Synthesis of isomaltooligosaccharides using 4-O-α-d-isomaltooligosaccharylmaltooligosaccharide 1,4-α-isomaltooligosaccharohydrolase.

Isomaltooligosaccharides (IMOs), including isomaltose, are valuable oligosaccharides, and the development of methods to synthesize high-purity IMOs has long been underway. We recently discovered a novel enzyme, 4-O-α-d-isomaltooligosaccharylmaltooligosaccharide 1,4-α-isomaltooligosaccharohydrolase (IMM-4IH), that showed promise for improving the synthesis process. In this study, we establish methods for synthesizing isomaltose and IMOs consisting of a variety of degrees of polymerization from starch using IMM-4IH. With 5% substrate, by combining IMM-4IH with 1,4-α-glucan 6-α-glucosyltransferase from Bacillus globisporus N75, the yield of isomaltose was 63.0%; incorporating isoamylase and cyclomaltodextrin glucanotransferase increased the yield to 75.3%. On the other hand, by combining IMM-4IH with 1,4-α-glucan 6-α-glucosyltransferase from Paenibacillus sp. PP710, IMOs were synthesized. The inclusion of isoamylase and α-amylase led to the 136 mM IMOs, consisting of oligosaccharides from isomaltose to isomaltodecaose, from 10% starch. The development of these efficient methods will be an important contribution to the industrial production of IMOs.

Cloning and sequence analysis of 4-O-α-d-isomaltooligosaccharylmaltooligosaccharide 1,4-α-isomaltooligosaccharohydrolase from Sarocladium kiliense U4520.

A novel enzyme, 4-O-α-d-isomaltooligosaccharylmaltooligosaccharide 1,4-α-isomaltooligosaccharohydrolase (IMM-4IH), was previously discovered from Sarocladium kiliense U4520. In order to identify the factors underlying the unique substrate specificity of IMM-4IH, we endeavored to determine the amino acid sequence of the enzyme. By comparing the partial amino acid sequence of the enzyme to whole genome sequencing data of S. kiliense U4520, the IMM-4IH gene was estimated. The putative gene was expressed in Pichia pastoris, and its activity and properties were found to be consistent with those of the native enzyme. Comparing the amino acid sequence of IMM-4IH with those in the CAZy database led to classification in the glycoside hydrolase family 49 (GH49). Several amino acids important for catalysis (Asp406, Asp425, and Asp426) and substrate recognition at subsites + 1 and -3 were estimated by multiple sequence alignment analysis. These results provide important information for characterizing IMM-4IH and other GH49 enzymes.

Discovery of a novel glucanohydrolase, 4-α-isomaltooligosylglucose 4-glucanohydrolase, that can be used for efficient production of isomaltose.

We discovered a novel enzyme in our pursuit of an improved method for the production of isomaltose. The enzyme, 4-α-isomaltooligosylglucose 4-glucanohydrolase from Sarocladium kiliense U4520, recognizes the panose motif (α-d-Glcp-(1 â†’ 6)-α-d-Glcp-(1 â†’ 4)-d-Glcp) and hydrolyzes the α-1,4-glucosidic bond on the reducing end side with respect to the α-1,6-glucosidic bond. The structure on the non-reducing end of the panose motif is important for the recognition of the substrate by the enzyme, and the substrate specificity is unique and distinguished from previously reported enzymes. The enzyme catalyzes the hydrolysis of panose with a kcat/Km of 31.2 s-1mM-1, and catalysis results in anomeric inversion. These enzymatic properties suggest that this enzyme will pair well with 1,4-α-glucan 6-α-glucosyltransferase from Bacillus globisporus N75 in the efficient production of isomaltose from starch.

Development of a method for preparing cyclic nigerosylnigerose syrup and investigation of its value as a dietary fiber.

