isomaltotetraose and isomaltotriose

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.

Topics: Carbohydrate Conformation; Carbohydrate Sequence; Crystallography, X-Ray; Fungal Polysaccharides; Fungal Proteins; Glucan 1,4-alpha-Glucosidase; Hydrogen Bonding; Ligands; Models, Molecular; Molecular Sequence Data; Oligosaccharides; Protein Binding; Protein Structure, Secondary; Rhizopus; Trisaccharides

Banana is one of the important crops native to tropical Southeast Asia. Since overproduction frequently leads to excessive waste of produce, alternative uses are continuously sought in order to utilise fruits at all stages of maturity. The aim of this study was to investigate the production of isomaltooligosaccharides (IMOs) from banana flour.. Banana slurries liquefied by Termamyl SC and saccharified by either Fungamyl 800 L or barley β-amylase were used for IMO synthesis by Transglucosidase L. After 12 h of transglucosylation, maximum IMO yields of 76.67 ± 2.71 and 70.74 ± 4.09 g L(-1) respectively were achieved. Although the yields were comparable, the IMO profiles obtained through the use of the two saccharification enzymes were different. Glucose and maltose were removed by 10 g L(-1) bakers' yeast fermentation for 12 h. Regarding total sugars, the final IMO mixture was composed of 53% isomaltotriose, 21% isomaltotetraose and 26% maltooligoheptaose and larger oligomers.. Banana flour could be used as a potential raw material for IMO synthesis.

Topics: alpha-Amylases; beta-Amylase; Fermentation; Flour; Fruit; Glucose; Glucosidases; Hordeum; Isomaltose; Maltose; Musa; Oligosaccharides; Saccharomyces cerevisiae; Trisaccharides

A gene encoding a special thermophilic multifunctional amylase OPMA-N was cloned from Bacillus sp. ZW2531-1. OPMA-N has an additional 124-residue N-terminal domain compared with typical amylases and forms a relatively independent domain with a β-pleated sheet and random coil structure. Here we reported an unusual substrate and product specificities of OPMA-N and the impact of the additional N-terminal domain (1-124 aa) on the function and properties of OPMA-N. Both OPMA-N (12.82 U/mg) and its N-terminal domain-truncated ΔOPMA-N (12.55 U/mg) only degraded starch to produce oligosaccharides including maltose, maltotriose, isomaltotriose, and isomaltotetraose, but not to produce glucose. Therefore, the N-terminal domain did not determine its substrate and product specificities that were probably regulated by its C-terminal β-pleated sheet structure. However, the N-terminal domain of OPMA-N seemed to modulate its catalytic feature, leading to the production of more isomaltotriose and less maltose, and it seemed to contribute to OPMA-N's thermostability since OPMA-N showed higher activity than ΔOPMA-N in a temperature range from 40 to 80°C and the half-life (t(1/2)) was 5 h for OPMA-N and 2 h for ΔOPMA-N at 60°C. Both OPMA-N and ΔOPMA-N were Ca(2+)-independent, but their activities could be influenced by Cu(2+), Ni(2+), Zn(2+), EDTA, SDS (1 mM), or Triton-X100 (1%). Kinetic analysis and starch-adsorption assay indicated that the N-terminal domain of OPMA-N could increase the OPMA-N-starch binding and subsequently increase the catalytic efficiency of OPMA-N for starch. In particular, the N-terminal domain of OPMA-N did not determine its oligomerization, because both OPMA-N and ΔOPMA-N could exist in the forms of monomer, homodimer, and homooligomer at the same time.

Topics: Amino Acid Sequence; Amylases; Bacillus; Bacterial Proteins; Binding Sites; Biocatalysis; Enzyme Stability; Hydrogen-Ion Concentration; Kinetics; Maltose; Metals; Models, Molecular; Molecular Sequence Data; Mutation; Oligosaccharides; Protein Structure, Secondary; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Starch; Substrate Specificity; Temperature; Trisaccharides

Maltogenic amylase and alpha-glucanotransferase (alpha-GTase) were employed in an effort to develop an efficient process for the production of isomaltooligosaccharides (IMOs). Bacillus stearothermophilus maltogenic amylase (BSMA) and alpha-GTase from Thermotoga maritima were overexpressed in Escherichia coli using overexpression vectors. An IMO mixture containing 58% of various IMOs was produced from liquefied corn syrup by the hydrolyzing and transglycosylation activities of BSMA alone. When BSMA and alpha-GTase were reacted simultaneously, the IMO content increased to 68% and contained relatively larger IMOs compared with the products obtained by the reaction without alpha-GTase. Time course analysis of the IMO production suggested that BSMA hydrolyzed maltopentaose and maltohexaose most favorably into maltose and maltotriose and transferred the resulting molecules simultaneously to acceptor molecules to form IMOs. alpha-GTase transferred donor sugar molecules to the hydrolysis products such as maltose and maltotriose to form maltopentaose, which was then rehydrolyzed by BSMA as a favorable substrate.

Topics: Escherichia coli; Gene Expression; Glycogen Debranching Enzyme System; Glycoside Hydrolases; Isomaltose; Oligosaccharides; Recombinant Proteins; Restriction Mapping; Trisaccharides