Characterization of dextranase from Penicillium purpurogenum (Ftoll)]

An extracellular dextranase (E. C. 3.2.1.11) was purified from cell-free culture filtrates of Penicillium purpurogenum (Ftoll). The enzyme was most active at pH 5,5. The dextranase was endo-type, it split quickly isomaltotetraose into two isomaltose molecules, slowly degraded isomaltotriose, and did not act on isomaltose. The rate of isomaltooligosaccharides hydrolysis was increased with the increase of the polymerization degree. Polyols obtained from isomaltooligosaccharides were split more slowly than the respective sugars. The isomaltopentaitol was split at two glucosidic linkages, 38% of hydrolyzed linkages being the second linkage from the sorbitol end of the molecule and 62% being the third one. The degree of degradation of dextrans depended on amount of 1,6 linkages. Isomaltose and tetrasaccharides of two types, 2(2)-alpha-D-glucosylmaltotriose and linear tetrasaccharide(s), are the lowest molecular weight products of exhaustive hydrolysis of branched dextrans.     

Kinetics, equilibria, and modeling of the formation of oligosaccharides from D-glucose with Aspergillus niger glucoamylases I and II

Near-homogeneous forms of glucoamylases I and II, previously purified from an industrial Aspergillus niger preparation, were incubated with D-glucose at a number of temperatures and pH values. Kinetics and equilibria of the formation of alpha,beta-trehalose, kojibiose, nigerose, maltose, isomaltose, panose, and isomaltotriose, which with isomaltotetraose were the only products formed, were determined. There was no difference in the abilities of GA I and GA II to form these products. Activation energies for the formation of maltose and panose were lower than those of the other Oligosaccharides. Relative rates of oligosaccharide production based on glucoamylase hydrolytic activity did not vary significantly between pH 3.5 and 4.5 but were lower at pH 5.5. Maltose was formed much faster than any other product. Equilibrium concentrations at higher dissolved solids concentrations decreased in the order isomaltose, isomaltotriose, kojibiose, nigerose, maltose, alpha, beta-Mrehalose, panose, and isomaltotetraose. They were not appreciably affected by changes in temperature or pH. A kinetic model based on adsorption of D-glucose and the seven di- and trisaccharides by the first three glucoamylase subsites was formulated. Oligosaccharide formation was simulated with the model, using equilibrium data gathered for this article and subsite binding energies and kinetic parameters for oligosaccharide hydrolysis measured earlier. Agreement of simulated and actual oligosaccharide formation data through the course of the reaction was excellent except at very high solid concentrations.     

Transisomaltosylation Activity of a Bacterial Isomalto-dextranase

A bacterial isomalto-dextranase, described previously as a new type of dextranase different from the known 1,6-α-d-glucan 6-glucanohydrolase [EC 3.2.1.11], was found to be a configuration-retaining exo-glucanase so far as judged from the downward mutarotation shown by products in a dextran digest, and from the lower activity of the enzyme on lesspolymerized isomaltodextrins according to one of the criteria proposed by Reese et al. The dextranase was observed to cause not only transisomaltosylation (isomaltotetraose formation in dextran digests, isomaltotriose formation in dextran digests containing glucose and transisomaltosylation among isomaltodextrins) but also isomaltose condensation to isomaltotetraose in concentrated solutions. These activities shown by the isomalto-dextranase are in keeping with the novel concept that carbohydrases are catalysts of glycosylation (glycosylhydrogen interchange), proposed by Hehre and his coworkers. The relative ease of isomaltose condensation catalyzed by the enzyme appears due to the exergonic nature of the reaction. A free energy change value of ca. ?1200 cal/mole was obtained for the condensation.     

Hydrolysis of low molecular weight isomaltosaccharides by a p-nitrophenyl-alpha-D-glucopyranoside-hydrolyzing alpha-glucosidase from a thermophile, Bacillus thermoglucosidius KP 1006.