Cyclic nigerosylnigerose (CNN) syrup, containing 76% water-soluble dietary fiber, was prepared from starch on an industrial scale, using isoamylase, 6-α-glucosyltransferase, 3-α-isomaltosyltransferase, and cyclodextrin glucanotransferase. CNN syrup has a unique linkage pattern, consisting mainly of α-1,3 and α-1,6 glucoside linkages, and is characterized by its low weight average molecular weight (807) and moderate sweetness (relative sweetness = 25), unlike in well-known dietary fiber materials. The glass transition temperature of CNN is higher than that of the straight chain structures, maltotetraose and maltosyltrehalose. Even when 40% of normally added sucrose was replaced with CNN syrup, sponge cake puffed up sufficiently. The no observed adverse effect level for a single dose of CNN syrup was 0.88 and 0.89 g dry solid/kg body weight for men and women, respectively. The increase in blood glucose and insulin concentrations during consumption of CNN syrup was lower than that of glucose.

Efficient production of isomaltose and isomaltooligosaccharides from starch using 1,4-α-glucan 6-α-glucosyltransferase and isopullulanase.

We attempted to develop an efficient method for producing isomaltose, a disaccharide consisting of an α-(1→6)-linkage, from starch by combining enzymes of known activity. We found that the combination of 1,4-α-glucan 6-α-glucosyltransferase from Bacillus globisporus N75 and isopullulanase from Aspergillus brasiliensis ATCC 9642 led to the efficient synthesis of isomaltose. Inclusion of isoamylase and cyclomaltodextrin glucanotransferase resulted in increased efficiency, with production yields exceeding 70%. Furthermore, we considered that isomaltooligosaccharides could be synthesized from starch by combining 1,4-α-glucan 6-α-glucosyltransferase from Paenibacillus sp. PP710 and isopullulanase. In reactions that additionally utilized isoamylase and α-amylase, the total concentration of product, which included a series of isomaltooligosaccharides from isomaltose to isomaltodecaose, was 131 m m, and the ratio of 6-linked glucopyranosyl bonds to all bonds was 91.7% at a substrate concentration of 10%. The development of these manufacturing methods will accelerate the industrial production of isomaltose and isomaltooligosaccharides.

Cloning of the cycloisomaltotetraose-forming enzymes using whole genome sequence analyses of Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006.

We performed whole genome sequence analyses of Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006 that secrete enzymes to produce cyclo-{→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→} (CI4) from dextran. Full-length amino acid sequences of CI4-forming enzymes were identified by matching known N-terminal amino acid sequences with products of the draft genome. Domain searches revealed that the CI4-forming enzymes are composed of Glycoside Hydrolase family 66 (GH66) domain, Carbohydrate Binding Module family 35 (CBM35) domain, and CBM13 domain, categorizing the CI4-forming enzymes in the GH66. Furthermore, the amino acid sequences of the two CI4-forming enzymes were 71% similar to each other and up to 51% similar to cycloisomaltooligosaccharide glucanotransferases (CITases) categorized in GH66. Differences in sequence between the CI4-forming enzymes and the CITases suggest mechanisms to produce specific cycloisomaltooligosaccharides, and whole genome sequence analyses identified a gene cluster whose gene products likely work in concert with the CI4-forming enzymes.

A novel dextrin produced by the enzymatic reaction of 6-α-glucosyltransferase. â ¡. Practical advantages of the novel dextrin as a food modifier.

High-molecular-weight dextrin (WS-1000) was produced from waxy corn starch and enzymatically modified to link glucose by α-1,6 glycosidic bond at the terminal point of the glucose chain, forming MWS-1000. In this study, the physical properties of MWS-1000 were characterized, and the advantages of its use as a food modifier were described. From rheological and calorimetric studies, it was found that MWS-1000 does not undergo retrogradation, but it does not prevent the retrogradation of WS-1000, suggesting that they have no intermolecular interaction in solution. Investigation of the effect of MWS-1000 on the viscoelasticity of gelatinized wheat starch showed that in the linear viscoelastic region, storage modulus decreased and tan δ increased with increase in replaced MWS-1000 content. In addition, it was confirmed that gelatinized starch containing MWS-1000 showed viscoelastic behavior similar to that of commercially available custard cream.

A novel dextrin produced by the enzymatic reaction of 6-α-glucosyltransferase. I. The effect of nonreducing ends of glucose with by α-1,6 bonds on the retrogradation inhibition of high molecular weight dextrin.

