Physicochemical functionality of chimeric isomaltomegalosaccharides with α-(1 → 4)-glucosidic segments of various lengths.

Isomaltomegalosaccharide (IMS) is a long chimeric glucosaccharide composed of α-(1 â†’ 6)- and α-(1 â†’ 4)-linked segments at nonreducing and reducing ends, respectively; the hydrophilicity and hydrophobicity of these segments are expected to lead to bifunctionality. We enzymatically synthesized IMS with average degrees of polymerization (DPs) of 15.8, 19.3, and 23.5, where α-(1 â†’ 4)-segments had DPs of 3, 6, and 9, respectively. IMS exhibited considerably higher water solubility than maltodextrin because of the α-(1 â†’ 6)-segment and an identical resistance to thermal degradation as short dextran. Interaction of IMS with a fluorescent probe of 2-p-toluidinylnaphthalene-6-sulfonate demonstrated that IMS was more hydrophobic than maltodextrin, where the degree of hydrophobicity increased as DP of α-(1 â†’ 4)-segment increased (9 > 6 > 3). Fluorescent pyrene-estimating polarity of IMS was found to be similar to that of methanol or 1-butanol. The bifunctional IMS enhanced the water solubility of quercetin-3-O-glucoside and quercetin: the solubilization of less-soluble bioactive substances is beneficial in carbohydrate industry.

A practical approach to producing isomaltomegalosaccharide using dextran dextrinase from Gluconobacter oxydans ATCC 11894.

Dextran dextrinase (DDase) catalyzes formation of the polysaccharide dextran from maltodextrin. During the synthesis of dextran, DDase also generates the beneficial material isomaltomegalosaccharide (IMS). The term megalosaccharide is used for a saccharide having DP = 10-100 or 10-200 (DP, degree of polymerization). IMS is a chimeric glucosaccharide comprising α-(1 → 6)- and α-(1 → 4)-linked portions at the nonreducing and reducing ends, respectively, in which the α-(1 → 4)-glucosyl portion originates from maltodextrin of the substrate. In this study, IMS was produced by a practical approach using extracellular DDase (DDext) or cell surface DDase (DDsur) of Gluconobacter oxydans ATCC 11894. DDsur was the original form, so we prepared DDext via secretion from intact cells by incubating with 0.5% G6/G7 (maltohexaose/maltoheptaose); this was followed by generation of IMS from various concentrations of G6/G7 substrate at different temperatures for 96 h. However, IMS synthesis by DDext was limited by insufficient formation of α-(1 → 6)-glucosidic linkages, suggesting that DDase also catalyzes elongation of α-(1 → 4)-glucosyl chain. For production of IMS using DDsur, intact cells bearing DDsur were directly incubated with 20% G6/G7 at 45 °C by optimizing conditions such as cell concentration and agitation efficiency, which resulted in generation of IMS (average DP = 14.7) with 61% α-(1 → 6)-glucosyl content in 51% yield. Increases in substrate concentration and agitation efficiency were found to decrease dextran formation and increase IMS production, which improved the reaction conditions for DDext. Under modified conditions (20% G6/G7, agitation speed of 100 rpm at 45 °C), DDext produced IMS (average DP = 14.5) with 65% α-(1 → 6)-glucosyl content in a good yield of 87%. KEY POINTS: • Beneficial IMS was produced using thermostabilized DDase. • Optimum conditions for reduced dextran formation were successfully determined. • A practical approach was established to provide IMS with a great yield of 87%.

Structural insights reveal the second base catalyst of isomaltose glucohydrolase.

