A fructan:fructan 1-fructosyltransferase (1-FFT) was purified from edible burdock for the first time, and a cDNA encoding 1-FFT was isolated from the plant. Two 1-FFTs named 1-FFTa and 1-FFTb were purified from extract of edible burdock by ammonium sulfate precipitation, followed by chromatographies on DEAE-Sepharose CL-6B, Toyopearl HW-55S and Sephadex G-100 columns. Inulin-type fructan such as nystose and fructooligosaccharides with higher DP were produced from 1-kestose by purified 1-FFTs. The general properties of both purified enzymes were very similar to each other. The purified 1-FFTa and 1-FFTb showed a single band by native PAGE and two bands, relative molecular masses of about 46,000 and 17,500 for 1-FFTa and 46,000 and 17,000 for 1-FFTb, by SDS-PAGE, respectively. The N-terminal sequences of the 46,000 peptides of both enzymes were the same, and those of the 17,500 and the 17,000 peptides were also identical. Based on the sequences, the 1-FFT cDNA named alft1 was cloned. The alft1 encoded a polypeptide of 617 amino acids. The relative molecular mass and pI of the mature protein region of deduced polypeptide were calculated to be 60,213 and 4.89, respectively. The characteristics of recombinant protein produced by Pichia pastoris closely resembled those of the native 1-FFTs purified from edible burdock. In this study, we demonstrated that alft1 encoding edible burdock 1-FFT was involved in the elongation of fructosyl chains of fructooligosaccharides in edible burdock.
The particular effect of vegetable sera having different charged free amino acid contents prepared from tomato, carrot and red bell pepper on the gelatinization and retrogradation behavior of potato starch was analyzed from the results evaluated by differential scanning calorimetry, rapid viscoanalysis and transparency. The gelatinization temperature (GT) of potato starch increased with increasing added amounts of each vegetable serum, and this increased GT was almost completely dependent on the charged amino acid content of the vegetable serum. A small addition of each vegetable serum markedly reduced the peak viscosity and breakdown, and increased the pasting temperature due to the inhibition of swelling. Adding vegetable sera resulted in increased setback and turbidness, and in reduced enthalpy change for re-gelatinization to about 50-60% of that of the control without any vegetable serum, suggesting that the vegetable sera reduced reconstitution of the ordered structure and caused subsequent entanglement and aggregation of the dispersed starch chains during cooling.
An endoglucanase gene of Paenibacillus sp. strain KSM-N546 was cloned and sequenced. The gene for the endoglucanase (Egl-546H) had an open reading frame of 1710 bp that encoded a putative signal sequence (37 amino acids) and mature enzyme (533 amino acids: 61,348 Da). The mature enzyme was most homologous to a cellulase of Geobacillus sp. Y412MC10 with 68% identity. The optimal pH and temperature of the recombinant enzyme for degrading carboxymethyl cellulose (CMC) were around pH 5.3 and 40°C, respectively. The enzyme efficiently degraded CMC and lichenan as well as glucomannan. It was crystallized with a rod shape by a hanging drop vapor-diffusion method. Egl-546H is comprised of two conserved domains, a glycosyl hydrolase family 5 domain and a DUF291 domain of unknown function. Several mutant proteins deleted of a DUF291-like domain were produced by Escherichia coli as inclusion bodies with no enzymatic activity.
Two endopolygalacturonases (EndoPG IVa and IVb) were purified from the culture filtrate of Stereum purpureum (Chondrostereum purpureum) , the causative fungus of apple silver-leaf disease, by three steps of column chromatography. Respective molecular masses were determined at 36,017 and 37,266 Da using ESI-MS. Deglycosylation with endo-β-N-acetylglucosaminidase reduced molecular masses to 34,987 and 35,202 Da, reductions corresponding to the loss of one and two high-mannose type M5 sugar chains, respectively. From this result, EndoPG IVa and IVb are assumed to share a common primary structure. Their thermal stabilities were up to 55℃, considerably lower than that of EndoPG Ia (up to 70℃). Melting temperature (Tm) values for EndoPG IVb and EndoPG Ia were 62 and 79.5℃ respectively. Specific activities and Km values measured for EndoPG IVa and IVb were almost identical to those of EndoPG Is. Amino acid sequences were deduced by 3′ and 5′ RACEs cloning of cDNA based on both EndoPG IVs sharing an identical N-terminus (Accession No. AB252456). No large deletion of C-terminal amino acid residues (44 residues) was observed in EndoPG IV, as has been reported for mature EndoPG I. Amino acid sequence homology of EndoPG IV with EndoPG I was found to be 72%.