A p-nitrophenyl-alpha-D-glucopyranoside-hydrolyzing alpha-glucosidase of a thermophile, Bacillus thermoglucosidius KP 1006, was purified to an electrophoretically-homogeneous state. Its molecular weight was estimated as 60 000 by gel electrophoresis. The molecular activity (ko) and the Km value at 60 degrees C and pH 6.8 for p-nitrophenyl-alpha-D-glucopyranoside were 233 s-1 and 0.24 mM, respectively. The enzyme cleft the non-reducing terminal alpha-1,6-glucosidic bonds of isomaltose, panose, isomaltotriose, isomaltotetraose, and isomaltopentaose. The ko values were 72.4, 194, 208, 233 and 167 s-1, and the Km values were 3.3, 9.5, 11, 13 and 21 mM, respectively. Each isomaltosaccharide was hydrolyzed to glucose by the cleavage of single glucose units from its nonreducing end. The present study suggests that the enzyme is an oligo-1,6-glucosidase (dextrin 6-alpha-glucanohydrolase, EC 3.2.1.10) and an exo-glucosidase.     

Quantitative Study of the Anomeric Forms of Isomaltose and Glucose Produced by Isomalto-dextranase and Glucodextranase

A gas-liquid chromatographic method was applied to the determination of anomeric forms of isomaltose and glucose produced by Arthrobacter globiformis isomalto-dextranase and glucodex- tranase. The anomeric forms of products released from isomaltotriose, panose and dextran were quantitatively determined. The isomalto-dextranase that was also capable of splitting the α-1,4- glucosidic linkage of panose was found to exclusively produce α-isomaltose from these substrates, and the glucodextranase, ^-glucose.     

Linkage analysis of (1→6)-linked oligosaccharides by alkaline degradation

2-C-Methylglyceric acid was formed by sequential degradation of the chain in isomaltose oligosaccharides in aqueous barium hydroxide. The products of the stopping reaction, namely 6-O-substituted 3-deoxy-d-hexonic acids, were also obtained in considerable yield. The separation and determination of these products is of importance for the analysis of the linkage sequence in oligosaccharides.     

Expression and characterization of an <Emphasis Type="Italic">α</Emphasis>-glucosidase from <Emphasis Type="Italic">Thermoanaerobacter ethanolicus</Emphasis> JW200 with potential for industrial application

In this study, a new α-glucosidase gene from Thermoanaerobacter ethanolicus JW200 was cloned and expressed in Escherichia coli by a novel heat-shock vector pHsh. The recombinant α-glucosidase exhibited its maximum hydrolytic activity at 70°C and pH 5.0∼5.5. With p-nitrophenyl-α-D-glucoside as a substrate and under the optimal condition (70°C, pH 5.5), K _m and V _max of the enzyme was 1.72 mM and 39 U/mg, respectively. The purified α-glucosidase could hydrolyze oligosaccharides with both α-1,4 and α-1,6 linkages. The enzyme also had strong transglycosylation activity when maltose was used as sugar donor. The transglucosylation products towards maltose are isomaltose, maltotriose, panose, isomaltotriose and tetrasaccharides. The enzyme could convert 400 g/L maltose to oligosaccharides with a conversion rate of 52%, and 83% of the oligosaccharides formed were prebiotic isomaltooligosaccharides (containing isomaltose, panose and isomaltotriose).     

Purification and substrate specificity of an endo-dextranase of Streptococcus mutans K1-R

An extracellular endo-dextranase has been isolated from Streptococcus mutans K1-R. Incubation of cell-free culture fluid with sucrose permitted the removal of a large proportion of the extracellular d-glucosyltransferases by irreversible adsorption onto the insoluble glucans that these enzymes synthesize from sucrose. The remaining d-glucosyltransferases were separated from dextranase by precipitation with ammonium sulphate, chromatography on hydroxylapatite and DEAE-cellulose, followed by filtration on Ultrogel. The major products of action of the purified dextranase on (1→6)-α-d-glucans were isomaltotriose (IM₃), isomaltotetraose (IM₄), and isomaltopentaose (IM₅). Further hydrolysis of IM₄ and IM₅ occurred after prolonged incubation with excess of enzyme, to give d-glucose, IM₂, and IM₃. The relative rate of hydrolysis of isomaltose saccharides fell sharply with decreasing chainlength from IM₁₂ to IM₅. The hydrolysis of dextrans containing 96% or more of (1→6)-α-d-glucosidic linkages, expressed as apparent conversion into IM₃, was virtually complete, and substrates such as Streptococcus sanguis glucan, containing sequences of (1→6)-α-d-glucosidic linkages, were also effectively hydrolyzed. Dextranase activity towards the soluble glucan of Streptococcus mutans was limited, and there was no action on the insoluble glucan synthesized by S. mutans sucrose 3-d-glucosyltransferase.     