Glycoside hydrolase family 15 (GH15) inverting enzymes contain two glutamate residues functioning as a general acid catalyst and a general base catalyst, for isomaltose glucohydrolase (IGHase), Glu178 and Glu335, respectively. Generally, a two-catalytic residue-mediated reaction exhibits a typical bell-shaped pH-activity curve. However, IGHase is found to display atypical non-bell-shaped pH-kcat and pH-kcat /Km profiles, theoretically better-fitted to a three-catalytic residue-associated pH-activity curve. We determined the crystal structure of IGHase by the single-wavelength anomalous dispersion method using sulfur atoms and the cocrystal structure of a catalytic base mutant E335A with isomaltose. Although the activity of E335A was undetectable, the electron density observed in its active site pocket did not correspond to an isomaltose but a glycerol and a ß-glucose, cryoprotectant, and hydrolysis product. Our structural and biochemical analyses of several mutant enzymes suggest that Tyr48 acts as a second catalytic base catalyst. Y48F mutant displayed almost equivalent specific activity to a catalytic acid mutant E178A. Tyr48, highly conserved in all GH15 members, is fixed by another Tyr residue in many GH15 enzymes; the latter Tyr is replaced by Phe290 in IGHase. The pH profile of F290Y mutant changed to a bell-shaped curve, suggesting that Phe290 is a key residue distinguishing Tyr48 of IGHase from other GH15 members. Furthermore, F290Y is found to accelerate the condensation of isomaltose from glucose by modifying a hydrogen-bonding network between Tyr290-Tyr48-Glu335. The present study indicates that the atypical Phe290 makes Tyr48 of IGHase unique among GH15 enzymes.

Molecular insight into regioselectivity of transfructosylation catalyzed by GH68 levansucrase and ß-fructofuranosidase.

Glycoside hydrolase family 68 (GH68) enzymes catalyze ß-fructosyltransfer from sucrose to another sucrose, the so-called transfructosylation. Although regioselectivity of transfructosylation is divergent in GH68 enzymes, there is insufficient information available on the structural factor(s) involved in the selectivity. Here, we found two GH68 enzymes, ß-fructofuranosidase (FFZm) and levansucrase (LSZm), encoded tandemly in the genome of Zymomonas mobilis, displayed different selectivity: FFZm catalyzed the ß-(2→1)-transfructosylation (1-TF), whereas LSZm did both of 1-TF and ß-(2→6)-transfructosylation (6-TF). We identified His79FFZm and Ala343FFZm and their corresponding Asn84LSZm and Ser345LSZm respectively as the structural factors for those regioselectivities. LSZm with the respective substitution of FFZm-type His and Ala for its Asn84LSZm and Ser345LSZm (N84H/S345A-LSZm) lost 6-TF and enhanced 1-TF. Conversely, the LSZm-type replacement of His79FFZm and Ala343FFZm in FFZm (H79N/A343S-FFZm) almost lost 1-TF and acquired 6-TF. H79N/A343S-FFZm exhibited the selectivity like LSZm but did not produce the ß-(2→6)-fructoside-linked levan and/or long levanooligosaccharides that LSZm did. We assumed Phe189LSZm to be a responsible residue for the elongation of levan chain in LSZm and mutated the corresponding Leu187FFZm in FFZm to Phe. An H79N/L187F/A343S-FFZm produced a higher quantity of long levanooligosaccharides than H79N/A343S-FFZm (or H79N-FFZm), although without levan formation, suggesting that LSZm has another structural factor for levan production. We also found that FFZm generated a sucrose analog, ß-D-fructofuranosyl α-D-mannopyranoside, by ß-fructosyltransfer to d-mannose and regarded His79FFZm and Ala343FFZm as key residues for this acceptor specificity. In summary, this study provides insight into the structural factors of regioselectivity and acceptor specificity in transfructosylation of GH68 enzymes.

Megalo-type α-1,6-glucosaccharides induce production of tumor necrosis factor α in primary macrophages via toll-like receptor 4 signaling.

The term "megalo-saccharide" is used for saccharides with ten or more saccharide units, whereas the term "oligo-saccharide" is used for saccharides containing fewer than ten monosaccharide units. Megalo-type α-1,6-glucosaccharide (M-IM) is a non-digestible saccharide and not utilized by intestinal bacteria, suggesting that ingested M-IM may encounter ileum Peyer's patches that contains immune cells such as macrophages. Macrophages are responsible for antigen incorporation and presentation during the initial step of immune responses. We investigated whether M-IMs modulate macrophage functions such as cytokine production, nitric oxide production, cell viability, and phagocytosis. Primary macrophages collected from the rats were cultured with the existence of M-IM or lipopolysaccharides (LPS). M-IM and LPS induced the production of tumor necrosis factor α (TNFα), interleukin 6 (IL6), and nitric oxide in the primary macrophages. The gene expression profile of inflammatory factors including TNFα, IL6, and ILlß in M-IM-stimulated cells was similar to that of LPS-stimulated cells. The M-IM did not affect phagocytosis in the primary macrophages. The M-IM-induced TNFα production was suppressed in the cells treated with a tolllike receptor 4 (TLR4) inhibitor called TAK-242. In conclusion, the M-IM modulates cytokine expression via TLR4 signaling and may play a role in the modulation of immune responses.

