Search

Glucose, a common monosaccharide in nature, is dominated by the d-enantiomer. Meanwhile, the discovery of l-glucose-utilizing bacteria and the elucidation of their metabolic pathways 10 years ago suggests that l-glucose exists naturally. Most carbohydrates exist as glycosides rather than monosaccharides; therefore, we expected that nature also contains l-glucosides. Sequence analysis within glycoside hydrolase family 29 led us to identify two α-l-glucosidases, ClAgl29A and ClAgl29B, derived from

Thoracoscopic bullectomy is a common treatment modality for spontaneous pneumothorax but can result in a high frequency of postoperative recurrent pneumothorax in young patients. This retrospective study compared the recurrence rate of pneumothorax following conventional thoracoscopic bullectomy to that following bullectomy using a low-density polyglycolic acid mesh to cover the staple line. Group A comprised 237 patients who experienced 294 episodes of pneumothorax and underwent thoracoscopic bullectomy alone, and Group B comprised 130 patients who experienced 155 episodes of pneumothorax and underwent bullectomy with polyglycolic acid mesh used to cover the visceral pleura. To compare the postoperative inflammatory response between the two groups, we measured three inflammatory parameters: highest body temperature after surgery, C-reactive protein level on postoperative day 3, and change in eosinophil count from the day before the surgery to postoperative day 3. The recurrence rate was significantly lower in Group B than in Group A (2.6% vs. 24.8%, P

Isomaltomegalosaccharide (IMS) is a long chimeric glucosaccharide composed of α-(1 → 6)- and α-(1 → 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 → 4)-segments had DPs of 3, 6, and 9, respectively. IMS exhibited considerably higher water solubility than maltodextrin because of the α-(1 → 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 → 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.

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 alpha-(1 -> 6)- and alpha-(1 -> 4)-linked portions at the nonreducing and reducing ends, respectively, in which the alpha-(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 alpha-(1 -> 6)-glucosidic linkages, suggesting that DDase also catalyzes elongation of alpha-(1 -> 4)-glucosyl chain. For production of IMS using DDsur, intact cells bearing DDsur were directly incubated with 20% G6/G7 at 45 degrees C by optimizing conditions such as cell concentration and agitation efficiency, which resulted in generation of IMS (average DP =14.7) with 61% alpha-(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 degrees C), DDext produced IMS (average DP =14.5) with 65% alpha-(1 -> 6)-glucosyl content in a good yield of 87%.

Paenibacillus sp. 598K produces cycloisomaltooligosaccharides (CIs) in culture from dextran and starch. CIs are cyclic oligosaccharides consisting of seven or more α-(1 → 6)-linked-d-glucose residues. The extracellular enzyme CI glucanotransferase (PsCITase), which is the member of glycoside hydrolase family 66, catalyzes the final stage of CI production and produces mainly cycloisomaltoheptaose. We have discovered a novel intracellular CI-degrading dextranase (PsDEX598) from Paenibacillus sp. 598K. The 69.7-kDa recombinant PsDEX598 does not digest isomaltotetraose or shorter isomaltooligosaccharides, but digests longer ones of at least up to isomaltoheptaose. It also digests oligoCIs of cycloisomaltoheptaose, cycloisomaltooctaose, and cycloisomaltononaose better than it does with megaloCIs of cycloisomaltodecaose, cycloisomaltoundecaose, and cycloisomaltododecaose, as well as an α-(1 → 6)-d-glucan of dextran 40. PsDEX598 is produced intracellularly when culture medium is supplemented with cycloisomaltoheptaose or dextran, but not with isomaltooligosaccharides (a mixture of isomaltose, isomaltotriose, and panose), starch, or glucose. The whole genomic DNA sequence of the strain 598K implies that it harbors two genes for enzymes belonging to glycoside hydrolase family 66 (PsCITase and PsDEX598), and PsDEX598 is the only dextranase in the strain. PsDEX598 does not have any carbohydrate-binding modules (CBMs) and has a low similarity (< 30%) with other family 66 dextranases, and the catalytic amino acids of this enzyme are predicted to be Asp191, Asp303, and Glu368. The strain Paenibacillus sp. 598K appears to take up CI-7, so these findings indicate that this bacterium can degrade CIs using a dextranase within the cells and so utilize them as a carbon source for growth.

Makgeolli is a traditional Korean alcoholic rice beverage. It is brewed of ingredients containing starch, Nuruk, and water. In order to improve the quality and functionality of Makgeolli, the Rhizopus oligosporus fermented buckwheat containing 18.7 mg/kg of l‐carnitine were utilized to brew l‐carnitine fortified Makgeolli with rice. Makgeolli was prepared in two‐stage fermentation method and total rutin and quercetin in each fermented buckwheat Makgeolli were increased 1.8‐fold greater than buckwheat Makgeolli. DPPH antioxidant activity was enhanced in fermented buckwheat Makgeolli than buckwheat Makgeolli (21.9%–65.7%). The amounts of l‐carnitine in rice Makgeolli, buckwheat Makgeolli, and fermented buckwheat Makgeolli were 0.9, 0.8–1.0, and 1.0–1.9 mg/L, respectively. The fermented buckwheat Makgeolli not only promoted health benefit by increasing l‐carnitine and flavonols, but also made effective alcohol production (2.8%–8.4%) compared to common buckwheat Makgeolli, indicating the potential industrial application with health benefits.