We prepared a high-molecular-weight modified dextrin (MWS-1000) from a partial hydrolysate of waxy corn starch with a weight average molecular weight of 1 × 106 (WS-1000) using Paenibacillus alginolyticus PP710 α-glucosyltransferase. The gel permeation chromatography showed that the weight average molecular weight of MWS-1000 was almost the same as that of WS-1000. The side chain lengths of WS-1000 and MWS-1000 after isomaltodextranase digestion were also shown to be similar to each other by high-performance anion exchange chromatography with pulsed amperometric detection. Since MWS-1000 confirmed the presence of α-1,6 bonds by enzyme digestibility, methylation, and 1H-NMR analyses, it was presumed that the structure of MWS-1000 was based on the introduction of α-1,6 glucosyl residues at the nonreducing ends of the partial hydrolysate of waxy corn starch. Furthermore, the MWS-1000 solution was not retrograded even during refrigerated storage or after repeated freeze-thaw cycles.

Purification and characterization of cycloisomaltotetraose-forming glucanotransferases from Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006.

Glucanotransferases that can synthesize cyclo-{→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→} (CI4) from dextran were purified to homogeneity from the culture supernatant of Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006. The molecular mass of both enzymes was estimated to be 86 kDa by SDS-PAGE. The glucanotransferase, named CI4-forming enzyme, from Agreia sp. exhibited the highest activity at pH 6.0 and 40 °C. The enzyme was stable on the pH range of 4.6-9.9 and up to 40 °C. On the other hand, the enzyme from M. trichothecenolyticum exhibited the highest activity at pH 5.7 and 40 °C. The enzyme was stable on the pH range of 5.0-6.9 and up to 35 °C. Both enzymes catalyzed 4 reactions, namely, intramolecular α-1,6-transglycosylation (cyclization), intermolecular α-1,6-transglycosylation, hydrolysis of CI4, and coupling reaction. Furthermore, the CI4-forming enzyme produced CI4 from α-1,6-linked glucan synthesized from starch by 6-α-glucosyltransferase. These findings will enable the production of CI4 from starch.

A cyclic tetrasaccharide, cycloisomaltotetraose, was enzymatically produced from dextran and its crystal structure was determined.

Two bacterial strains isolated from soil, namely Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006, were found to produce a novel oligosaccharide. The oligosaccharide was enzymatically produced from dextran using the culture supernatant of Agreia sp. D1110 or M. trichothecenolyticum D2006. LC-MS and NMR analysis identified the novel oligosaccharide as cyclo-{→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→}, which was named cycloisomaltotetraose, and abbreviated as CI4. CI4 was subsequently crystalized and its X-ray crystallographic structure was determined. CI4 crystals were shown to be pentahydrate, with the CI4 molecules in the crystal structure displaying a unique 3D structure, in which two glucosyl residues in the molecule were facing each other. This unique 3D structure was quite different from the 3D structure of known cyclic tetrasaccharides. This is the first report of CI4 molecules and their unique crystal structure.

Enzymatic properties of recombinant kojibiose phosphorylase from Caldicellulosiruptor saccharolyticus ATCC43494.

One kojibiose phoshorylase (KP) homolog gene was cloned from Caldicellulosiruptor saccharolyticus ATCC43494. Recombinant KP from C. saccharolyticus (Cs-KP) expressed in Escherichia coli showed highest activity at pH 6.0 at 85 °C, and was stable from pH 3.5 to 10.0 and up to 85 °C for phosphorolysis. Cs-KP showed higher productivity of kojioligosaccharides of DP ≧ 4 than KP from Thermoanaerobacter brockii ATCC35047.

Improved yields of cyclic nigerosylnigerose from starch by pretreatment with a thermostable branching enzyme.

Cyclic nigerosylnigerose (CNN) is produced enzymatically from starch by the combined action of 6-alpha-glucosyltransferase and 3-alpha-isomaltosyltransferase. In our previous study, alpha-1,6-branching chains found in the structure of amylopectin and glycogen were shown to be favorable for CNN formation by the two enzymes. Therefore, we examined whether the introduction of alpha-1,6-branch points into starch using the action of branching enzyme (BE) could improve the yield of CNN from starch. Thermostable BE from Geobacillus stearothermophilus TC-91 was prepared as a purified recombinant protein. Pretreatment of amylose with BE considerably increased the CNN yield from 5% to 38%. When BE acted on tapioca starch, the CNN yield was elevated from 47% to 60%. Conversely, BE treatment of waxy corn starch containing very little amylose resulted in a negligible increase in CNN yield. In addition, BE exerted a beneficial effect when starch with a lower degree of hydrolysis was used as a substrate. The present results indicate that the addition of alpha-1,6-glucosidic linkages to starch using BE is an effective strategy to improve the yield of CNN from starch.

