Makoto Shimosaka

Purification and characterization of a novel bacteriocin produced by Enterococcus faecalis strain RJ-11.

ABSTRACT Lactic acid bacteria exhibiting activity against the gram-positive bacterium Bacillus subtilis were isolated from rice bran. One of the isolates, identified as Enterococcus faecalis RJ-11, exhibited a wide spectrum of growth inhibition with various gram-positive bacteria. A bacteriocin purified from culture fluid, designated enterocin RJ-11, was heat stable and was not sensitive to acid and alkaline conditions, but it was sensitive to several proteolytic enzymes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that enterocin RJ-11 had a molecular weight of 5,000 in its monomeric form. The amino acid sequence determined for purified enterocin RJ-11 exhibited high levels of similarity to the sequences of enterocins produced by Enterococcus faecium.

Effects of methyl jasmonate and elicitor on the activation of phenylalanine ammonia-lyase and the accumulation of scopoletin and scopolin in tobacco cell cultures

Abstract The effects of methyl jasmonate (MJ) and elicitor on the phenylpropanoid metabolism in tobacco was studied. It was found that suspended cells of tobacco, responded to MJ and elicitor from the plant pathogenic fungus Fusarium solani with differential formation of phenolic compounds. Both MJ and elicitor induced phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity to comparable levels. Northern hybridization showed that the increase in PAL activity was preceded by PAL mRNA accumulation. PAL was induced transcriptionally and enzymatically faster in elicitor-treated cells as compared to MJ-treated cells. HPLC of extracts from tobacco cells and their culture filtrate treated with MJ, showed an induction of the syntheses of coumarin-scopoletin and scopolin. This effect was not seen in elicitor-treated or control cell cultures. Scopolin was mainly found within the cell (99%), whereas scopoletin accumulated in the culture filtrate (90%). We conclude that though MJ mimics stress, indicating its possible role in the plant defense metabolism, responses of tobacco cells to MJ and elicitor are distinct.

Effect of gene disruptions of the TCA cycle on production of succinic acid in Saccharomyces cerevisiae.

Succinate is the main taste component produced by yeasts during sake (Japanese rice wine) fermentation. The pathway leading to accumulation of succinate was examined in liquid culture in the presence of a high concentration (15%) of glucose under aerobic and anaerobic conditions using a series of Saccharomyces cerevisiae strains in which various genes that encode the expression of enzymes required in TCA cycle were disrupted. When cultured in YPD medium containing 15% glucose under aerobic conditions, the KGD1 (alpha-ketoglutarate dehydrogenase) gene disrupted mutant produced a lower level of succinate than the wild-type strain, while the SDH1 (succinate dehydrogenase) gene-disrupted mutant produced an increased level of succinate. On the other hand, the FUM1 (fumarase) gene disrupted mutant produced significantly higher levels of fumarate but did not form malate at all. These results indicate that succinate, fumarate and malate are mainly synthesized through the TCA cycle (oxidative direction) even in the presence of glucose at a concentration as high as 15%. When the growth condition was shifted from aerobic to anaerobic, the increased level of succinate in SDH1 disruptants was no longer observed, whereas the decreased level of succinate in the KGD1 diruptant was still observed. A double mutant of the two fumarate reductase isozyme genes (OSM1 and FRDS) showed a succinate productivity of 50% as compared to the parent when cells were incubated in glucose-buffered solution. These results indicate that succinate could be synthesized through two pathways, namely, alpha-ketoglutarate oxidation via the TCA cycle and fumarate reduction under anaerobic conditions.