Herein, we investigated enzymatic properties and reaction specificities of Streptococcus mutans dextranase, which hydrolyzes α-(1→6)-glucosidic linkages in dextran to produce isomaltooligosaccharides. Reaction specificities of wild-type dextranase and its mutant derivatives were examined using dextran and a series of enzymatically prepared p-nitrophenyl α-isomaltooligosaccharides. In experiments with 4-mg·mL−1 dextran, isomaltooligosaccharides with degrees of polymerization (DP) of 3 and 4 were present at the beginning of the reaction, and glucose and isomaltose were produced by the end of the reaction. Increased concentrations of the substrate dextran (40 mg·mL−1) yielded isomaltooligosaccharides with higher DP, and the mutations T558H, W279A/T563N, and W279F/T563N at the −3 and −4 subsites affected hydrolytic activities of the enzyme, likely reflecting decreases in substrate affinity at the −4 subsite. In particular, T558H increased the proportion of isomaltooligosaccharide with DP of 5 in hydrolysates following reactions with 4-mg·mL−1 dextran.Abbreviations CI: cycloisomaltooligosaccharide; CITase: CI glucanotransferase; CITase-Bc: CITase from Bacillus circulans T-3040; DP: degree of polymerization of glucose unit; GH: glycoside hydrolase family; GTF: glucansucrase; HPAEC-PAD: high performance anion-exchange chromatography-pulsed amperometric detection; IG: isomaltooligosaccharide; IGn: IG with DP of n (n, 2‒5); PNP: p-nitrophenol; PNP-Glc: p-nitrophenyl α-glucoside; PNP-IG: p-nitrophenyl isomaltooligosaccharide; PNP-IGn: PNP-IG with DP of n (n, 2‒6); SmDex: dextranase from Streptococcus mutans; SmDexTM: S. mutans ATCC25175 SmDex bearing Gln100‒Ile732

Megalo-type isomaltosaccharides are an enzymatically synthesized foodstuff produced by transglucosylation from maltodextrin, and they contain a mid-chain length polymer of D-glucose with α-1,6-glycoside linkages. The injection of a solution of megalo-type isomaltosaccharides (1–4%(w/v), average DP = 12.6), but not oligo-type isomaltosaccharides (average DP = 3.3), into the intestinal lumen dose-dependently reduced the transport rates of tight junction permeable markers in a ligated loop of the anesthetized rat jejunum. Application of the megalosaccharide also suppressed the transport of tight junction markers and enhanced transepithelial electrical resistance (TEER) in Caco-2 cell monolayers. Cholesterol sequestration by methyl-β-cyclodextrin in the Caco-2 monolayers abolished the effect of megalosaccharide. Treatment with anti-caveolin-1 and a caveolae inhibitor, but not clathrin-dependent endocytosis and macropinocytosis inhibitors, suppressed the increase in TEER. These results indicate that isomaltosaccharides promote the barrier function of tight junctions in the intestinal epithelium in a chain-length dependent manner and that caveolae play a role in the effect.

Glycoside hydrolase family 97 (GH97) is one of the most interesting glycosidase families, which contains inverting and retaining glycosidases. Currently, only two enzyme types, alpha-glucoside hydrolase and alpha-galactosidase, are registered in the carbohydrate active enzyme database as GH97 function-known proteins. To explore new specificities, BT3661 and BT3664, which have distinct amino acid sequences when compared with that of GH97 alpha-glucoside hydrolase and alpha-galactosidase, were characterized in this study. BT3664 was identified to be an alpha-galactosidase, whereas BT3661 exhibits hydrolytic activity toward both beta-L-arabinopyranoside and alpha-D-galactopyranoside, and thus we designate BT3661 as a beta-L-arabinopyranosidase/alpha-D-galactosidase. Since this is the first dual substrate specificity enzyme in GH97, we investigated the substrate recognition mechanism of BT3661 by determining its three-dimensional structure and based on this structural data generated a number of mutants to probe the enzymatic mechanism. Structural comparison shows that the active-site pocket of BT3661 is similar to GH97 alpha-galactosidase BT1871, but the environment around the hydroxymethyl group of the galactopyranoside is different. While BT1871 bears G1u361 to stabilize the hydroxy group of C6 through a hydrogen bond with its carboxy group, BT3661 has Asn338 at the equivalent position. Amino acid mutation analysis indicates that the length of the side chain at Asn338 is important for defining specificity of BT3661. The kcat/Km value for the hydrolysis of p-nitrophenyl alpha-galactoside decreases when Asn338 is substituted with Glu, whereas an increase is observed when the mutation is Ala. Interestingly, mutation of Asn338 to Ala reduces the kcat/Km value for hydrolysis of p-nitrophenyl beta-L-arabinopyranoside. (C) 2017 Published by Elsevier B.V.