Transglycosylation reaction and raw starch hydrolysis by novel carbohydrolase from<Emphasis Type="Italic">Lipomyces starkeyi</Emphasis>

A novel carbohydrolase, which is a DXAMase, containing both dextranase and amylase equivalent activities, was purified fromLipomyces starkeyi KSM22. The purified DXAMase was also found to hydrolyze cellobiose, gentiobiose, trehalose and melezitose, while disproportionation reactions were exhibited with various di-and tri-saccharides, such as maltose, isomaltose, gentiobiose, kojibiose, sophorose, panose, maltotriose, and isomaltotriose with various kinds of oligosaccharides produced as acceptor reaction products. Furthermore, the purified DXAMase hydrolyzed raw waxy rice starch and produced maltodextrin to the extent of 50% as a glucose equivalent.     

Characterization of Two Novel α-Glucosidases from Bifidobacterium breve UCC2003

Two α-glucosidase-encoding genes (agl1 and agl2) from Bifidobacterium breve UCC2003 were identified and characterized. Based on their similarity to characterized carbohydrate hydrolases, the Agl1 and Agl2 enzymes are both assigned to a subgroup of the glycosyl hydrolase family 13, the α-1,6-glucosidases (EC 3.2.1.10). Recombinant Agl1 and Agl2 into which a His₁₂ sequence was incorporated (Agl1_His and Agl2_His, respectively) exhibited hydrolytic activity towards panose, isomaltose, isomaltotriose, and four sucrose isomers—palatinose, trehalulose, turanose, and maltulose—while also degrading trehalose and, to a lesser extent, nigerose. The preferred substrates for both enzymes were panose, isomaltose, and trehalulose. Furthermore, the pH and temperature optima for both enzymes were determined, showing that Agl1_His exhibits higher thermo and pH optima than Agl2_His. The two purified α-1,6-glucosidases were also shown to have transglycosylation activity, synthesizing oligosaccharides from palatinose, trehalulose, trehalose, panose, and isomaltotriose.     

The synthesis of 1,6-anhydro-β-d-glucopyranose and d-glucosyl oligosaccharides from maltose by a fungal glucosyltransferase

The formation of 1,6-anhydro-β-d-glucopyranose and several d-glucosyl oligosaccharides has been observed during the action of a purified, fungal glucosyltransferase (EC 2.4.1.24) on maltose. Such products are synthesized by a transglucosylation mechanism involving the formation of a d-glucosyl-enzyme complex and the displacement of the d-glucosyl group by appropriate acceptor-substrates. The formation of the 1,6-anhydro bond is a novel type of transfer reaction and occurs by displacement of the enzyme from the d-glucosyl-enzyme complex by the proton of the primary hydroxyl group of the same glucosyl group. This reaction is characterized by inversion of configuration at the position of glucosidic bond-cleavage of the substrate. Synthesis of the d-glucosyl oligosaccharides occurs by displacement of the d-glucosyl groups from the enzyme by suitable acceptor-substrates. In these cases, the reactions are characterized by retention of configuration of the d-glucosidic bonds of the substrate. The list of oligosaccharides produced from maltose includes nigerose, kojibiose, isomaltose, maltotriose, panose, isomaltotriose, and 6-O-d-glucosyl-panose. The identity of these compounds has been established by methylation analysis and enzymic hydrolysis. d-Glucose is also a product of the reaction and arises from both the reducing and the non-reducing groups of maltose.     