Two Novel Glycoside Hydrolases Responsible for the Catabolism of Cyclobis-(1→6)-α-nigerosyl.

The actinobacterium Kribbella flavida NBRC 14399(T) produces cyclobis-(1→6)-α-nigerosyl (CNN), a cyclic glucotetraose with alternate α-(1→6)- and α-(1→3)-glucosidic linkages, from starch in the culture medium. We identified gene clusters associated with the production and intracellular catabolism of CNN in the K. flavida genome. One cluster encodes 6-α-glucosyltransferase and 3-α-isomaltosyltransferase, which are known to coproduce CNN from starch. The other cluster contains four genes annotated as a transcriptional regulator, sugar transporter, glycoside hydrolase family (GH) 31 protein (Kfla1895), and GH15 protein (Kfla1896). Kfla1895 hydrolyzed the α-(1→3)-glucosidic linkages of CNN and produced isomaltose via a possible linear tetrasaccharide. The initial rate of hydrolysis of CNN (11.6 s(-1)) was much higher than that of panose (0.242 s(-1)), and hydrolysis of isomaltotriose and nigerose was extremely low. Because Kfla1895 has a strong preference for the α-(1→3)-isomaltosyl moiety and effectively hydrolyzes the α-(1→3)-glucosidic linkage, it should be termed 1,3-α-isomaltosidase. Kfla1896 effectively hydrolyzed isomaltose with liberation of ß-glucose, but displayed low or no activity toward CNN and the general GH15 enzyme substrates such as maltose, soluble starch, or dextran. The kcat/Km for isomaltose (4.81 ± 0.18 s(-1) mm(-1)) was 6.9- and 19-fold higher than those for panose and isomaltotriose, respectively. These results indicate that Kfla1896 is a new GH15 enzyme with high substrate specificity for isomaltose, suggesting the enzyme should be designated an isomaltose glucohydrolase. This is the first report to identify a starch-utilization pathway that proceeds via CNN.

Extracellular and cell-associated forms of Gluconobacter oxydans dextran dextrinase change their localization depending on the cell growth.

Gluconobacter oxydans ATCC 11894 produces dextran dextrinase (DDase, EC 2.4.1.2), which synthesizes dextran from the starch hydrolysate, dextrin and is known to cause ropy beer. G. oxydans ATCC 11894 was believed to possess both a secreted DDase (DDext) and an intracellular DDase (DDint), expressed upon cultivation with dextrin and glucose, respectively. However, genomic Southern blot, peptide mass fingerprinting and reaction product-pattern analyses revealed that both DDext and DDint were identical. The activity in the cell suspension and its liberation from the spheroplast cells indicated that DDint was localized on the cell surface. The localization of DDase was altered during the culture depending on the growth phase. During the early growth stage, DDase was exclusively liberated into the medium (DDext), and the cell-associated form (DDint) appeared after depletion of glucose from the medium.

Different molecular complexity of linear-isomaltomegalosaccharides and ß-cyclodextrin on enhancing solubility of azo dye ethyl red: towards dye biodegradation.

Intermolecular interaction of linear-type α-(1 → 6)-glucosyl megalosaccharide rich (L-IMS) and water-insoluble anionic ethyl red was firstly characterized in a comparison with inclusion complexation by cyclodextrins (CDs) to overcome the problem of poor solubility and bioavailability. Phase solubility studies indicated an enhancement of 3- and 9-fold over the solubility in water upon the presence of L-IMS and ß-CD, respectively. (1)H NMR and circular dichrosim spectra revealed the dye forms consisted of 1:1 stoichiometric inclusion complex within the ß-CD cavity, whereas they exhibited non-specific hydrophobic interaction, identified by solvent polarity changes, with L-IMS. The inclusion complex delivered by ß-CD showed an uncompetitive inhibitory-type effect to azoreductase, particularly with high water content that did not promote dye liberation. Addition of the solid dye dispersed into coupled-enzyme reaction system supplied by L-IMS as the dye solubilizer provided usual degradation rate. The dye intermission in series exhibited successful removal with at least 5 cycles was economically feasible.