Paenibacillus sp. 598K α-1,6-glucosyltransferase (Ps6TG31A), a member of glycoside hydrolase family 31, catalyzes exo-α-glucohydrolysis and transglucosylation and produces α-1,6-glucosyl-α-glucosaccharides from α-glucan via its disproportionation activity. The crystal structure of Ps6TG31A was determined by an anomalous dispersion method using a terbium derivative. The monomeric Ps6TG31A consisted of one catalytic (β/α)8-barrel domain and six small domains, one on the N-terminal and five on the C-terminal side. The structures of the enzyme complexed with maltohexaose, isomaltohexaose, and acarbose demonstrated that the ligands were observed in the catalytic cleft and the sugar-binding sites of four β-domains. The catalytic site was structured by a glucose-binding pocket and an aglycon-binding cleft built by two sidewalls. The bound acarbose was located with its non-reducing end pseudosugar docked in the pocket, and the other moieties along one sidewall serving three subsites for the α-1,4-glucan. The bound isomaltooligosaccharide was found on the opposite sidewall, which provided the space for the acceptor molecule to be positioned for attack of the catalytic intermediate covalent complex during transglucosylation. The N-terminal domain recognized the α-1,4-glucan in a surface-binding mode. Two of the five C-terminal domains belong to the carbohydrate-binding modules family 35 and one to family 61. The sugar complex structures indicated that the first family 35 module preferred α-1,6-glucan, whereas the second family 35 module and family 61 module preferred α-1,4-glucan. Ps6TG31A appears to have enhanced transglucosylation activity facilitated by its carbohydrate-binding modules and substrate-binding cleft that positions the substrate and acceptor sugar for the transglucosylation.

Paenibacillus sp. 598K produces cycloisomaltooligosaccharides (cyclodextrans) from starch even in the absence of dextran. Cycloisomaltooligosaccharide glucanotransferase synthesizes cycloisomaltooligosaccharides exclusively from an α-(1 → 6)-consecutive glucose chain consisting of at least four molecules. Starch is not a substrate of this enzyme. Therefore, we predicted that the bacterium possesses another enzyme system for extending α-(1 → 6)-linked glucoses from starch, which can be used as the substrate for cycloisomaltooligosaccharide glucanotransferase, and identified the transglucosylation enzyme Ps6GT31A. We purified Ps6GT31A from the bacterial culture supernatant, cloned its corresponding gene, and characterized the recombinant enzyme. Ps6GT31A belongs to glycoside hydrolase family 31, and it liberates glucose from the non-reducing end of the substrate in the following order of activity: α-(1 → 4)-> α-(1 → 2)- > α-(1 → 3)- > α-(1 → 6)-glucobiose and maltopentaose > maltotetraose > maltotriose > maltose. Ps6GT31A catalyzes both hydrolysis and transglucosylation. The resulting transglucosylation compounds were analyzed by high-performance liquid chromatography and mass spectrometry. Analysis of the initial products by 13C nuclear magnetic resonance spectroscopy revealed that Ps6GT31A had a strong α-(1 → 4) to α-(1 → 6) transglucosylation activity. Ps6GT31A elongated α-(1 → 6)-linked glucooligosaccharide to at least a degree of polymerization of 10 through a successive transglucosylation reaction. Eventually, cycloisomaltooligosaccharide glucanotransferase creates cycloisomaltooligosaccharides using the transglucosylation products generated by Ps6GT31A as the substrates. Our data suggest that Ps6GT31A is the key enzyme to synthesize α-(1 → 6)-glucan for cycloisomaltooligosaccharide production in dextran-free environments.

α-Glucosidase from Aspergillus niger (AgdA; typical α-1,4-glucosidase) is known to industrially produce α-(1→6)-glucooligosaccharides. This fungus also has another α-glucosidase-like protein, AgdB. To learn its function, wild-type AgdB was expressed in Pichia pastoris. However, the enzyme displayed two electrophoretic forms due to heterogeneity of N-glycosylation at Asn354. The deglycosylation mutant N354D shared the same properties with wild-type AgdB. N354D demonstrated hydrolytic specificity toward α-(1→3)- and α-(1→4)-glucosidic linkages, indicating that AgdB is an α-1,3-/α-1,4-glucosidase. N354D-catalyzed transglucosylation from maltose was analyzed in short- and long-term reactions, enabling us to learn the transglucosylation specificity and product accumulation, respectively. A short-term reaction (

