Weeranuch Lang

Biosorption of nonylphenol on dead biomass of Rhizopus arrhizus encapsulated in chitosan beads

The nonylphenol (NP) biosorption and desorption potential for fungal biomass used under batch conditions was investigated using kinetics and isotherm models. Fungal biomass of Rhizopus arrhizus TISTR 3610 exhibited preferential uptake of NP, an endocrine disrupting chemicals. Sporangiospores, asexual spores, were immobilised in chitosan beads. The biosorption data of NP on the moist heat inactivated R. arrhizus-chitosan beads were analyzed using four popular adsorption isotherms and, by using non-linear least-regression with the solver add-in in Microsoft Excel, correlated in order with the Fritz-Schluender>Redlich-Peterson>Freundlich>Langmuir isotherms. The pseudo first-order kinetics was found to have the best fit with the experimental data. The diffusivity of NP in the R. arrhizus-chitosan beads was calculated using the shrinking core model, and the diffusivity values were in the ranges of 2.3736x10(-4)-1.8950x10(-4) cm(2) s(-1). Desorption to recover the adsorbed NP from the beads was performed in methanol and was best described using a pseudo second-order kinetic model.

Characterization of a new oxygen-insensitive azoreductase from Brevibacillus laterosporus TISTR1911: Toward dye decolorization using a packed-bed metal affinity reactor

This study reports the identification of a new bacterial azoreductase from Brevibacillus laterosporus TISTR1911, its heterologous production in Escherichia coli, the biochemical characterization and immobilization for use in dye biodegradation processes. The recombinant azoreductase (BrAzo) is a monomeric FMN oxygen-insensitive enzyme with a molecular mass of 23 kDa showing a broad specificity for the reduction of synthetic azo dyes. Double hexahistidine-tagged BrAzo was immobilized onto a nickel chelating column and methyl orange was used to assess its degradation potential using a packed-bed reactor. The dye degradation is described by an exponential model in a downstream batchwise continuous flow mode operated with recycling. The complete degradation of methyl orange (170 μM at 600 mL/h) was achieved in 3 h and continued over 9 cycles. Coupling the immobilized BrAzo with glucose dehydrogenase for NADH regeneration yielded a shorter 1.5 h-degradation period that was maintained throughout 16 cycles.

A novel mechanism for the promotion of quercetin glycoside absorption by megalo α-1,6-glucosaccharide in the rat small intestine

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.

Novel dextranase catalyzing cycloisomaltooligosaccharide-formation and identification of catalytic amino acids and their functions using chemical rescue approach

Background: Catalytic residues and molecular mechanism of GH-66 enzymes were hitherto unknown. Results: Novel dextranase produced isomaltotetraose and cyclo-isomaltosaccharides. Its nucleophile (Asp340) and acid/base catalyst (Glu412) were identified by a chemical rescue approach. Conclusion: Three GH-66 enzyme types were newly classified for the first time. Significance: This work elucidates production of isomaltotetraose and cycloisomaltosaccharides, classification of GH-66, identification of catalytic residues, and novel dextran-forming type chemical rescue. A novel endodextranase from Paenibacillus sp. (Paenibacillus sp. dextranase; PsDex) was found to mainly produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides (CIs) with a degree of polymerization of 7–14 from dextran. The 1,696-amino acid sequence belonging to the glycosyl hydrolase family 66 (GH-66) has a long insertion (632 residues; Thr451–Val1082), a portion of which shares identity (35% at Ala39–Ser1304 of PsDex) with Pro32–Ala755 of CI glucanotransferase (CITase), a GH-66 enzyme that catalyzes the formation of CIs from dextran. This homologous sequence (Val837–Met932 for PsDex and Tyr404–Tyr492 for CITase), similar to carbohydrate-binding module 35, was not found in other endodextranases (Dexs) devoid of CITase activity. These results support the classification of GH-66 enzymes into three types: (i) Dex showing only dextranolytic activity, (ii) Dex catalyzing hydrolysis with low cyclization activity, and (iii) CITase showing CI-forming activity with low dextranolytic activity. The fact that a C-terminal truncated enzyme (having Ala39–Ser1304) has 50% wild-type PsDex activity indicates that the C-terminal 392 residues are not involved in hydrolysis. GH-66 enzymes possess four conserved acidic residues (Asp189, Asp340, Glu412, and Asp1254 of PsDex) of catalytic candidates. Their amide mutants decreased activity (11,500 to 140,000 times), and D1254N had 36% activity. A chemical rescue approach was applied to D189A, D340G, and E412Q using α-isomaltotetraosyl fluoride with NaN3. D340G or E412Q formed a β- or α-isomaltotetraosyl azide, respectively, strongly indicating Asp340 and Glu412 as a nucleophile and acid/base catalyst, respectively. Interestingly, D189A synthesized small sized dextran from α-isomaltotetraosyl fluoride in the presence of NaN3.