Hydrolysis of Nothogenia erinacea xylan by xylanases from families 10 and 11

The structures of several enzymatic hydrolysis products of Nothogenia erinacea seaweed xylan, a linear homopolymer with mixed beta-(1-->3)/beta-(1-->4) linkages, were analysed by physicochemical and biochemical techniques. With the glycoside hydrolase family 10 beta-(1-->4)-xylanase from Cryptococcus adeliae, hydrolysis proceeds to a final mixture of products containing a mixed linkage-type triose as a major compound, whereas with the family 11 xylanase from Thermomyces lanuginosus this is a mixed linkage tetraose. The Cryptococcus xylanase is shown to be capable of also catalysing the hydrolysis of beta-(1-->3) linkages, that is this of a mixed type tetraose intermediary formed, in accordance with the broader substrate specificity of family 10 enzymes. From a partial degradation experiment with the T. lanuginosus xylanase, a series of higher mixed oligosaccharides were isolated and identified. The observed oligosaccharide intermediates and splicing pattern indicate an irregular beta-(1-->3)/beta-(1-->4) linkage distribution within the linear d-xylose polymer. Similar results were obtained with rhodymenan, the seaweed xylan from Palmares palmata.     

Sephadex-binding RNA ligands: rapid affinity purification of RNA from complex RNA mixtures

Sephadex-binding RNA ligands (aptamers) were obtained through in vitro selection. They could be classified into two groups based on their consensus sequences and the aptamers from both groups showed strong binding to Sephadex G-100. One of the highest affinity aptamers, D8, was chosen for further characterization. Aptamer D8 bound to dextran B512, the soluble base material of Sephadex, but not to isomaltose, isomaltotriose and isomaltotetraose, suggesting that its optimal binding site might consist of more than four glucose residues linked via alpha-1,6 linkages. The aptamer was very specific to the Sephadex matrix and did not bind appreciably to other supporting matrices, such as Sepharose, Sephacryl, cellulose or pustulan. Using Sephadex G-100, the aptamer could be purified from a complex mixture of cellular RNA, giving an enrichment of at least 60 000-fold, compared with a non-specific control RNA. These RNA aptamers can be used as affinity tags for RNAs or RNA subunits of ribonucleoproteins to allow rapid purification from complex mixtures of RNA using only Sephadex.     

Preparation of isomaltose oligosaccharides labelled with 14C in the non-reducing terminal unit, and their use in studies of dextranase activity

A series of end-labelled isomaltose oligosaccharides was prepared by the reaction of dextran-sucrase with sucrose-¹⁴C in the presence of excess of unlabelled isomaltose saccharides as alternative acceptor. The main product of each reaction contained one more D-glucose residue than the acceptor substrate, and the label was located at the non-reducing end. The end-labelled saccharides were used to determine the specificity of a bacterial dextranase that required five or more consecutive α-(1→6)-D-glucosidic linkages in the substrate. The third linkage from the reducing end of isomaltohexaose (IM₆) and of other substrates with longer chains (IM₇ and IM₈) was the most susceptible to attack, and the products from higher oligosaccharides were IM₃, IM₄, and IM₅. Isomaltopentaose (IM₅) was further hydrolysed to IM₃ and IM₂ when a 35-fold excess of enzyme was added, but there was no action on IM₄, IM₃, or IM₂ under these conditions. It was concluded that the dextranase hydrolysed linkages penultimate to either end of the chain only with difficulty, and that end linkages were completely resistant to attack.     

Digestibility Characteristics of Isomaltooligosaccharides in Comparison with Several Saccharides Using the Rat Jejunum Loop Method

Isomaltooligosaccharides (IMO) are a mixture of isomaltose, isomaltotriose, panose, isomaltotetraose, etc. IMO and its hydrogenated derivative (IMH) were characterized for their luminal clearance from rat jujunum loops as the indication of their digestibility. They were compared with a disaccharide fraction (IM2) and a higher oligosaccharide fraction (IM3) prepared from IMO, typical digestible saccharides (maltose, maltotriose, and sucrose), and typical nondigestible saccharides (maltitol, raffinose, and fructooligosaccharides (FO)). The clearance rate of IMO was significantly smaller than that of IM2, which was mainly composed of isomaltose (64.3%), and digestible saccharides, and significantly larger than that of nondigestible saccharides. That of IM2 was almost the same as that of sucrose or maltotriose but significantly smaller than that of maltose. That of IM3 tended to be smaller than that of IMO, and larger than that of nondigestible saccharides. That of IMH was significantly smaller than that of IMO and similar to that of maltitol. These results seem to indicate that IMO is slowly digested in the jejunum, that the components having higher degree of polymerization of IMO are less digestible, and that IMH is nondigestible.     

