Starches and their amyloses and amylopectins were prepared from three domestic wheat cultivars (Haruyutaka, Chihokukomugi and Norin No. 61) produced in Hokkaido and from a foreign cultivar, 1CW, imported from Canada. The results of Rapid Visco Analyser and differential scanning calorimeter (DSC) showed that the patterns of gelatinization and retrogradation of these starches were different, although the amylose content of the domestic cultivars (21.0–22.2%) and 1CW (24.0%) were similar. The outer branches of the molecules of wheat amylopectins were obtained by partial hydrolysis with pullulanase and analyzed by high performance anion exchange chromatography with pulsed amperometric detection. When the degree of polymerization (DP) ranged from 9 to 11, the ratio of glucosyl chains to the total of partial hydrolysates from amylopectins was lower for all the domestic cultivars than that for 1CW, but higher than those of 1CW when DP ≥12. Subtotal of the ratio of the hydrolysates from domestic wheat amylopectins, revealed as Fraction A (Fr. A) (DP 6–12) and Fr. B1 (DP 13–24), were different from that of 1CW. A negative correlation was observed between Fr. B1 and Tp (peak temperature in DSC), whereas a positive correlation was found between Fr. A and Tp. The retrogradation properties of wheat amylopectins tended to be different, although their starches tended to have similar properties. These results suggest that small differences in outer glucosyl chain-lengths of the amylopectin molecules have a very slight influence on gelatinization, but exert a somewhat greater influence on retrogradation of wheat amylopectins and their starches.
A series of β-(1→4)-thiooligosaccharide analogs, O-β-D-glucopyranosyl-(1→4)-S-β-D- glucopyranosyl-(1→4)-4-deoxy-4-thio-D-glucopyranose (1: Glc-O-Glc-S-Glc), S-β-D-glucopyranosyl- (1→4)-O-(4-deoxy-4-thio-β-D-glucopyranosyl)-(1→4)-D-glucopyranose (2: Glc-S-Glc-O-Glc), S-β-D-glucopyranosyl-(1→4)-4-deoxy-4-thio-D-glucopyranose (3: Glc-S-Glc), O-β-D-galactopyranosyl-(1→4)-S-β-D-glucopyranosyl-(1→4)-4-deoxy-4-thio-D-glucopyranose (4: Gal-O-Glc-S-Glc) and O-β-D-glucopyranosyl-(1→4)-S-β-D-glucopyranosyl-(1→4)-O- (4-deoxy-4-thio-β-D-glucopyranosyl)-(1→4)-D-glucopyranose (5: Glc-O-Glc-S-Glc-O-Glc), including novel compounds were synthesized for the substrates and/or the inhibitors of cellobiohydrolases for the evaluation of cellulolytic activities. The triflated acceptors were constructed in two reaction steps, regioselective benzoylation and triflation. After S-glycosylation of these triflated acceptors, acyl protecting group was deprotected to yield target compounds. In this way, all target compounds were successfully synthesized in short-step (four reaction steps).
Physicochemical and structural properties of starch isolated from tuberous roots of apios (Apios americana Medikus) were examined and these features were compared with those from arrowroot starch, potato starch and normal maize starch. The results obtained from the examination of apios starch were similar to those of arrowroot starch in X-ray diffraction pattern, solubility and swelling power, their behavior in the RVA, the susceptibility of starch granules to pancreatin digestion and the FE-SEM appearance as the digestion proceeded. The starch granules from apios showed Ca type in the X-ray diffractogram and had significantly lower swelling power at 70 and 80 °C than starch granules from potato. Apios starch granules had a rapid increase in solubility from 70 to 80 °C, and at 80 °C, it was higher than those of potato and normal maize starch granules. The susceptibility of apios starch granules to pancreatin digestion was higher than that of potato starch granules and lower than that of normal maize starch granules. The elution patterns of isoamylase debranched materials of the apios starch granules were similar to arrowroot and normal maize starch granules. The amount of amylose present in the apios starch granules (25.7%) was similar to the levels found in potato starch granules. Digestion of the granules was monitored by FE-SEM and varied between the different plant species. The differences in the structure of the starches lead to different susceptibilities to pancreatin digestion and the observed changes in the structural appearance of the granules as the digestion proceeds.
