Katsuichi Saito

Difructose Anhydrides: Their Mass-Production and Physiological Functions

Difructose anhydrides (DFAs) are the smallest cyclic disaccharides consisting of two fructose residues, and are expected to have novel physiological functions from their unique structures and properties. For mass-production of α-D-fructofuranose-β-D- fructofuranose-2′,1:2,3′-dianhydride (DFA III) and β-D-fructofuranose-β-D-fructofuranose-2′,6:2,6′-dianhydride (DFA IV), Arthrobacter sp. H65-7 and A. nicotinovorans GS-9 were selected as the best producers of inulase II, which produced DFA III from inulin and LFTase, which produced DFA IV from levan. The enzymes were purified and their genes were subsequently cloned and expressed in E. coli at higher levels than in the original bacteria. Thus, it became possible to provide a large amount of DFA III and DFA IV for evaluating their physiological properties. DFA III and DFA IV have half the sweetness of sucrose, but cannot be digested by the digestive system of rats. Their use by the intestinal microorganisms was observed in vivo even though their assimilation could not be detected in vitro. This implied that they were degraded by an unknown system in the intestine. It was also found that they affected calcium absorption mainly in the small intestine through mechanisms different from the known stimulants such as fructooligosaccharides and raffinose.

Lactic acid fermentation of potato pulp by the fungus Rhizopus oryzae

Thirty-eight strains of the fungus Rhizopus oryzae were grown on potato pulp, an agricultural by-product of the starch industry. Either lactic acid or fumaric acid and ethanol were formed, and the ratio differed among the strains tested. The highest amount of L(+)-lactic acid (10 mg/g fresh matter) was observed in the pulp fermented for six days by Rhizopus oryzae IFO 4707. The IFO 4707 strain rapidly reduced the hardness and pH of potato pulp within one day followed by the gradual synthesis of lactic acid. A composition analysis showed that the enzymes secreted from the fungal cells hydrolyzed starch efficiently with partial degradation of the cell wall. Rhizopus oryzae may be used as an inoculant for ensiling potato pulp and other agricultural by-products containing starch.

Endophytes as Producers of Xylanase

One hundred and sixty-nine endophytic fungi and 81 endophytic bacteria were isolated from 14 plants in total. Among them, 155 fungi (91.7%) and 52 bacteria (64%) were found to produce xylanase. The inside part of plants is a novel and good source for isolating xylanase producers in comparison with soil.

rDNA ITS sequence of Rhizopus oryzae: its application to classification and identification of lactic acid producers.

Rhizopus oryzae is an important organism for its production of organic acids such as lactic acid, fumaric acid, etc. To date, there were no easy methods to classify strains according to their acid production. The sequences of the ribosomal RNA-encoding DNA (rDNA) internal transcribed spacer (ITS) region of 64 strains of R. oryzae were analyzed and found to conserve mutations correspond to acid production. We have devised a way to use these mutations for a novel method to identify lactic-acid-producing Rhizopus oryzae, by designing specific polymerase chain reaction (PCR) primers on them. Touch down PCR using these primers amplified the ITS DNA of lactic acid producers specifically. By this method, we could isolate lactic acid producing strains from Indonesian fermented foods.

Genetic diversity in Rhizopus oryzae strains as revealed by the sequence of lactate dehydrogenase genes

Twenty-seven strains of Rhizopus oryzae accumulating predominantly lactic acid were shown to possess two ldh genes, ldhA and ldhB, encoding NAD-dependent lactate dehydrogenases. Variation in nucleotide sequence was identified for each gene from different strains, and similar phylogenetic trees were obtained based on the nucleotide sequences of both genes. The other 21 strains of R. oryzae accumulating predominantly fumaric and malic acids contained a single ORF of ldhB. Compared to the strains accumulating predominantly lactic acid, a lower degree of sequence divergence was found in ldhB, resulting in a separate cluster in the phylogenetic tree. The high similarity (>90%) spanning the ORF and adjacent regions demonstrates that ldhA and ldhB are derived from the same ancestor gene. The strains accumulating predominantly fumaric and malic acids lack functional ldhA, which plays a role in lactic acid synthesis and may form a lineage separated from the strains accumulating predominantly lactic acid in the genus Rhizopus.