Acceptor recognition of kojibiose phosphorylase from Thermoanaerobacter brockii: syntheses of glycosyl glycerol and myo-inositol.

The glucosyl transfer reaction of kojibiose phosphorylase (KP; EC 2.4.1.230) was examined using glycerol or myo-inositol as an acceptor. In the case of glycerol, KP produced two main transfer products: saccharides A and B. The structure of saccharide A was O-alpha-D-glucopyranosyl-(1-->1)-glycerol and that of saccharide B was O-alpha-D-glucopyranosyl-(1-->2)-O-alpha-D-glucopyranosyl-(1-->1)-glycerol. These results show that KP transferred a glucose residue to the hydroxyl group at position 1 of glycerol. On the other hand, when myo-inositol was used as an acceptor, KP produced four transfer products: saccharides 1-4. The structures of saccharides 1 and 2 were O-alpha-D-glucopyranosyl-(1-->1)- and O-alpha-D-glucopyranosyl-(1-->5)-myo-inositol, respectively; those of saccharides 3 and 4 were O-alpha-D-glucopyranosyl-(1-->2)-O-alpha-D-glucopyranosyl-(1-->1)- and O-alpha-D-glucopyranosyl-(1-->2)-O-alpha-D-glucopyranosyl-(1-->5)-myo-inositol, respectively. KP transferred a glucose residue to the hydroxyl group at position 1 or 5 of myo-inositol. On the basis of the structures of their glucosyl transfer products, glycerol and myo-inositol were found to have a common structure with three hydroxyl groups corresponding to the hydroxyl group of the glucose molecule at positions 2, 3 and 4. The conformation of these three hydroxyl groups in the structure is equatorial. This structure is the substrate recognition site of KP. It has been suggested that KP strictly recognizes the structures of glycerol and myo-inositol, and catalyzes the transfer reaction of a glucose residue to the hydroxyl group at position 1 in glycerol, and at position 1 or 5 in myo-inositol, corresponding to position 2 in glucose.

Enzymatic synthesis of a novel cyclic pentasaccharide consisting of alpha-D-glucopyranose with 6-alpha-glucosyltransferase and 3-alpha-isomaltosyltransferase.

A novel cyclic pentasaccharide (CPS) and a branched cyclic pentasaccharide (6G-CPS) consisting of d-glucopyranose were synthesized with 6-alpha-glucosyltransferase (6GT) and 3-alpha-isomaltosyltransferase (IMT) from Bacillus globisporus N75. The structure of CPS was cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]. The other, 6G-CPS, had the structure cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-[alpha-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]. The formation of CPS was presumed to occur after the following four successive reactions: a 6-glucosyltransfer reaction with 6GT, a 4-glucosyltransfer reaction with 6GT, a 3-isomaltosyltransfer reaction with IMT, and a cyclization reaction with IMT.

Enzymatic synthesis of a 2-O-alpha-D-glucopyranosyl cyclic tetrasaccharide by kojibiose phosphorylase.

The glucosyl transfer reaction of kojibiose phosphorylase (KPase) from Thermoanaerobacter brockii ATCC35047 was examined using cyclo-{-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->} (CTS) as an acceptor. KPase produced four transfer products, saccharides 1-4. The structure of a major product, saccharide 4, was 2-O-alpha-d-glucopyranosyl-CTS, cyclo-{-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-[alpha-d-Glcp-(1-->2)]-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->}. The other transfer products, saccharides 1-3, were 2-O-alpha-kojibiosyl-, 2-O-alpha-kojitriosyl-, and 2-O-alpha-kojitetraosyl-CTS, respectively. These results showed that KPase transferred a glucose residue to the C-2 position at the ring glucose residue of CTS. This enzyme also catalyzed the chain-extending reaction of the side chain of 2-O-alpha-d-glycopyranosyl-CTS.