Purification and characterization of chitosanase and exo-β-D-glucosaminidase from a Koji mold, Aspergillus oryzae IAM2660

Chitosan-degrading activity was detected in the culture fluid of Aspergillus oryzae, A. sojae, and A. flavus among various fungal strains belonging to the genus Aspergillus. One of the strong producers, A. oryzae IAM2660 had a higher level of chitosanolytic activity when N-acetylglucosamine (GlcNAc) was used as a carbon source. Two chitosanolytic enzymes, 40 kDa and 135 kDa in molecular masses, were purified from the culture fluid of A. oryzae IAM2660. Viscosimetric assay and an analysis of reaction products by thin-layer chromatography clearly indicated the endo- and exo-type cleavage manner for the 40-kDa and 135-kDa enzymes, respectively. The 40-kDa enzyme, designated chitosanase, catalyzed a hydrolysis of glucosamine (GlcN) oligomers larger than pentamer, glycol chitosan, and chitosan with a low degree of acetylation (0-30%). The 135-kDa enzyme, named exo-β-D-glucosaminidase, released a single GlcN residue from the GlcN oligomers and chitosan, but did not release GlcNAc residues from either GlcNAc oligomer or colloidal chitin.

Chitosanase from the Plant Pathogenic Fungus, Fusarium solani f. sp. Phaseoli—Purification and Some Properties

Among 162 strains of the genus Fusarium tested, 22 strains, mainly from F. solani and F. splendens, formed halos on chitosan-containing agar medium. Chitosanase secreted in the culture by the most effective producer, F. solani f. sp. phaseoli SUF386, was further investigated. N-Acetylglucosamine (GlcNAc) used as a carbon source was most effective for production of chitosanase. Chitosan, a substrate for chitosanase, inhibited cell growth completely and abolished production of chitosanase when used as a carbon source in the liquid medium. Chitosanase purified from the culture filtrate had a molecular mass of 36 kDa, and showed a maximum activity at pH 5.6 and 40°C. The enzyme catalyzed the hydrolysis of chitopentaose, chitosan (70% and 100% deacetylation), and glycolchitosan, but showed little activity toward chitobiose, chitotriose, chitotetraose, glycol chitin, and carboxymethyl cellulose. A rapid reduction in the viscosity of chitosan solutions suggested that the enzyme catalyzed an endo-type cleavage reaction.

Cloning and Characterization of a Chitosanase Gene from the Plant Pathogenic Fungus Fusarium solani

The plant pathogenic fungus Fusarium solani f. sp. phaseoli SUF386 secretes a chitosanase in the absence of exogenous chitosan. Based on partial amino acid sequences of the purified chitosanase, two degenerate oligonucleotides were synthesized and used as reverse transcription-mediated PCR (RT-PCR) primers to amplify a 500-bp fragment of corresponding cDNA. The PCR product was used as a probe to isolate the genomic copy of the gene (csn). F. solani csn has an open reading frame encoding a polypeptide of 304 amino acid residues with a calculated molecular mass of 31,876 Da and containing a putative 19-amino acid residue signal sequence. Comparison between the genomic and cDNA sequences revealed that three introns are present in the coding region. Southern blot analysis results indicated that csn is present as a single copy in the genome of F. solani SUF386. The cDNA fragment corresponding to the mature enzyme was introduced into E. coli using an expression vector driven by the T7 promoter. The resulting E. coli transformant overproduced proteins with chitosanolytic activity.

Isolation of sake yeast strains possessing various levels of succinate- and/or malate-producing abilities by gene disruption or mutation

Succinate and malate are the main taste components produced by yeast during sake (Japanese alcohol beverage) fermentation. Sake yeast strains possessing various organic acid productivities were isolated by gene disruption. Sake fermented using the aconitase gene (ACO1) disruptant contained a two-fold higher concentration of malate and a two-fold lower concentration of succinate than that made using the wild-type strain K901. The fumarate reductase gene (OSM1) disruptant produced sake containing a 1.5-fold higher concentration of succinate as compared to the wild-type, whereas the alpha-ketoglutarate dehydrogenase gene (KGD1) and fumarase gene (FUMI) disruptants gave lower succinate concentrations. The Deltakgd1 disruptant exhibited lower succinate productivity in the earlier part of the sake fermentation, while the Deltafum1 disruptant showed lower succinate productivity later in the fermentation, indicating that succinate is mainly produced by an oxidative pathway of the TCA cycle in the early phase of sake fermentation and by a reductive pathway in the later phases. Sake yeasts with low succinate productivity and/or high malate productivity was bred by isolating mutants unable to assimilate glycerol as a carbon source. Low malate-producing yeasts were also obtained from phenyl succinate-resistant mutants. The mutation of one of these mutant strains with low succinate productivity was found to occur in the KGD1 gene. These strains possessing various succinate- and/or malate-producing abilities are promising for the production of sake with distinctive tastes.