Hydrolysis of the potato glycoalkaloid α-chaconine by filamentous fungi

Abstract Three strains of filamentous fungi have been isolated from potato sprouts to obtain an enzyme degrading the glycoalkaloids. All of the strains hydrolyzed α-chaconine and not α-solanine when grown on the sprouts. From strain HP341, identified as Plectosphaerella cucumerina, the enzyme hydrolyzing α-chaconine was purified on columns of DEAE-Toyopearl and Phenyl-Toyopearl. The partially purified enzyme hydrolyzed α-chaconine to β 1 -chaconine but not to β 2 - or γ-chaconine, suggesting that the enzyme is a rhamnosidase specific for the hydrolysis of the rhamnose (C1–C4) glucose linkage in α-chaconine. Conversion of α-chaconine to β 1 -chaconine may be the first step of detoxification for filamentous fungi to grow on potato sprouts that accumulated antifungal α-chaconine.

Purification and characterization of 2,6-β-D-fructan 6-levanbiohydrolase from Streptomyces exfoliatus F3-2

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

Purification of Levan Fructotransferase from Arthrobacter nicotinovorans GS-9 and Production of DFA IV from Levan by the Enzyme

A bacterial strain, GS-9, isolated from soil as a levan-degrading microorganism produced an extracellular enzyme that converted levan into DFA IV. This strain was identified as Arthrobacter nicotinovorvans. The DFA IV-producing enzyme was specifically induced by levan. The enzyme was purified 60-fold from culture supernatant to give a single band on SDS-PAGE. The molecular weight of this enzyme was 52,000 by SDS-PAGE and a monomer by gel filtration. The enzyme gave DFA IV as a main product (> 75%), and fructose, levanbiose, and two unidentified oligosaccharides as minor products, and was identified as a novel levan fructotransferase.

Molecular cloning and high-level expression in Escherichia coli of fungal α-galactosidase from Absidia corymbifera IFO 8084

A cDNA encoding the alpha-galactosidase of Absidia corymbifera IFO 8084 was cloned and sequenced. The cloned DNA has a single open-reading frame consisting of 2190 base pairs, and the deduced amino acid sequence revealed that the mature enzyme consisted of 730 amino acid residues with a molecular mass of 82,712 Da. The native structure of the alpha-galactosidase of A. corymbifera IFO 8084 was determined to be a tetramer. Comparison with amino acid sequences of other alpha-galactosidase showed high homology with sequences of members of family 36. An expression vector, pET32Trx/galalpha, was constructed by introducing the cDNA coding region into a thioredoxin fusion system, pET32-Ek/LIC. The resulting transformant, pET32Trx/galalpha, overproduced the active enzyme as a thioredoxin fused form in the host Escherichia coli. By using His-binding metal affinity chromatography, recombinant alpha-galactosidase was purified to homogeneity in a single step. The purified recombinant fusion alpha-galactosidase showed properties very similar to the native alpha-galactosidase from A. corymbifera IFO 8084.

Changes in rutin concentration and flavonol-3-glucosidase activity during seedling growth in tartary buckwheat (Fagopyrum tataricum Gaertn.)

To investigate the physiological roles of rutin (quercetin 3-O-rutinoside) and flavonol-3-glucosidase (f3g, a rutin-degrading enzyme) during cotyledon growth in tartary buckwheat, changes in rutin and quercetin (aglycone of rutin) concentrations, and f3g activities were measured. The rutin concentration gradually increased (about 50 mg g-1 DW cotyledons) up to 12 d after germination (DAG). The quercetin concentration in the tartary buckwheat cotyledon increased immediately after germination, reached a maximum at 4 DAG, and then remained around 1.5 mg g-1 DW until 12 DAG. The f3g activity in the cotyledon and testa decreased gradually after germination. However, it retained 80% activity at 4 DAG compared with dry seed. In addition, the f3g activity was high on the surface of the cotyledon. We also investigated spatial distribution of rutin and f3g activity in 4 DAG seedlings. While rutin was mostly present in the cotyledon, f3g activity was observed in the testa, the surface of the testa, and root. These r...