Ethanol fermentation from starch by a mushroom, Flammulina velutipes NBRC33210, was investigated. When the mycelia were anaerobically incubated with 5% of starch, only a small amount of ethanol was produced (less than 6% of the theoretical ethanol recovery rate). Aerobic culture was effective to produce amylolytic enzymes from F. velutipes and a significant amount of ethanol was produced (32.4% of the theoretical ethanol recovery rate) when the mycelia were anaerobically incubated with 5% of starch after 7 days of aerobic seed culture. Addition of α-amylase into the culture did not influence ethanol fermentation from starch, however, α-glucosidase and glucoamylase were effective for the ethanol fermentation by F. velutipes NBRC33210. The maximum ethanol conversion rate for the starch was 72.6 % when 0.1 U/mL of glucoamylase was added to the culture.
The chitinolytic bacterial strain SN184, which produces chitin oligosaccharide deacetylase (COD), was identified a species of the genus Vibrio. The COD gene was cloned from the chromosomal DNA of SN184 and its nucleotide sequence was analyzed. The gene encoded a 427 amino acid protein, including the 22 amino acid signal sequence. The deduced amino acid sequence of this protein showed high similarity to those of several bacterial CODs belonging to carbohydrate esterase family 4. An expression plasmid containing the COD gene of strain SN184 was constructed and inserted into Escherichia coli cells; the recombinant enzyme was secreted into the culture medium with the aid of the signal sequence. The recombinant enzyme was purified from the culture supernatant and its substrate specificity was investigated. These studies confirmed that the recombinant COD hydrolyzed N-acetyl groups of the second N-acetyl-D-glucosamine residue from the nonreducing-ends of di-N-acetylchitobiose and tri-N-acetylchitotriose.
β-Glucuronidase from Aspergillus niger, which showed transglycosylation activity, was purified from a commercial enzyme preparation. Upon addition of the previously reported acceptors such as D-glucose, the enzyme transferred the glucuronyl residues to 2-deoxy-, 3-deoxy-D-glucose, 2-deoxy-D-galactose, D-galactosamine and D-glucosamine. D-Allose, D-gulose and D-mannosamine were not good acceptors. The enzyme produced three products with D-glucose by transglycosylation, and the structure of the main product was β-D-glucosyluronic acid-(1→4)-D-glucose. Furthermore β-D-glucosyluronic acid-(1→2)-D-glucuronic acid and β-D-glucosyluronic acid-(1→4)-D-glucose were hydrolyzed by the enzyme. The enzyme did not degrade methyl β-glucosiduronic acid or α, β-trehalose dicarboxylate having β-(1→4) glucuronyl linkage.
To manufacture low-malt beer using potato starch, we selected the potato starch suited for low-malt beer brewing from several potato starches. Moreover, the componential and functional characteristics of the low-malt beer obtained thus were evaluated. As extremely fine potato starch obtained by air-classification (median size 13.6 μm) had high phosphorus content but low peak viscosity of Rapid Visco Analyser (RVA), we regarded it as the most suitable material for low-malt beer brewing of the 10 potato starches examined. Phosphoryl-oligosaccharides, which are obtainable after digestion of a starch with phosphate, were obviously found (600 ppm) in low-malt beer using such potato starch. In contrast, no or very little phosphoryl-oligosaccharide was found in low-malt beers using corn or sweet potato starches. Thus, novel type functional low-malt beer would be manufactured by using extremely fine potato starch.
Ten cultivars of potatoes called tasting potatoes (Andes Red, Inkanomezame, Sherry, Jaga Kids Purple, Cynthia, Tawara Murasaki, Destroyer, Tokachi Kogane, Violetta and Yodel) that were collected from different areas of the world were cultivated in Aomori to be adapted to a Japanese climate. After harvest, the potatoes were stored for 2 and 7 months at 2°C. This study examined physicochemical properties of starches isolated from these potatoes and investigated the effects of the low temperature storage. The starch properties determined in this study were particle size distribution, X-ray diffractography, pasting properties using a Rapid Visco Analyzer (RVA), thermal properties using differential scanning calorimetry, the contents of phosphorus and amylose, swelling power, solubility, digestibility by pancreatin and observation using scanning electron microscopy. Starches from tasting potatoes showed type B X-ray diffraction patterns, and the patterns were not affected by increasing the duration of the storage period under low temperature. Setback by RVA, digestibility by pancreatin and the contents of amylose tended to increase during the storage. But the other physicochemical properties changed variously and inconsistently with the cultivars or the places of origin.
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