Effects of mutation of Asn694 in Aspergillus niger α-glucosidase on hydrolysis and transglucosylation

Aspergillus niger α-glucosidase (ANG), a member of glycoside hydrolase family 31, catalyzes hydrolysis of α-glucosidic linkages at the non-reducing end. In the presence of high concentrations of maltose, the enzyme also catalyzes the formation of α-(1→6)-glucosyl products by transglucosylation and it is used for production of the industrially useful panose and isomaltooligosaccharides. The initial transglucosylation by wild-type ANG in the presence of 100 mM maltose [Glc(α1–4)Glc] yields both α-(1→6)- and α-(1→4)-glucosidic linkages, the latter constituting ~25% of the total transfer reaction product. The maltotriose [Glc(α1–4)Glc(α1–4)Glc], α-(1→4)-glucosyl product disappears quickly, whereas the α-(1→6)-glucosyl products panose [Glc(α1–6)Glc(α1–4)Glc], isomaltose [Glc(α1–6)Glc], and isomaltotriose [Glc(α1–6)Glc(α1–6)Glc] accumulate. To modify the transglucosylation properties of ANG, residue Asn694, which was predicted to be involved in formation of the plus subsites of ANG, was replaced with Ala, Leu, Phe, and Trp. Except for N694A, the mutations enhanced the initial velocity of the α-(1→4)-transfer reaction to produce maltotriose, which was then degraded at a rate similar to that by wild-type ANG. With increasing reaction time, N694F and N694W mutations led to the accumulation of larger amounts of isomaltose and isomaltotriose than achieved with the wild-type enzyme. In the final stage of the reaction, the major product was panose (N694A and N694L) or isomaltose (N694F and N694W).

     

Structural elements in dextran glucosidase responsible for high specificity to long chain substrate

Dextran glucosidase from Streptococcus mutans (SMDG) and Bacillus oligo-1,6-glucosidases, members of glycoside hydrolase family 13 enzymes, have the high sequence similarity. Each of them is specific to alpha-1,6-glucosidic linkage at the non-reducing end of substrate to liberate glucose. The activities toward long isomaltooligosaccharides were different in both enzymes, in which SMDG and oligo-1,6-glucosidase showed high and low activities, respectively. We determined the structural elements essential for high activity toward long-chain substrate. From conformational comparison between SMDG and B. cereus oligo-1,6-glucosidase (three-dimensional structure has been solved), Trp238 and short beta-->alpha loop 4 of SMDG were considered to contribute to the high activity to long-chain substrate. W238A had similar kcat/Km value for isomaltotriose to that for isomaltose, suggesting that the affinity of subsite +2 was decreased by Trp238 replacement. Trp238 mutants as well as the chimeric enzyme having longer beta-->alpha loop 4 of B. subtilis oligo-1,6-glucosidase showed lower preference for long-chain substrates, indicating that both Trp238 and short beta-->alpha loop 4 were important for high activity to long-chain substrates.     

