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.

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.

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.

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.