The enzyme cycloisomaltooligosaccharide glucanotransferase (CITase) was isolated from Bacillus circulans U-155, which had a yield of 25.8%, and purified to homogeneity. The Mr was approximately 98,000, similar to that for all known CITases. Specific activity of the purified enzyme was 2.11 U/mg protein, with maximal activity at approximately pH 6.0. The enzyme was stable at pH 4.5 up to pH 9.0 and at temperatures up to 50°C. The main product of the initial enzyme reaction was cycloisomalto-heptaose. The cit gene has a 2,895-bp open reading frame and encodes CITase in B. circulans U-155. We cloned this gene into a recombinant plasmid pCI811 and expressed it in Escherichia coli. A comparison between DNA sequence data from the transformant and the N-terminal amino acid sequence of the purified enzyme from B. circulans U-155 suggested that CITase was translated as a secretory precursor with a 30-amino-acid signal peptide. The mature enzyme contained 934 residues with a predicted molecular mass of 103.93 kDa. The enzyme activity in the transformants was approximately 3.0 mU/mL, similar to that of the purified enzyme secreted by B. circulans U-155. The enzyme also showed 72 and 67% identity with CITase from Paenibacillus sp. 598K and B. circulans T-3040, respectively. These results suggest that the enzyme isolated from B. circulans U-155 shares a greater similarity with CITase expressed by Paenibacillus sp. 598K than B. circulans T-3040.
We have previously observed that the Super Ohtaka®, produced by fermenting extracts from 50 types of fruits and vegetables, contained the disaccharide, α-D-fructofuranosyl-(2→6)-D-glucose (α-Ff2→6G), which was produced during the fermentation process. α-Ff2→6G was also formed from equal amounts of D-glucose and D-fructose under melting conditions at 130°C for 45 min or at 140°C for 30 min. This disaccharide was isolated from the reaction mixture by carbon-Celite column chromatography and preparative- high performance liquid chromatography. It was confirmed to be α-Ff2→6G by matrix-assisted laser desorption ionization/time of flight mass spectrometry analysis and nuclear magnetic resonance measurements. The characteristics of α-Ff2→6G were investigated. The saccharide showed low digestibility and was 0.25 times as sweet as sucrose. Furthermore, unfavorable bacteria such as Enterobacter cloacae 1180, Escherichia coli 1099 and Clostridium perfringens 1211 that produce mutagenic substances did not break down the synthetic oligosaccharide.
The water-insoluble fraction of Zea mays L. hybrid B73×Mo17 shoot cell walls, pretreated with purified Bacillus subtilis (1→3), (1→4)-β-D-glucan 4-glucanohydrolase and purified B. subtilis endo-(1→4)-β-D-xylanase, was subsequently treated with a glucuronoxylan xylanohydrolase preparation, all of which were obtained from a commercially available B. subtilis α-amylase (Novo Ban 120). Carbohydrates (about 16% of the original water-insoluble fraction of Zea shoot cell-walls) derived from the enzyme treatment contained significant amounts of galactan and (1→4)-β-D-galactobiose in addition to glucuronoarabinoxylan and neutral sugar residues-containing rhamnogalacturonan fragments. Methylation analysis and partial acid-hydrolysis of the isolated galactan followed by analysis of the hydrolyzate showed that the galactan consisted of about 14 (1→4)-β-consecutively linked galactose moieties.
A cross-linked (CL) collagen peptide (CP)-potato starch (PS) compound (CL-CP-PS) was prepared by autoclaving PS and CP and subsequently cross-linking with a microbial transglutaminase (MTGase). CP-compounded PS (CP-PS) was prepared by autoclaving a mixture of PS and CP at 120°C for 120 min. After suspending in an MTGase solution, CP-PS was cross-linked with MTGase at room temperature for 24 h while shaking. The reaction product was washed three times with distilled water, and then air-dried to obtain CL-CP-PS. CL-CP-PS showed a clear polarized image almost the same as that of PS, and had a 0.7% CP content. The median diameter of CL-CP-PS was significantly larger than that of CP-PS or of PS, suggesting the formation of multiple granules through cross-linking among the compounded CP moieties. CL-CP-PS exhibited a grater thermal structural stability, lower swelling index and solubility, as well as higher heat resistance for maintaining the swollen starch granules at 120°C for 20 min than those of CP-PS and PS. Cross-linking of CP-PS with MTGase should thus be valuable for providing a starch material having high rigidity, low swelling index and solubility, and enhanced heat resistance.
A simple turbidimetric assay using chitin nanofiber as the substrate was employed to measure chitinase activity. The higher dispersive properties of chitin nanofibers enabled the degradation of chitin to be monitored turbidimetrically. When non-processive chitinases, a family GH18 chitinase from the tobacco plant and a GH19 chitinase from rye seeds, were added to the β-chitin nanofiber suspension, no significant changes were observed in the turbidity of the suspension, however, the amounts of reducing sugars produced were significantly high and small amounts of GlcNAc and (GlcNAc)2 were detected by HPLC in the reaction mixture. However, the addition of a processive family GH18 chitinase, Serratia marcescence chitinase B or chitinase from Autographa californica multiple nucleopolyhedrovirus, resulted in a significant decrease in the turbidity of the chitin nanofiber suspension, and produced larger amounts of reducing sugars including GlcNAc and (GlcNAc)2. The rate of decreases in turbidity was clearly dependent upon the enzyme concentration. We concluded that the turbidimetric assay using β-chitin nanofibers as the substrate was useful for measuring the activities of processive chitinases.
Spectrophotometric quantification method of carbohydrates is useful for processing multiple samples. In this study, we established colorimetric quantification for 4-O-β-D-mannosyl-D-glucose (Man-Glc) and β-(1→4)-mannobiose (Man2). For quantification of Man-Glc, phosphorolysis of Man-Glc catalyzed by 4-O-β-D-mannosyl-D-glucose phosphorylase (MGP) was coupled with quantification of D-glucose by the glucose oxidase-peroxidase method. In addition to MGP, cellobiose 2-epimerase (CE) was added for quantification of Man2. In both quantifications, a good linear relationship was obtained between A505 and the sample concentration (0-0.5 mM). The A505 values obtained at various concentrations of Man2 and Man-Glc were almost identical to those with equivalent D-glucose concentrations. Kinetic parameters of Ruminococcus albus and Rhodothermus marinus CEs for the epimerization of Man2 were determined using the quantification method for Man-Glc. Both enzymes showed 5-15-fold higher kcat/Km values than those for cellobiose and lactose, which supports the prediction that these enzymes utilize Man2 as a substrate in the β-mannan metabolic pathway.