Production of Gentiobiose from Hydrothermally Treated Aureobasidium pullulans β-1,3-1,6-Glucan

We report production of the functional disaccharide gentiobiose β-D-Glcp-(1→6)-D-Glc by a hydrolysis reaction of hydrothermally treated Aureobasidium pullulans β-1,3-1,6-glucan as the substrate and Kitalase as the enzyme. Gentiobiose was produced over the pH range 4−6 and the concentration of gentiobiose produced decreased above pH 7. The maximum value of gentiobiose production was unaffected by the enzyme concentration. The maximum concentration of gentiobiose produced was dependent on the substrate concentration whereas the maximum ratio of gentiobiose to glucose was not. The production of gentiobiose from yeast β-1,3-1,6-glucan was lower than that from A. pullulans β-1,3-1,6-glucan.     

Analysis of Transglucosylation Products of Aspergillus niger α-Glucosidase that Catalyzes the Formation of α-1,2- and α-1,3-Linked Oligosaccharides

According to whole-genome sequencing, Aspergillus niger produces multiple enzymes of glycoside hydrolases (GH) 31. Here we focus on a GH31 α-glucosidase, AgdB, from A. niger . AgdB has also previously been reported as being expressed in the yeast species, Pichia pastoris ; while the recombinant enzyme (rAgdB) has been shown to catalyze tranglycosylation via a complex mechanism. We constructed an expression system for A. niger AgdB using Aspergillus nidulans . To better elucidate the complicated mechanism employed by AgdB for transglucosylation, we also established a method to quantify glucosidic linkages in the transglucosylation products using 2D NMR spectroscopy. Results from the enzyme activity analysis indicated that the optimum temperature was 65 °C and optimum pH range was 6.0–7.0. Further, the NMR results showed that when maltose or maltopentaose served as the substrate, α-1,2-, α-1,3-, and small amount of α-1,1-β-linked oligosaccharides are present throughout the transglucosylation products of AgdB. These results suggest that AgdB is an α-glucosidase that serves as a transglucosylase capable of effectively producing oligosaccharides with α-1,2-, α-1,3-glucosidic linkages.     

A Novel α-Glucosidase of the Glycoside Hydrolase Family 31 from Aspergillus sojae

We characterized an α-glucosidase belonging to the glycoside hydrolase family 31 from Aspergillus sojae. The α-glucosidase gene was cloned using the whole genome sequence of A. sojae, and the recombinant enzyme was expressed in Aspergillus nidulans. The enzyme was purified using affinity chromatography. The enzyme showed an optimum pH of 5.5 and was stable between pH 6.0 and 10.0. The optimum temperature was approximately 55 °C. The enzyme was stable up to 50 °C, but lost its activity at 70 °C. The enzyme acted on a broad range of maltooligosaccharides and isomaltooligosaccharides, soluble starch, and dextran, and released glucose from these substrates. When maltose was used as substrate, the enzyme catalyzed transglucosylation to produce oligosaccharides consisting of α-1,6-glucosidic linkages as the major products. The transglucosylation pattern with maltopentaose was also analyzed, indicating that the enzyme mainly produced oligosaccharides with molecular weights higher than that of maltopentaose and containing continuous α-1,6-glucosidic linkages. These results demonstrate that the enzyme is a novel α-glucosidase that acts on both maltooligosaccharides and isomaltooligosaccharides, and efficiently produces oligosaccharides containing continuous α-1,6-glucosidic linkages.     

GH-16 Type β-1,3-Glucanase from Lysobacter sp. MK9-1 Enhances Antifungal Activity of GH-19 Type Chitinase,and Its Glucan-binding Domain Binds to Fungal Cell-wall

The GH-16 type β-1,3-glucanase (BgluC16MK) gene of Lysobacter sp. MK9-1 was cloned to study its antifungal activities. BgluC16MK displays amino acid sequence similarity with GluC from L. enzymogenes strain N4-7. BgluC16MK includes a signal sequence, a catalytic domain and carbohydrate-binding module family 6-type β-glucan binding domain (B-GBD). The expression of the BgluC16MK gene in Escherichia coli without the signal sequence resulted in antifungal activity at a dose of 0.6-0.8 nmol/disk. However, BgluC16MK displayed antifungal activity at a dose of 0.025 nmol/disk in combination with Chi19MK. Substrate-specific assay revealed that purified BgluC16MK hydrolyzed insoluble curdlan more readily than the soluble substrate. Furthermore, to explore the binding selectivity of B-GBD of BgluC16MK, we constructed a fusion protein (B-GBD-GFP) using the B-GBD and green fluorescent protein. The activity of the fusion protein against various substrates indicates that B-GBD was selective for glucans with β-1,3-linkages. An additional study demonstrated the binding ability of B-GBD-GFP to the cell-wall of living fungi, such as T. reesei and Aspergillus oryzae. These findings suggest that BgluC16MK can be utilized to generate antifungal enzyme preparations and that the fusion protein B-GBD-GFP can be used to identify the fungal cell surface structure using β-glucans.     