Background: Production of wheat bran (WB) for human consumption is estimated to be about 90 million tons per year. WB contains an abundant source of dietary fiber, minerals, vitamins, and bioactive compounds. WB is a by-product of milling and contains an abundant source of carbohydrate (60%), protein (12%), fat (0.5%), minerals (2%), and bioactive compounds such as phenolic acids, arabinoxylans, flavonoids, caroteinoids alkylresorcinol and phytosterols. These are known for health promoting properties such as controlling glycemic index, reducing plasma cholesterol level, antioxidant, anti-inflammatory, and anticarcinogenic activities. Several terpene glycosides such as mogroside V, paenoiflorin, geniposide, rubusoside (Ru), stevioside (Ste), rebaudioside A (RebA), steviol monoside, and stevioside glucoside have been discovered to enhance the solubility of a number of pharmaceutically and medically important compounds that normally show poor solubility in water. Context and purpose of this study: In this study, in order to increase soluble extraction of polyphenol compounds of WB using Ru, the expression of β-galactosidase from Thermus thermophilus (T. thermophilus) was optimized using different E. coli hosts and a different concentration of lactose inducer rather than of isopropyl-1- thio-β-D-galactopyranoside (IPTG) for industrial production. Additionally, the effect of different steviol glucosides (Ru, Ste, RebA, and SG) on the enhancement of polyphenol compounds extraction from wheat bran was studied. Results: β-galactosidase from T. thermophilus was used for the specific conversion of stevioside (Ste) to rubusoside (Ru) with 92% productivity. The enzyme was optimized to be expressed in E. coli. With 7 mM lactose, the β-galactosidase activity expressed was 34.3, 14.2, or 34.4 ± 0.5 U/mL in E. coli BL21(DE3)pLysS, Rosetta(DE3)pLysS, or BL21(DE3) at 37°C, and 9.8 ± 0.2, 7.0 ± 0.5, or 7.4 ± 0.2 U/mL at 28°C respectively. The expression of β-galactosidase was dependent on the lactose concentration and the highest activity was obtained with the conditions of 5 mM lactose in E. coli Rosetta(DE3)pLysS, 53.3 ± 1.5 U/mL. 78% of the mesophilic proteins was eliminated by heating at 70°C for 15 min with 89% β-galactosidase activity recovery. The total polyphenol content of WB extracted by water, Ru, Ste, rebaudioside A (RebA), and steviol glucosides (SG) were 533.8 ± 9.6 µg/mL, 633.3 ± 1.25 µg/mL, 604.4 ± 10.1 µg/mL, 654.8 ± 26.5 µg/mL, and 601.2 ± 33.4 µg/mL, respectively. The DPPH radical scavenging activity prepared by water, Ru, Ste, RebA, and SG extraction were 8.76 ± 0.3 mg/mL, 4.87 ± 0.3 mg/mL, 5.34 ± 0.22 mg/mL, 7.27 ± 0.1 mg/mL, and 7.82 ± 0.02 mg/mL respectively. Conclusions: To increase soluble extraction of polyphenol compounds of WB using Ru, the expression of β-galactosidase from T. thermophilus was optimized using different E. coli hosts and a different concentration of lactose inducer rather than isopropyl-1- thio-β-D- galactopyranoside (IPTG) for industrial production of the enzyme. The highest antioxidant activity was shown in WB extracted by Ru. The number of glucosyl units attached to steviol can possibly affect the efficiency of antioxidant activity of WB extracted by steviol glucosides.

Streptomyces thermolilacinus mannanase (StMan), which requires Ca2+ for its enhanced thermal stability and hydrolysis activity, possesses two Ca2+-binding sites in loop6 and loop7. We evaluated the function of the Ca2+-binding site in loop7 and the hydrogen bond between residues Ser247 in loop6 and Asp279 in loop7. The Ca2+-binding in loop7 was involved only in thermal stability. Mutations of Ser247 or Asp279 retained the Ca2+-binding ability; however, mutants showed less thermal stability than StMan. Phylogenetic analysis indicated most glycoside hydrolase family 5 subfamily 8 mannanases could be stabilized by Ca2+; however, the mechanism of StMan thermal stability was found to be quite specific in some actinomycete mannanases. This article is protected by copyright. All rights reserved.

The actinobacterium Kribbella flavida NBRC 14399(T) produces cyclobis-(1 -> 6)-alpha-nigerosyl (CNN), a cyclic glucotetraose with alternate alpha-(1 -> 6)- and alpha-(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-alpha-glucosyl-transferase and 3-alpha-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 alpha-(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 alpha-(1 -> 3)-isomaltosyl moiety and effectively hydrolyzes the alpha-(1 -> 3)-glucosidic linkage, it should be termed 1,3-alpha-isomaltosidase. Kfla1896 effectively hydrolyzed isomaltose with liberation of beta-glucose, but displayed low or no activity toward CNN and the general GH15 enzyme substrates such as maltose, soluble starch, or dextran. The k(cat)/K-m 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.

