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)-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

Partial depolymerization of tamarind seed xyloglucan and its functionality toward enhancing the solubility of curcumin

Polysaccharides of tamarind seed, a byproduct of the tamarind pulp industry, displayed a potential solubility improvement of lipophilic bioactive molecules but their textural characteristics hinder the dietary formulation. In contrast, the commonly available xyloglucan oligosaccharides (XOSs) with degrees of polymerization (DPs) of 7, 8, and 9 were too short to maintain their ability. The binding capacity of the between sizes is unknown due to a lack of appropriate preparation. We prepared xyloglucan megalosaccharides (XMSs) by partial depolymerization, where term megalosaccharide (MS) defines the middle chain-length saccharide between DPs 10 and 100. Digestion with fungal cellulase enabled reproducible active XMSs. Further identification of pure XMS segments indicated that XMS-B has an average DP of 17.2 (Gal3Glc8Xyl6) with a branched dimer of XOS 8 and 9 and was free of side-chain arabinose, the residue influencing high viscosity. Curcumin, a bioactive pigment, has poor bioavailability because of its water insolubility. XMSs with average DPs of 15.4-24.3 have similarly sufficient capacities to solubilize curcumin. The solubility of curcumin was improved 180-fold by the addition of 50 %, w/ v, XMSs, which yielded a clear yellow liquid. Our findings indicated that XMSs were a promising added-value agent in foods and pharmaceuticals for the oral intake of curcumin

Nonreducing terminal chimeric isomaltomegalosaccharide and its integration with azoreductase for the remediation of soil-contaminated lipophilic azo dyes

Lipophilic azo dyes are practically water-insoluble, and their dissolution by organic solvents and surfactants is harmful to biological treatment with living cells and enzymes. This study aimed to evaluate the feasibility of a newly synthesized nonreducing terminal chimeric isomaltomegalosaccharide (N-IMS) as a nontoxic solubilizer of four simulated lipophilic azo dye wastes for enzymatic degradation. N-IMS bearing a helical α-(1 → 4)-glucosidic segment derived from a donor substrate α-cyclodextrin was produced by a coupling reaction of cyclodextrin glucanotransferase. Inclusion complexing by N-IMS overcame the solubility issue with equilibrium constants of 1786-242 M-1 (methyl yellow > ethyl red > methyl red > azo violet). Circular dichroism spectra revealed the axial alignment of the aromatic rings in the N-IMS cavity, while UV-visible absorption quenching revealed that the azo bond of methyl yellow was particularly induced. Desorption of the dyes from acidic and neutral soils was specific to aqueous organic over alkali extraction. The dissolution kinetics of the incorporated dyes followed a sigmoid pattern facilitating the subsequent decolorization process with azoreductase. It was demonstrated that after soil extraction, the solid dyes dissolved with N-IMS assistance and spontaneously digested by coupled azoreductase/glucose dehydrogenase (for a cofactor regeneration system) with the liberation of the corresponding aromatic amine

Formulation and evaluation of a novel megalomeric microemulsion from tamarind seed xyloglucan-megalosaccharides for improved high-dose quercetin delivery