A colloidal particle rejection and recovery system using extended and shrunken dextran was proposed. Dextransucrase forms a complex with dextran in an enzymatic reaction with sucrose; therefore dextran is easily formed on membrane pores. A dextransucrase solution was permeated through a Shirasu porous glass membrane at various permeation rates. A sucrose solution was permeated to generate dextran from the active sites of DSase immobilized on the membrane pores, at an immobilized density of 1.9-23 U/g, by controlling the dextran density; the immobilized density of DSase was high when immobilization was performed at a low permeation rate, resulting in a high generated-dextran density of 9.6-28 mg/g. A colloidal particle solution was permeated through the membrane for rejection and recovery. The high-density dextran rejected colloidal particles efficiently. In 50% methanol solution, the rejected colloidal particles leaked through the shrunken dextran structure to achieve smart recovery of colloidal particles.
Stereum purpureum endopolygalacturonase (EndoPG) IV was expressed in Aspergillus oryzae. Recombinant forms of EndoPG IV (rEndoPGs IV0, IV1, and IV2) were purified from the culture filtrate by ammonium sulfate precipitation and two-step column chromatography with DE52 and hydroxyapatite. Each of these three recombinants was shown to be homogeneous by SDS-PAGE. Since it was predicted that the differences between them would be based on the number of sugar chains, the changes in molecular mass after enzymatic cleavage of the N-linked oligosaccharide with EndoH were determined by MALDI-TOF MS. From the data, the numbers of N-linked oligosaccharides were estimated to be 0, 1, and 2 for rEndoPG IV0, IV1, and IV2, respectively. These sugar chains appeared to be a mixture of GlcNAc2Hexose5-10 and to include galactose as well as mannose. Mass analyses of IV0, IV1 and IV2 after deglycosylation with EndoH demonstrated a hexose carbohydrate, suggesting the presence of an O-linked oligosaccharide that is lacking in native EndoPG IVs. The thermal denaturation curves of rEndoPG IV0 and IV1 based on CD analysis indicated Tm values of 60 and 59 °C, with a one-step thermal denaturation curve, similar to that of native EndoPG IV.
D-xylose isomerase was cloned and characterized from a newly isolated actinobacteria strain, Thermobifisa fusca MBL 10003. There was only one base difference between the xylose isomerase genes from T. fusca XY, a cellulosic bacteria, and T. fusca MBL10003. The structural gene (xylA) is predicted to encode a polypeptide of 387 amino acids with an estimated molecular mass of 43,900. The deduced amino acid sequence of the gene showed high identity with homologous enzymes from the species of Streptomyces, Actinoplanes and Arthrobacter. The optimal temperature and pH were 75°C and 10, respectively. The enzyme was stable between 70 and 75°C, and between pH 4 and 12. Unlike other known xylose isomerases, the T. fusca enzyme had very similar Km values for xylose and glucose, 264 and 274 mM, respectively. In contrast, kcat and, therefore, kcat/Km for xylose was approximately 18-fold larger than that for glucose. It required divalent metal ions for activity. Of these, Mg2+ was the most effective activator, and Co2+ functioned as a co-activator of Mg2+.
Recombinant class V chitinases from Nicotiana tabacum and Arabidopsis thaliana (NtChiV and AtChiC) were produced by the Escherichia coli expression system, and the antifungal activity of the enzymes was investigated using the hyphal extension inhibition assay on agar plates with Trichoderma viride as the test fungus. The activity of NtChiV was found to be much higher than that of AtChiC. The inactive mutants of both enzymes, in which the individual catalytic acids were mutated to glutamine, were also tested by the same assay system. The activity was impaired by the mutation, indicating that the hydrolytic activity contributes to the antifungal action of the enzymes. However, the activity of the enzymes toward glycol chitin substrate was not proportional to the antifungal activity, indicating that the hydrolytic activity does not exclusively contribute to the antifungal action. X-ray crystal structures of these enzymes revealed that the aglycon-binding region of NtChiV consists of a number of polar side chains but not in AtChiC. Polarity of the surface of substrate-binding cleft could be another factor controlling the antifungal action of class V chitinases.
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