Plant β-glucosidases are usually members of the glucosyl hydrolase 1 (GH1) or 3 (GH3) families. Previously, a β-glucosidase (torvosidase) was purified from Solanum torvum leaves that specifically catalyzed hydrolysis of two furostanol 26-O-β-glucosides, torvosides A and H. Furostanol glycoside 26-O-β-glucosides have been reported as natural substrates of some plant GH1 enzymes. However, torvosidase was classified as a GH3 β-glucosidase, but could not hydrolyze β-oligoglucosides, the natural substrates of GH3 enzymes. Here, the full-length cDNA encoding S. torvum β-glucosidase (SBgl3) was isolated by the rapid amplification of cDNA ends method. The 1887bp ORF encoded 629 amino acids and showed high homology to other plant GH3 β-glucosidases. Internal peptide sequences of purified native Sbgl3 determined by LC-MS/MS matched the deduced amino acid sequence of the Sbgl3 cDNA, suggesting that it encoded the natural enzyme. Recombinant SBgl3 with a polyhistidine tag (SBgl3His) was successfully expressed in Pichia pastoris. The purified SBgl3His showed the same substrate specificity as natural SBgl3, hydrolyzing torvoside A with much higher catalytic efficiency than other substrates. It also had similar biochemical properties and kinetic parameters to the natural enzyme, with slight differences, possibly attributable to post-translational glycosylation. Quantitative real-time PCR (qRT-PCR) showed that SBgl3 was highly expressed in leaves and germinated seeds, suggesting a role in leaf and seedling development. To our knowledge, a recombinant GH3 β-glucosidase that hydrolyzes furostanol 26-O-β-glucosides, has not been previously reported in contrast to substrates of GH1 enzymes.

α-Glucosidases (AGases) and α-1,4-glucan lyases (GLases) catalyze the degradation of α-glucosidic linkages at the non-reducing ends of substrates to release α-glucose and anhydrofructose, respectively. The AGases belong to glycoside hydrolase (GH) families 13 and 31, and the GLases belong to GH31 and share the same structural fold with GH31 AGases. GH13 and GH31 AGases show diverse functions upon the hydrolysis of substrates, having linkage specificities and size preferences, as well as upon transglucosylation, forming specific α-glucosidic linkages. The crystal structures of both enzymes were determined using free and ligand-bound forms, which enabled us to understand the important structural elements responsible for the diverse functions. A series of mutational approaches revealed features of the structural elements. In particular, amino-acid residues in plus subsites are of significance, because they regulate transglucosylation, which is used in the production of industrially valuable oligosaccharides. The recently solved three-dimensional structure of GLase from red seaweed revealed the amino-acid residues essential for lyase activity and the strict recognition of the α-(1 → 4)-glucosidic substrate linkage. The former was introduced to the GH31 AGase, and the resultant mutant displayed GLase activity. GH13 and GH31 AGases hydrate anhydrofructose to produce glucose, suggesting that AGases are involved in the catabolic pathway used to salvage unutilized anhydrofructose.

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 ILlβ 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.

Cycloisomaltooligosaccharide glucanotransferase (CITase) is a member of glycoside hydrolase family 66 and it produces cycloisomaltooligosaccharides (CIs). Small CIs (CI-7-9) and large CIs (CI-≥10) are designated as oligosaccharide-type CIs (oligo-CIs) and megalosaccharide-type CIs (megalo-CIs) respectively. CITase from Bacillus circulans T-3040 (BcCITase) produces mainly CI-8 with little megalo-CIs. It has two family 35 carbohydrate-binding modules (BcCBM35-1 and BcCBM35-2). BcCBM35-1 is inserted in a catalytic domain of BcCITase and BcCBM35-2 is located at the C-terminal region. Our previous studies suggested that BcCBM35-1 has two substrate-binding sites (B-1 and B-2) [Suzuki et al. (2014) J. Biol. Chem. 289, 12040-12051]. We implemented site-directed mutagenesis of BcCITase to explore the preference for product size on the basis of the 3D structure of BcCITase. Mutational studies provided evidence that B-1 and B-2 contribute to recruiting substrate and maintaining product size respectively. A mutant (mutant-R) with four mutations (F268V, D469Y, A513V and Y515S) produced three times as much megalo-CIs (CI-10-12) and 1.5 times as much total CIs (CI-7-12) as compared with the wild-type (WT) BcCITase. The 3D structure of the substrate-enzyme complex of mutant-R suggested that the modified product size specificity was attributable to the construction of novel substrate-binding sites in the B-2 site of BcCBM35-1 and reactivity was improved by mutation on subsite -3 on the catalytic domain.

Glycoside hydrolase family 13 contains exo-glucosidases specific for alpha-(1 -> 4)- and alpha-(1 -> 6)-linkages including alpha-glucosidase, oligo-1,6-glucosidase, and dextran glucosidase. The alpha-(1 -> 6)-linkage selectivity of Streptococcus mutans dextran glucosidase was altered to alpha-(1 -> 4)-linkage selectivity through site-directed mutations at Val195, Lys275, and Glu371. V195A showed 1300-fold higher k(cat)/K-m for maltose than wild-type, but its k(cat)/K-m for isomaltose remained 2-fold higher than for maltose. K275A and E371A combined with V195A mutation only decreased isomaltase activity. V195A/K275A, V195A/E371A, and V195A/K275A/E371A showed 27-, 26-, and 73-fold higher k(cat)/K-m for maltose than for isomaltose, respectively. Consequently, the three residues are structural elements for recognition of the alpha-(1 -> 6)-glucosidic linkage. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The α-glucosidase from sugar beet (SBG) is an exo-type glycosidase. The enzyme has a pocket-shaped active site, but efficiently hydrolyzes longer maltooligosaccharides and soluble starch due to lower Km and higher kcat/Km for such substrates. To obtain structural insights into the mechanism governing its unique substrate specificity, a series of acarviosyl-maltooligosaccharides was employed for steady-state kinetic and structural analyses. The acarviosyl-maltooligosaccharides have a longer maltooligosaccharide moiety compared with the maltose moiety of acarbose, which is known to be the transition state analog of α-glycosidases. The clear correlation obtained between log Ki of the acarviosyl-maltooligosaccharides and log(Km/kcat) for hydrolysis of maltooligosaccharides suggests that the acarviosyl-maltooligosaccharides are transition state mimics. The crystal structure of the enzyme bound with acarviosyl-maltohexaose reveals that substrate binding at a distance from the active site is maintained largely by van der Waals interactions, with the four glucose residues at the reducing terminus of acarviosyl-maltohexaose retaining a left-handed single-helical conformation, as also observed in cycloamyloses and single helical V-amyloses. The kinetic behavior and structural features suggest that the subsite structure suitable for the stable conformation of amylose lowers the Km for long-chain substrates, which in turn is responsible for higher specificity of the longer substrates.