Enzymatic synthesis of a beta-D-galactopyranosyl cyclic tetrasaccharide by beta-galactosidases.

The galactosyl transfer reaction to cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] (CTS) was examined using lactose as a donor and beta-galactosidases from Aspergillus oryzae and Bacillus circulans. The A. oryzae beta-galactosidase produced three galactosyl derivatives of CTS. The main galactosyl derivative produced by the A. oryzae enzyme was identified as 6-O-beta-D-galactopyranosyl-CTS, cyclo-[-->6)-alpha-D-Glcp-(1-->3)-[beta-D-Galp-(1-->6)]-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. The B. circulans beta-galactosidase also synthesized three galactosyl-transfer products to CTS. The structure of main transgalactosylation product was 3-O-beta-D-galactopyranosyl-CTS, cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-[beta-D-Galp-(1-->3)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. These results showed that beta-galactosidase transferred galactose directly to the ring glucose residue of CTS.

Enzymatic synthesis of glycosyl cyclic tetrasaccharide with 6-alpha-glucosyltransferase and 3-alpha-isomaltosyltransferase.

Transglycosylation reactions to cyclic tetrasaccharide (CTS, cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]) and its derivatives were investigated. An enzyme, 6-alpha-glucosyltransferase, which is involved in CTS synthesis from starch, from Bacillus globisporus C11 produced 4-O-alpha-glucosyl-CTS (4G-CTS) from a mixture containing CTS and maltopentaose. Another enzyme, 3-alpha-isomaltosyltransferase, synthesized 3-O-alpha-isomaltosyl-CTS (3IM-CTS) from CTS and panose. Two novel branched CTSs, 3-O-alpha-isomaltosyl-4-O-alpha-glucosyl-CTS (3IM-4G-CTS) and 3-O-alpha-isomaltosyl-(4-O-alpha-glucosyl)-CTS [3IM-(4G)-CTS], were synthesized by the isomaltosyl transfer of IMT into 4G-CTS. IMT also produced a novel saccharide, 3-O-alpha-isomaltosyl-3-O-alpha-isomaltosyl-CTS (3IM-3IM-CTS) from 3IM-CTS. It was confirmed that the oligosaccharides, including 4G-CTS, 3IM-CTS, 3IM-4G-CTS, 3IM-(4G)-CTS and 3IM-3IM-CTS, remaining in the reaction mixture during the production of CTS from starch were the transfer products of 6GT and IMT into CTS.

Transglycosylation of glycosyl residues to cyclic tetrasaccharide by Bacillus stearothermophilus cyclomaltodextrin glucanotransferase using cyclomaltodextrin as the glycosyl donor.

Cyclomaltodextrin glucanotransferase (EC 2.4.1.19, abbreviated as CGTase) derived from Bacillus stearothermophilus produced a series of transfer products from a mixture of cyclomaltohexaose and cyclic tetrasaccharide (cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->], CTS). Of the transfer products, only two components, saccharides A and D, remained and accumulated after digestion with glucoamylase. The total combined yield of the saccharides reached 63.4% of total sugars, and enzymatic and instrumental analyses revealed the structures of both saccharides. Saccharide A was identified as 4-mono-O-alpha-glucosyl-CTS, [-->6)-[alpha-D-Glcp-(1-->4)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->], and sachharide D was 4,4'-di-O-alpha-glucosyl-CTS, [-->6)-[alpha-D-Glcp-(1-->4)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-[alpha-D-Glcp-(1-->4)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. These structures led us to conclude that the glycosyltransfer catalyzed by CGTase was specific to the C4-OH of the 6-linked glucopyranosyl residues in CTS.

Synthesis of 3-O-beta-N-acetylglucosaminyl cyclic tetrasaccharide through a lysozyme-catalyzed transfer reaction.

Egg white lysozyme was found to catalyze the transfer of N-acetylglucosamine to cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] (CTS). Structural analysis showed that the transfer product was 3-O-beta-N-acetylglucosaminyl CTS, cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-[beta-GlcNAc-(1-->3)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. This branched saccharide is anticipated to be a model compound of the sugar chains of glycoproteins.