Molecular cloning and characterization of a chitosanase from the chitosanolytic bacterium Burkholderia gladioli strain CHB101

Abstract A chitosanase was purified from the culture fluid of the chitino- and chitosanolytic bacterium Burkholderia gladioli strain CHB101. The purified enzyme (chitosanase A) had a molecular mass of 28 kDa, and catalyzed the endo-type cleavage of chitosans having a low degree of acetylation (0–30%). The enzyme hydrolyzed glucosamine oligomers larger than a pentamer, but did not exhibit any activity toward N-acetyl-glucosamine oligomers and colloidal chitin. The gene coding for chitosanase A (csnA) was isolated and its nucleotide sequence determined. B. gladioli csnA has an ORF encoding a polypeptide of 355 amino acid residues. Analysis of the N-terminal amino acid sequence of the purified chitosanase A and comparison with that deduced from the csnA ORF suggests post-translational processing of a putative signal peptide and a possible substrate-binding domain. The deduced amino acid sequence corresponding to the mature protein showed 80% similarity to the sequences reported from Bacillus circulans strain MH-K1 and Bacillus ehimensis strain EAG1, which belong to family 46 glycosyl hydrolases.

Scopoletin uptake from culture medium and accumulation in the vacuoles after conversion to scopolin in 2,4-D-treated tobacco cells.

Tobacco (Nicotiana tabacum L. Bright Yellow) T-13 cell line has the ability to produce scopoletin endogenously and release some of it into the culture medium. We investigated the mechanism of scopoletin uptake following treatment of a tobacco culture with 2,4-dichlorophenoxyacetic acid (2,4-D). Addition of [14C]-labeled scopoletin showed that scopoletin was taken up by 2,4-D-treated cells and converted to scopolin, a 7-O-glucoside of scopoletin. This uptake of scopoletin began 6 h after 2,4-D addition to the cells. Experiments using several inhibitors showed that this uptake was energy-dependent. The phenomenon of 2,4-D-stimulated uptake was observed only for 7-hydroxycoumarins, such as scopoletin, umbelliferone and esculetin. To further investigate the site for scopoletin accumulation, we separated the vacuoles from T-13 cells and quantified the coumarin contents in this fraction. Most of the scopoletin in the vacuoles was present as glucoconjugate, scopolin. Moreover, glucosylation activity was absent from isolated vacuoles and, therefore, is likely to be located in the cytosol. Therefore, we can state that 2,4-D treatment of tobacco cells stimulated scopoletin uptake. The scopoletin was converted into scopolin in the cytoplasm, and then transferred into the vacuoles.

Isolation and characterization of a gene coding for chitin deacetylase specifically expressed during fruiting body development in the basidiomycete Flammulina velutipes and its expression in the yeast Pichia pastoris

Fv-pda, a gene coding for chitin deacetylase (CDA), was isolated from the basidiomycete Flammulina velutipes by differential display targeted for genes specifically expressed during fruiting body development. The fv-pda ORF comprises 250 amino acid residues and is interrupted by 10 introns. The fv-pda cDNA was expressed in the yeast Pichia pastoris, and the resulting recombinant FV-PDA was used for enzymatic characterization. The recombinant FV-PDA catalyses deacetylation of N-acetyl-chitooligomers, from dimer to pentamer, glycol chitin and colloidal chitin. The fv-pda was specifically expressed through the entire stage of fruiting body development, and the transcript was abundant in stipes of mature fruiting bodies. These results suggest that CDA plays an important role in the process of fruiting of F. velutipes.