Megalomeric microemulsion is a new term referring to lipid-based formulation using amphiphilic megalosaccharide as a coexcipient. Quercetin is a dose-dependent bioactive compound and has promising therapeutic potential, but its low water solubility and permeability restrict its treatment efficacy. We aimed to formulate high-dose quercetin loaded into colloidal micelles by the self-micro emulsifying system (SMES) in combination with Tween 80, isopropyl myristate, and xyloglucan megalosaccharide (X-MS). X-MS is a moderate-size heterologous saccharide obtained from enzymatic cleavage of tamarind seed xyloglucan. X-MSs with an average degree of polymerization of 16 and 56 were investigated to bearing their surface hydrophobic interaction with a fluorescence probe 6-(p-toluidino)-2-naphthalene-6-sulfonate yielded the binding constant values of 127 and 180 M-1, respectively and X-MS itself displayed a slight effect on quercetin binding. However, the implementation of X-MSs toward SMES was highly compatible because X-MS molecules were confined in micellular solutions. Consequently, X-MSs improved the quercetin loading from 1 to 2 to 12.5-17.7 mg/mL based on the composition ratio, X-MS chain lengths, and X-MS concentrations (0.15-3.0%, w/v) and stabilized the quercetin-loaded oil-in-water SMES. The optical appearances were transparent yellow containing uniformly fine droplets with diameters of 11-12 nm. In vitro radical scavenging activity tests with 2,2-diphenyl-1-picrylhydrazyl showed that the megalomeric microemulsions improved the half-maximal inhibitory concentration (IC50 = 22-24 μg/mL) over that of the X-MS-free microemulsion. This study provided a new approach of liquid supplementation from commercially unavailable-size xyloglucan to be a promising added-value agent for oral uptake of quercetin

Effects of mutation of Asn694 in Aspergillus niger α-glucosidase on hydrolysis and transglucosylation

Aspergillus niger α-glucosidase (ANG), a member of glycoside hydrolase family 31, catalyzes hydrolysis of alpha-glucosidic linkages at the non-reducing end. In the presence of high concentrations of maltose, the enzyme also catalyzes the formation of α-(1 -> 6)-glucosyl products by transglucosylation and it is used for production of the industrially useful panose and isomaltooligosaccharides. The initial transglucosylation by wild-type ANG in the presence of 100 mM maltose [Glc(α 1-4)Glc] yields both α-(1 -> 6)- and α-(1 -> 4)-glucosidic linkages, the latter constituting similar to 25% of the total transfer reaction product. The maltotriose [Glc(α 1-4)Glc(α 1-4)Glc], α-(1 -> 4)-glucosyl product disappears quickly, whereas the α-(1 -> 6)-glucosyl products panose [Glc(α 1-6)Glc(α 1-4)Glc], isomaltose [Glc(α 1-6)Glc], and isomaltotriose [Glc(α 1-6)Glc(α 1-6)Glc] accumulate. To modify the transglucosylation properties of ANG, residue Asn694, which was predicted to be involved in formation of the plus subsites of ANG, was replaced with Ala, Leu, Phe, and Trp. Except for N694A, the mutations enhanced the initial velocity of the α-(1 -> 4)-transfer reaction to produce maltotriose, which was then degraded at a rate similar to that by wild-type ANG. With increasing reaction time, N694F and N694W mutations led to the accumulation of larger amounts of isomaltose and isomaltotriose than achieved with the wild-type enzyme. In the final stage of the reaction, the major product was panose (N694A and N694L) or isomaltose (N694F and N694W)

Engineered dextranase from Streptococcus mutans enhances the production of longer isomaltooligosaccharides

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

Increased serum malondialdehyde concentration in cows with subclinical ketosis

The purpose of this study is to compare the assessment of pre- and postpartum oxidative stress-related causal indicators and other metabolites in cows with postpartum subclinical ketosis (SCK). The prepartum serum malondialdehyde concentration and body condition score (BCS) were elevated in the SCK cows (n=17) compared to healthy controls (n=12), while the insulin sensitivity check index was lower in the SCK cows than in the controls. Oxidative stress is enhanced in cows with prepartum higher BCS, causing decreased insulin sensitivity, and may be associated with onset of postpartum SCK. However, paraoxonase alone might be insufficient to assess the antioxidant state because of no difference in pre- and postpartum activities between the two groups

Kinetic properties and substrate inhibition of α-galactosidase from Aspergillus niger

The recombinant AglB produced by Pichia pastoris exhibited substrate inhibition behavior for the hydrolysis of p-nitrophenyl -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 k(cat)/K-m values for hydrolysis of the branched -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 -galactosides, but not linear raffinose family oligosaccharides