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 β-CD, respectively. (1)H NMR and circular dichrosim spectra revealed the dye forms consisted of 1:1 stoichiometric inclusion complex within the β-CD cavity, whereas they exhibited non-specific hydrophobic interaction, identified by solvent polarity changes, with L-IMS. The inclusion complex delivered by β-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.

Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase belongs to the glycoside hydrolase family 66 and catalyzes an intramolecular transglucosylation reaction that produces cycloisomaltooligosaccharides from dextran. The crystal structure of the core fragment from Ser-39 to Met-738 of B. circulans T-3040 cycloisomaltooligosaccharide glucanotransferase, devoid of its N-terminal signal peptide and C-terminal nonconserved regions, was determined. The structural model contained one catalytic (β/α)8-barrel domain and three β-domains. Domain N with an immunoglobulin-like β-sandwich fold was attached to the N terminus; domain C with a Greek key β-sandwich fold was located at the C terminus, and a carbohydrate-binding module family 35 (CBM35) β-jellyroll domain B was inserted between the 7th β-strand and the 7th α-helix of the catalytic domain A. The structures of the inactive catalytic nucleophile mutant enzyme complexed with isomaltohexaose, isomaltoheptaose, isomaltooctaose, and cycloisomaltooctaose revealed that the ligands bound in the catalytic cleft and the sugar-binding site of CBM35. Of these, isomaltooctaose bound in the catalytic site extended to the second sugar-binding site of CBM35, which acted as subsite -8, representing the enzyme·substrate complex when the enzyme produces cycloisomaltooctaose. The isomaltoheptaose and cycloisomaltooctaose bound in the catalytic cleft with a circular structure around Met-310, representing the enzyme·product complex. These structures collectively indicated that CBM35 functions in determining the size of the product, causing the predominant production of cycloisomaltooctaose by the enzyme. The canonical sugar-binding site of CBM35 bound the mid-part of isomaltooligosaccharides, indicating that the original function involved substrate binding required for efficient catalysis.

This study reports the characterization of the ability of Dermacoccus spp. isolated from the deepest point of the world's oceans for azo dye decolorization. A detailed investigation of Dermacoccus abyssi MT1.1(T) with respect to the azoreductase activity and enzymatic mechanism as well as the potential role of the bacterial strain for biocleaning of industrial dye baths is reported. Resting cells with oxygen-insensitive azoreductase resulted in the rapid decolorization of the polysulfonated dye Brilliant Black BN (BBN) which is a common food colorant. The highest specific decolorization rate (vs) was found at 50 °C with a moderately thermal tolerance for over 1 h. Kinetic analysis showed the high rates and strong affinity of the enzymatic system for the dye with a Vmax = 137 mg/g cell/h and a Km = 19 mg/L. The degradation of BBN produces an initial orange intermediate, 8-amino-5-((4-sulfonatophenyl)diazenyl)naphthalene-2-sulfonic acid, identified by mass spectrometry which is later converted to 4-aminobenzene sulfonic acid. Nearly 80% of the maximum vs is possible achieved in resting cell treatment with the salinity increased up to 5.0% NaCl in reaction media. Therefore, this bacterial system has potential for dye decolorization bioprocesses occurring at high temperature and salt concentrations e.g. for cleaning dye-containing saline wastewaters.

For Podospora anserina, several studies of cellulolytic enzymes have been established, but characteristics of amylolytic enzymes are not well understood. When P. anserina grew in starch as carbon source, it accumulated glucose, nigerose, and maltose in the culture supernatant. At the same time, the fungus secreted α-glucosidase (PAG). PAG was purified from the culture supernatant, and was found to convert soluble starch to nigerose and maltose. The recombinant enzyme with C-terminal His-tag (rPAG) was produced with Pichia pastoris. Most rPAG produced under standard conditions lost its affinity for nickel-chelating resin, but the affinity was improved by the use of a buffered medium (pH 8.0) supplemented with casamino acid and a reduction of the cultivation time. rPAG suffered limited proteolysis at the same site as the original PAG. A site-directed mutagenesis study indicated that proteolysis had no effect on enzyme characteristics. A kinetic study indicated that the PAG possessed significant transglycosylation activity.