Crystal structures of starch binding domain from Rhizopus oryzae glucoamylase in complex with isomaltooligosaccharide: Insights into polysaccharide binding mechanism of CBM21 family

Glucoamylases are responsible for hydrolysis of starch and polysaccharides to yield β‐d ‐glucose. Rhizopus oryzae glucoamylase (RoGA) is composed of an N‐terminal starch binding domain (SBD) and a C‐terminal catalytic domain connected by an O‐glycosylated linker. Two carbohydrate binding sites in RoSBD have been identified, site I is created by three highly conserved aromatic residues, Trp47, Tyr83, and Tyr94, and site II is built up by Tyr32 and Phe58. Here, the two crystal structures of RoSBD in complex with only α‐(1,6)‐linked isomaltotriose (RoSBD‐isoG3) and isomaltotetraose (RoSBD‐isoG4) have been determined at 1.2 and 1.3 Å, respectively. Interestingly, site II binding is observed in both complexes, while site I binding is only found in the RoSBD‐isoG4 complex. Hence, site II acts as the recognition binding site for carbohydrate and site I accommodates site II to bind isoG4. Site I participates in sugar binding only when the number of glucosyl units of oligosaccharides is more than three. Taken together, two carbohydrate binding sites in RoSBD cooperate to reinforce binding mode of glucoamylase with polysaccharides as well as the starch. Proteins 2014; 82:1079–1085. © 2013 Wiley Periodicals, Inc.     

Newcastle disease virus contains a linkage-specific glycoprotein sialidase. Application to the localization of sialic acid residues in N-linked oligosaccharides of alpha 1-acid glycoprotein

Newcastle disease virus sialidase was found to exhibit strict specificity for hydrolysis of the NeuAc alpha 2 leads to 3Gal linkage contained in glycoprotein oligosaccharides both N-linked to asparagine and O-linked to threonine or serine under conditions that left oligosaccharides containing the NeuAc alpha 2 leads to 2 leads to 6Gal and NeuAc alpha 2 leads to 6GallNAc linkages intact. This was determined, in part, by examining the viral sialidase for its ability to hydrolyze glycoprotein oligosaccharides derivatized with purified sialyltransferases to contain the [14C]NeuAc alpha 2 leads to 3Gal, [14C]NeuAc alpha 2 leads to 6GalNAc, and [14C]NeuAc alpha 2 leads to 6Gal linkages. The viral sialidase was also tested for hydrolysis of the NeuAc alpha 2 leads to 3Gal and NeuAc alpha 2 leads to 6Gal linkages on the N-linked oligosaccharides of alpha 1-acid glycoprotein. Selective hydrolysis of the NeuAc alpha 2 leads to 3Gal linkage was shown by periodate oxidation and by 500-MHz 1H-NMR spectroscopy of native and sialidase-treated glycopeptides. The NMR spectra, together with composition data, further indicated that the NeuAc alpha 2 leads to 3Gal and NeuAc alpha 2 leads to 6Gal linkages were localized to specific branches of the major tri- and tetraantennary oligosaccharides of alpha 1-acid glycoprotein. The results indicate that the Newcastle disease virus sialidase can initiate the selective degradation of N-linked oligosaccharide branches containing the NeuAc alpha 2 leads to 3Gal linkage.     

Cloning and characterization of two α-glucosidases from <Emphasis Type="Italic">Bifidobacterium adolescentis</Emphasis> DSM20083

Two alpha-glucosidase encoding genes (aglA and aglB) from Bifidobacterium adolescentis DSM 20083 were isolated and characterized. Both alpha-glucosidases belong to family 13 of the glycosyl hydrolases. Recombinant AglA (EC 3.2.1.10) and AglB (EC 3.2.1.20), expressed in Escherichia coli, showed high hydrolytic activity towards isomaltose and pnp-alpha-glucoside. The K(m) for pnp-alpha-glucoside was 1.05 and 0.47 mM and the V(max) was 228 and 113 U mg(-1) for AglA and AglB, respectively. Using pnp-alpha-glucoside as substrate, the pH optimum for AglA was 6.6 and the temperature optimum was 37 degrees C. For AglB, values of pH 6.8 and 47 degrees C were found. AglA also showed high hydrolytic activity towards isomaltotriose and, to a lesser extent, towards trehalose. AglB has a high preference for maltose and less activity towards sucrose; minor activity was observed towards melizitose, low molecular weight dextrin, maltitol, and maltotriose. The recombinant alpha-glucosidases were tested for their transglucosylation activity. AglA was able to synthesize oligosaccharides from trehalose and sucrose. AglB formed oligosaccharides from sucrose, maltose, and melizitose.