The Delay in the Development of Experimental Colitis from Isomaltosyloligosaccharides in Rats Is Dependent on the Degree of Polymerization

Background Isomaltosyloligosaccharides (IMO) and dextran (Dex) are hardly digestible in the small intestine and thus influence the luminal environment and affect the maintenance of health. There is wide variation in the degree of polymerization (DP) in Dex and IMO (short-sized IMO, S-IMO; long-sized IMO, L-IMO), and the physiological influence of these compounds may be dependent on their DP. Methodology/Principal Findings Five-week-old male Wistar rats were given a semi-purified diet with or without 30 g/kg diet of the S-IMO (DP = 3.3), L-IMO (DP = 8.4), or Dex (DP = 1230) for two weeks. Dextran sulfate sodium (DSS) was administered to the rats for one week to induce experimental colitis. We evaluated the clinical symptoms during the DSS treatment period by scoring the body weight loss, stool consistency, and rectal bleeding. The development of colitis induced by DSS was delayed in the rats fed S-IMO and Dex diets. The DSS treatment promoted an accumulation of neutrophils in the colonic mucosa in the rats fed the control, S-IMO, and L-IMO diets, as assessed by a measurement of myeloperoxidase (MPO) activity. In contrast, no increase in MPO activity was observed in the Dex-diet-fed rats even with DSS treatment. Immune cell populations in peripheral blood were also modified by the DP of ingested saccharides. Dietary S-IMO increased the concentration of n-butyric acid in the cecal contents and the levels of glucagon-like peptide-2 in the colonic mucosa. Conclusion/Significance Our study provided evidence that the physiological effects of α-glucosaccharides on colitis depend on their DP, linkage type, and digestibility.

Preparation and characterization of polymeric host molecules, β-cyclodextrin linked chitosan derivatives having different linkers

Reductive alkylation of the amino group of chitosan with β-cyclodextrin (CD) aldehyde derivatives, i.e., 6-deoxy-6-(4-oxobutyramido)-β-CD and 6-oxo-β-CD, gave two β-CD-linked chitosan derivatives with C4 (4-butylamido) and C0 linking arms, respectively. Degree of substitution (D.S.) of both C4-β-CD and C0-β-CD linked chitosan was controlled by the ratio of starting materials. The structures of the products were confirmed by (1)H and (13)C NMR and FT-IR spectra. Their inclusion properties of C4-β-CD (D.S. 18%) and C0-β-CD linked chitosan (D.S. 17%) with a fluorescent probe, 6-(p-toluidino)-2-napthalene-6-sulfonate (TNS) were investigated in acetate buffer (pH 4.3) at 25°C. Continuous variation of Jobs method revealed that the stoichiometry of inclusion complex of C4-β-CD linked chitosan-TNS was 1:1, whereas that of C0-β-CD linked chitosan was not 1:1. The stability constant of C4-β-CD linked chitosan determined by Benesi-Hildebrand plot was 2.3×10(3)M(-1). These results suggested that length of the linking arms between CD and chitosan is influenced on their inclusion property.

Different molecular complexity of linear-isomaltomegalosaccharides and β-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 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.