Rice Os7BGlu26 is a GH1 family glycoside hydrolase with a threefold higherkcat/Kmvalue for 4-nitrophenyl β-D-mannoside (4NPMan) compared with 4-nitrophenyl β-D-glucoside (4NPGlc). To investigate its selectivity for β-D-mannoside and β-D-glucoside substrates, the structures of apo Os7BGlu26 at a resolution of 2.20 Å and of Os7BGlu26 with mannose at a resolution of 2.45 Å were elucidated from isomorphous crystals in space groupP212121. The (β/α)8-barrel structure is similar to other GH1 family structures, but with a narrower active-site cleft. The Os7BGlu26 structure with D-mannose corresponds to a product complex, with β-D-mannose in the1S5skew-boat conformation. Docking of the1S3,1S5,2SOand3S1pyranose-ring conformations of 4NPMan and 4NPGlc substrates into the active site of Os7BGlu26 indicated that the lowest energies were in the1S5and1S3skew-boat conformations. Comparison of these docked conformers with other rice GH1 structures revealed differences in the residues interacting with the catalytic acid/base between enzymes with and without β-D-mannosidase activity. The mutation of Tyr134 to Trp in Os7BGlu26 resulted in similarkcat/Kmvalues for 4NPMan and 4NPGlc, while mutation of Tyr134 to Phe resulted in a 37-fold higherkcat/Kmfor 4NPMan than 4NPGlc. Mutation of Cys182 to Thr decreased both the activity and the selectivity for β-D-mannoside. It was concluded that interactions with the catalytic acid/base play a significant role in glycon selection.

Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase (BcCITase) catalyses an intramolecular transglucosylation reaction and produces cycloisomaltooligosaccharides from dextran. BcCITase was overexpressed in Escherichia coli in two different forms and crystallized by the sitting-drop vapour-diffusion method. The crystal of BcCITase bearing an N-­terminal His6 tag diffracted to a resolution of 2.3 A and belonged to space group P3121, containing a single molecule in the asymmetric unit. The crystal of BcCITase bearing a C-terminal His6 tag diffracted to a resolution of 1.9 A and belonged to space group P212121, containing two molecules in the asymmetric unit.

Sugar beet α-glucosidase (SBG), a member of glycoside hydrolase family 31, shows exceptional long-chain specificity, exhibiting higher kcat/Km values for longer malto-oligosaccharides. However, its amino acid sequence is similar to those of other short chain-specific α-glucosidases. To gain structural insights into the long-chain substrate recognition of SBG, a crystal structure complex with the pseudotetrasaccharide acarbose was determined at 1.7 Å resolution. The active site pocket of SBG is formed by a (β/α)8 barrel domain and a long loop (N-loop) bulging from the N-terminal domain similar to other related enzymes. Two residues (Phe-236 and Asn-237) in the N-loop are important for the long-chain specificity. Kinetic analysis of an Asn-237 mutant enzyme and a previous study of a Phe-236 mutant enzyme demonstrated that these residues create subsites +2 and +3. The structure also indicates that Phe-236 and Asn-237 guide the reducing end of long substrates to subdomain b2, which is an additional element inserted into the (β/α)8 barrel domain. Subdomain b2 of SBG includes Ser-497, which was identified as the residue at subsite +4 by site-directed mutagenesis.

The presence of an α-1,6-glucosaccharide enhances absorption of water-soluble quercetin glycosides, a mixture of quercetin-3-O-β-d-glucoside (Q3G, 31.8%), mono (23.3%), di (20.3%) and more d-glucose adducts with α-1,4-linkage to a d-glucose moiety of Q3G, in a ligated small intestinal loop of anesthetized rats. We prepared α-1,6-glucosaccharides with different degrees of polymerization (DP) enzymatically and separated them into a megalo-isomaltosaccharide-containing fraction (M-IM, average DP=11.0) and an oligo-isomaltosaccharide-containing fraction (O-IM, average DP=3.6). Luminal injection of either saccharide fraction promoted the absorption of total quercetin-derivatives from the small intestinal segment and this effect was greater for M-IM than O-IM addition. M-IM also increased Q3G, but not the quercetin aglycone, concentration in the water-phase of the luminal contents more strongly than O-IM. The enhancement of Q3G solubilization in the luminal contents may be responsible for the increases in the quercetin glucoside absorption promoted by α-1,6-glucosaccharides, especially that by M-IM. These results suggest that the ingestion of α-1,6-glucosaccharides promotes Q3G bioavailability.

