccharides using sucrose and maltose as the donor and acceptor sugars, respectively via a hybrid technique of Response Surface Method (RSM) and Particle Swarm Optimization (PSO). The experimental design was performed using Central Composite Design and the tested parameters were enzyme concentration, acceptor:donor ratio and the reaction period. The optimization studies showed that enzyme concentration was the most effective parameter for the final oligosaccharides yields. The optimal values of the significant parameters determined for enzyme concentration and acceptor:donor ratio were 3.45?U and 0.62, respectively. Even the response surface plots for input parameters verified the PSO results, an experimental validation study was performed for the reverification. The experimental verification results obtained were also consistent with the PSO results. These findings will help our understanding in the role of different parameters for the production of oligosaccharides in the acceptor reactions of GTFs.     

Cloning, expression and functional characterization of a novel trehalose synthase from marine Pseudomonas sp. P8005

Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose and trehalose. A novel treS gene with a length of 3,369 bp, encoding a protein of 1,122 amino acid residues with a predicted molecular mass of 126 kDa, was cloned from a marine Pseudomonas sp. P8005 (CCTCC: M2010298) and expressed in Escherichia coli. The amino acid sequence identities between this novel TreS and other reported TreS is relatively low. The purified recombinant TreS showed an optimum pH and temperature of 7.2 and 37 °C, respectively. The enzyme displayed a high conversion rate (70 %) of maltose to trehalose during equilibrium and had a higher catalytic efficiency (k _cat/K _m) for maltose than for trehalose, suggesting its application in the production of trehalose. In addition to maltose and trehalose, this enzyme can also act on sucrose, although this activity is relatively low. Mutagenesis studies demonstrated that enzymatic activity was reduced dramatically by individually substitution with alanine for D78, Y81, H121, D219, E261, H331 or D332, which implied that these residues might be important in P8005-TreS. Experiments using isotope-labeled substrates showed that [²H₂]trehalose combined with unlabeled trehalose was converted to [²H₂]maltose and maltose, but without any production of [²H]maltose or [²H]trehalose and with no incorporation of exogenous [²H₇]glucose into the disaccharides during the conversion catalyzed by this enzyme. This finding indicated the involvement of an intramolecular mechanism in P8005-TreS catalyzing the reversible interconversion of maltose and trehalose.     

The mechanism of acceptor reactions of leuconostoc mesenteroides B-512F dextransucrase

Reactions of dextransucrase and sucrose in the presence of sugars (acceptors) of low molecular weight have been observed to give a dextran of low molecular weight and a series of oligosaccharides. The acceptor reaction of dextransucrase was examined in the absence and presence of sucrose by using d-[¹⁴C]glucose, d-[¹⁴C]fructose, and ¹⁴C-reducing-end labeled maltose as acceptors. A purified dextransucrase was pre-incubated with sucrose, and the resulting d-fructose and unreacted sucrose were removed from the enzyme by chromatography on columns of Bio-Gel P-6. The enzyme, which migrated at the void volume, was collected and referred to as “charged enzyme”. The charged enzyme was incubated with ¹⁴C-acceptor in the absence of sucrose. Each of the three acceptors gave two fractions of labeled products, a high molecular weight product, identified as dextran, and a product of low molecular weight that was an oligosaccharide. It was found that all three of the acceptors were incorporated into the products at the reducing end. Similar results were obtained when the reactions were performed in the presence of sucrose, but higher yields of labeled products were obtained and a series of homologous oligosaccharides was produced when d-glucose or maltose was the acceptor. We propose that the acceptor reaction proceeds by nucleophilic displacement of glucosyl and dextranosyl groups from a covalent enzyme-complex by a specific, acceptor hydroxyl group, and that this reaction effects a glycosidic linkage between the d-glucosyl and dextranosyl groups and the acceptor. We conclude that the acceptor reactions serve to terminate polymerization of dextran by displacing the growing dextran chain from the active site of the enzyme; the acceptors, thus, do not initiate dextran polymerization by acting as primers.     

Regioselective synthesis of neo-erlose by the β-fructofuranosidase from Xanthophyllomyces dendrorhous

The β-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous (Xd-INV) catalyzes the synthesis of neo-fructooligosaccharides (neo-FOS of the ⁶G-series), which contain a β(2 → 6) linkage between a fructose and the glucosyl moiety of sucrose. In this work we demonstrate that the enzyme is also able to fructosylate other carbohydrates that contain glucose, in particular disaccharides (maltose, isomaltulose, isomaltose, trehalose) and higher oligosaccharides (maltotriose, raffinose, maltotetraose), but not monosaccharides (glucose, fructose, galactose). With maltose as acceptor, the reaction in the presence of Xd-INV proceeded with high regioselectivity; the product was purified and chemically characterized, and turned out to be 6′-O-β-fructosylmaltose (neo-erlose). Using 100 g/L sucrose as fructosyl donor and 300 g/L maltose as acceptor, the maximum concentration of neo-erlose was 38.3 g/L. Thus, novel hetero-fructooligosaccharides with potential applications in the functional food and pharmaceutical industries can be obtained with Xd-INV.