Preparation and Analysis of α-1,6 Glucan as a Slowly Digestible Carbohydrate

Carbohydrate materials that produce lower postprandial blood glucose increase are required for diabetic patients. To develop slowly digestible carbohydrates, the effect of degree of polymerization (DP) of α-1,6 glucan on its digestibility was investigated in vitro and in vivo. We prepared four fractions of α-1,6 glucan composed primarily of DP 3–9, DP 10–30, DP 31–150, and DP 151+ by fractionating a dextran hydrolysate. An in vitro experiment using digestive enzymes showed that the glucose productions of DP 3–9, DP 10–30, DP 31–150, and DP 151+ were 70.3, 53.4, 28.2, and 19.2 % in 2 h, and 92.1, 83.9, 39.6, and 33.3 % in 24 h relative to dextrin, respectively. An in vivo glycemic response showed that the incremental area under the curve (iAUC) of blood glucose levels of α-1,6 glucan with DP 3–9, DP 10–30, DP 31–150, and DP 151+ were 99.5, 84.3, 65.4, and 40.1 % relative to dextrin, respectively. These results indicated that α-1,6 glucan with higher DP had stronger resistance to digestion and produced a smaller blood glucose response. DP 10–30 showed significantly lower maximum blood glucose levels than dextrin; however, no significant difference was observed in iAUC, indicating that DP 10–30 was slowly digestible. In addition, α-1,6 glucan was also produced using an enzymatic reaction with dextrin dextranase (DDase). This produced similar results to DP 10–30. The DDase product can be synthesized from dextrin at low cost. This glucan is expected to be useful as a slowly digestible carbohydrate source.     

Production of α‐amylase by Aspergillus oryzae As 3951 in solid state fermentation using spent brewing grains as substrate

BACKGROUND: The optimisation of nutrient levels for the production of α‐amylase by Aspergillus oryzae As 3951 in solid state fermentation (SSF) with spent brewing grains (SBG), an inexpensive substrate and solid support, was carried out using response surface methodology (RSM) based on Plackett–Burman design (PBD) and Box–Behnken design (BBD). RESULTS: In the first optimisation step a PBD was used to evaluate the influences of related factors. Corn steep liquor, CaCl₂ and MgSO₄ were found to be the most compatible supplements to the substrate of SBG and influenced α‐amylase activity positively. In the second step the concentrations of these three nutrients were optimised using a BBD. The final concentrations (g/g dry substrate basis) in the medium optimised with RSM were 1.8% corn steep liquor, 0.22% CaCl₂ and 0.2% MgSO₄ · 7H₂O using SBG as the solid substrate. The average α‐amylase activity reached 6186 U g^?1 dry substrate under the optimised conditions at 30 °C after 96 h. Under the optimised conditions of SSF an approximately 17.5% increase in enzyme yield was observed. CONCLUSION: SBG was found to be a good substrate for the production of α‐amylase by A. oryzae As 3951 under SSF. Copyright © 2007 Society of Chemical Industry     

Purification,Characterization, and cDNA Cloning of a Prominent β-Glucosidase from the Gut of the Xylophagous Cockroach Panesthia angustipennis spadica

Abstract: In this study, a β-glucosidase (PaBG1b) with high specific activity was purified from gut extracts of the wood-feeding cockroach Panesthia angustipennis spadica using Superdex 75 gel filtration chromatography and High-Trap phenyl hydrophobic chromatography. The protein was purified 14-fold to a single band identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with an apparent molecular mass of 56.7 kDa. The specific activity of the purified enzyme was 708 μmol/min/mg protein using cellobiose as substrate. To the best of our knowledge, this is the highest specific activity reported among β-glucosidases to date. The purified PaBG1b showed optimal activity at pH 5.0 and retained more than 65 % of the activity between pH 4.0 and 6.5. The activity was stable up to 50 °C for 30 min. Kinetic studies on cellobiose revealed that the K_m was 5.3 mM, and the V_max was 1,020 μmol/min/mg. The internal amino acid sequence of PaBG1b was analyzed, and two continuous sequences (a total of 39 amino acids) of the C-terminal region were elucidated. Based on these amino acid sequences, a full-length cDNA (1,552 bp) encoding 502 amino acids was isolated. The encoded protein showed high similarity to β-glucosidases from glycoside hydrolase family 1. Thus, the current study demonstrated the potential of PaBG1b for application in enzymatic biomass-conversion as a donor gene for heterologous recombination of cellulase-producing agents (fungi or bacteria) or an additive enzyme for cellulase products based on the high-performance of PaBG1b as a digestive enzyme in cockroaches.