The human fibroblast collagenase catalytic domain (MMP1ca) that is considered a prototype for all interstitial collagenase and plays an important role in the turnover of collagen fibrils in the matrix was expressed as an inclusion body in the Escherichia coli. The purified enzyme displayed activity with substrate Dnp-Pro-Leu-Ala-Leu-Trp-Ala-Arg-OH with a K(m) value of 26.61±1.42 μM. The inhibition activity of the nine flavonoid compounds and gallic acid against MMP1ca was examined. Among the compounds tested, the IC(50) of seven flavonoid compounds were determined and ranged from 14.13 to 339.21 μM. Epigallocatechin gallate (EGCG) showed the highest inhibition toward MMP1ca with IC(50) values of 14.13±0.49 μM. EGCG showed a competitive inhibition pattern with a K(i) value of 10.47±0.51 μM. The free binding energy of EGCG against MMP1ca was -13.07 kcal mol(-1), which was calculated by using Autodock 3.0.5 software and showed numerous hydrophobic and hydrogen bond interactions. The galloyl group of EGCG, gallocatechin gallate and epicatechin gallate was determined to be important for inhibitory activity against MMP1ca.

Glycoside hydrolase family 31 α-glucosidases (31AGs) show various specificities for maltooligosaccharides according to chain length. Aspergillus niger α-glucosidase (ANG) is specific for short-chain substrates with the highest k cat / K m for maltotriose, while sugar beet α-glucosidase (SBG) prefers long-chain substrates and soluble starch. Multiple sequence alignment of 31AGs indicated a high degree of diversity at the long loop (N-loop), which forms one wall of the active pocket. Mutations of Phe236 in the N-loop of SBG (F236A/S) decreased k cat / K m values for substrates longer than maltose. Providing a phenylalanine residue at a similar position in ANG (T228F) altered the k cat / K m values for maltooligosaccharides compared with wild-type ANG, i.e., the mutant enzyme showed the highest k cat / K m value for maltotetraose. Subsite affinity analysis indicated that modification of subsite affinities at + 2 and + 3 caused alterations of substrate specificity in the mutant enzymes. These results indicated that the aromatic residue in the N-loop contributes to determining the chain-length specificity of 31AGs.

The preparation of a glycosynthase, a catalytic nucleophile mutant of a glycosidase, is a well-established strategy for the effective synthesis of glycosidic linkages. However, glycosynthases derived from α-glycosidases can give poor yields of desired products because they require generally unstable β-glycosyl fluoride donors. Here, we investigate a transglycosylation catalyzed by a catalytic nucleophile mutant derived from a glycoside hydrolase family (GH) 97 α-galactosidase, using more stable β-galactosyl azide and α-galactosyl fluoride donors. The mutant enzyme catalyzes the glycosynthase reaction using β-galactosyl azide and α-galactosyl transfer from α-galactosyl fluoride with assistance of external anions. Formate was more effective at restoring transfer activity than azide. Kinetic analysis suggests that poor transglycosylation in the presence of the azide is because of low activity of the ternary complex between enzyme, β-galactosyl azide and acceptor. A three-dimensional structure of the mutant enzyme in complex with the transglycosylation product, β-lactosyl α-d-galactoside, was solved to elucidate the ligand-binding aspects of the α-galactosidase. Subtle differences at the β→α loops 1, 2 and 3 of the catalytic TIM barrel of the α-galactosidase from those of a homologous GH97 α-glucoside hydrolase seem to be involved in substrate recognitions. In particular, the Trp residues in β→α loop 1 have separate roles. Trp312 of the α-galactosidase appears to exclude the equatorial hydroxy group at C4 of glucosides, whereas the corresponding Trp residue in the α-glucoside hydrolase makes a hydrogen bond with this hydroxy group. The mechanism of α-galactoside recognition is conserved among GH27, 31, 36 and 97 α-galactosidases. Database The atomic coordinates (code: 5E1Q) have been deposited in the Protein Data Bank.

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 ILlβ 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.

The recombinant AglB produced by Pichia pastoris exhibited substrate inhibition behavior for the hydrolysis of p-nitrophenyl α-galactoside, whereas it hydrolyzed the natural substrates, including galactomanno-oligosaccharides and raffinose family oligosaccharides, according to the Michaelian kinetics. These contrasting kinetic behaviors can be attributed to the difference in the dissociation constant of second substrate from the enzyme and/or to the ability of the leaving group of the substrates. The enzyme displays the grater kcat/Km values for hydrolysis of the branched α-galactoside in galactomanno-oligosaccharides than that of raffinose and stachyose. A sequence comparison suggested that AglB had a shallow active-site pocket, and it can allow to hydrolyze the branched α-galactosides, but not linear raffinose family oligosaccharides.

Human intestinal maltase (HMA) is an α-glucosidase responsible for the hydrolysis of α-1,4-linkages from the non-reducing end of malto-oligosaccharides. HMA has become an important target in the treatment of type-2 diabetes. In this study, epigallocatechin gallate (EGCG) and EGCG glucoside (EGCG-G1) were identified as inhibitors of HMA by an in vitro assay with IC50 of 20 ± 1.0 and 31.5 ± 1.0 μM, respectively. A Lineweaver-Burk plot confirmed that EGCG and EGCG-G1 were competitive inhibitors of maltose substrate against HMA and inhibition kinetic constants (K i ) calculated from a Dixon plot were 5.93 ± 0.26 and 7.88 ± 0.57 μM, respectively. Both EGCG and EGCG-G1 bound to the active site of HMA with numerous hydrophobic and hydrogen bond interactions.