Expression of a gene encoding chitinase (pCA 8 ORF) from Aeromonas sp. no. 10S-24 in Escherichia coli and enzyme characterization

A gene encoding chitinase from Aeromonas sp. no. 10S-24 was expressed using pTrc99A in Escherichia coli JM 105 which yielded a 5-fold higher activity than when pUC19 was used. Three different truncated enzymes (SA-1, SA-2 and SA-3) were obtained after purification. Their isoelectric points were 7.0, 6.9, and 6.7, respectively. The enzymes showed two optimum pHs, 4.0 and 7.0, when incubated with ethylene glycol chitin as the substrate, and were stable over a wide pH range (3.0-9.0). The optimum temperature was 60 degrees C and the enzymes were stable up to 50 degrees C. The chitinases exhibited wide substrate specificities for chitin-related compounds.     

Application of microalgae hydrolysate as a fermentation medium for microbial production of 2-pyrone 4,6-dicarboxylic acid

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SEMICONTINUOUS ETHANOL FERMENTATION WITH SHOCHU DISTILLERY WASTE*

An effective utilization system using distillery waste discharged from Japanese traditional shochu factory was developed. Mugi (barley) shochu distillery waste discharged from a novel vacuum distillation procedure (35–40°C) contained a large number of viable yeast (7 × 10⁶ cells/ml), glucoamylase activity (19.7 units/ml), acid protease activity (940 units/ml), and neutral protease activity (420 units/ml). Ethanol fermentation was achieved with a mash composed of glucose as the sola carbon source and mugi shochu distillery waste. After ethanol fermentation was completed the fermented broth was again distilled at 35–40°C in vacuo and the non volatile residue used in the next ethanol fermentation. In this way, semicontinuous ethanol fermentation system of more than 10 cycles was developed. Even in the distillate of the mash of the 8th fermentation cycle, 7.9% of ethanol, 33.0 ppm of ethyl acetate, 28.5 ppm of isobutyl alcohol, and other aromatic compounds were present. A semicontinuous ethanol fermentation system has been developed for shochu distillery waste which conventionally is treated as wastewater.     

An anaerobic bacterium isolated from Japanese spiny lobster Panulirus japonicus

Eight strains of bacteria which could not grow under aerobic conditions, were isolated from Japanese spiny lobster. All the strains were Gram-positive, non-sporeforming cocci, and grew well under anaerobic conditions. The strains required a decreased oxygen tension rather than an increased CO₂ tension. Catalase, oxidase, nitrate reductase and urease were not produced. The strains fermented glucose, cellobiose, trehalose and levulose. Indole and H₂S were not produced. Macromolecule substances including starch, esculin, casein, gelatin and chitin were not hydrolyzed. Lactic acid was the sole product from peptone-yeast extract-Fildes solution-glucose broth. Fermentable carbohydrates were not required. The strains could grow at 20 to 37°C, and required NaCl for growth. From the above results, the strains were identified as anaerobic (aerotolerant) Streptococcus sp.     

Lipid-Lowering Effect of Eriocitrin, the Main Flavonoid in Lemon Fruit, in Rats on a High-Fat and High-Cholesterol Diet

ABSTRACT:  Eriocitrin (eriodictyol 7- O -β-rutinoside) is the main flavonoid in lemon fruit. In this study, eriocitrin was investigated for its lowering effect on serum and hepatic lipids in high-fat and high-cholesterol fed rats. Rats in the control group ( N = 6) were fed a 20% lard and 1% cholesterol diet for 21 d, and rats in the 0.35% eriocitrin group ( N = 6) and 0.70% eriocitrin group ( N = 6) were fed a diet supplemented with eriocitrin 0.35% and 0.70%, respectively. The content of hepatic total cholesterol and triglyceride in the eriocitrin group was no different from that of the control group. The total cholesterol, VLDL+LDL, triglyceride, and phospholipid in the serum of the 0.35% eriocitrin group showed significantly lower concentrations than the control group ( P < 0.05), although there was no difference in the HDL concentrations among the groups. The lowering effect of eriocitrin for serum total cholesterol was thought to be caused by a decrease in VLDL+LDL. The 0.35% eriocitrin group was shown to have a significant increase in excretion of fecal bile acid ( P < 0.05) and a tendency for enhanced hepatic m-RNA levels of LDL receptor in comparison with the control group.     

Simultaneous determination of five penicillins in muscle,liver and kidney from slaughtered animals using liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A simple and rapid method for the simultaneous determination of five penicillins (ampicillin, penicillin G, penicillin V, oxacillin and cloxacillin) in muscle, liver and kidney tissues using high-performance liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) was developed. Mass spectral acquisition was done in the negative ion mode by applying selected reaction monitoring (SRM). The five penicillins were extracted with water, and the extracted solution was cleaned up on a C18 cartridge. Phenethicillin was added as an internal standard, and the extract was diluted with water for injection into the LC-ESI-MS/MS. The recoveries of the five penicillins were in the range of 77.3-99.8% from muscle, liver and kidney fortified at 10-250 ng/g. The detection limits for ampicillin were 6 ng/g in muscle and kidney and 15 ng/g in liver. For penicillin G and penicillin V, the detection limits were 2 ng/g in muscle and kidney and 5 ng/g in liver. For oxacillin and cloxacillin, the detection limits were 4 ng/g in muscle and kidney and 10 ng/g in liver. Twenty-three muscle, fourteen liver and twenty-two kidney samples from the markets were analyzed by this method. No penicillins were detected in any sample.     

Establishment of a simple system to analyse the molecular interaction in the agglutination of Saccharomyces cerevisiae

Saccharomyces cerevisiae a-agglutinin, which is involved in mating and covalently anchoring to the cell wall, consists of two components, Aga1p and Aga2p, whose syntheses are individually regulated. To facilitate the analysis of the protein-protein interaction on agglutination between a- and alpha-agglutinins, the construction of a yeast strain (MATa) with the functional protein prepared by genetic fusion of Aga1p- and Aga2p-encoding genes and by the expression system using the UPR-ICL promoter derived from the n-alkane-assimilating yeast, Candida tropicalis, which is functional under the condition of lower glucose concentration was tried and the agglutination ability of the constructed strain was evaluated with a yeast strain (MATa) which expressed AGalpha1 encoding alpha-agglutinin under the control of the same promoter. The genes were integrated into the yeast chromosomes. Cell agglutination between both (MATa) strains was observed microscopically when these two strains were mix-cultured to a glucose-decreased concentration. The agglutination was further confirmed by the sedimentation test and by the quantification using a filter. These results proved that the constructed Aga1p-Aga2p fusion protein was enoughly functional for the interaction with the Agalpha1 protein, and that this phenomenon occurred dependent on glucose concentration, but independent of the peptide pheromones secreted by the cells of the opposite mating types. Using this system, the role of two disulphide linkages between Aga1p and Aga2p on the binding activity between Aga2p and Aga1p was first evaluated. Under the treatment by the SH-compound (dithiothreitol), in which Agalpha2p is easily released into the medium from the intact cell surface, the Aga1p and Aga2p fusion protein was a good tool to make clear the role of the disulphide linkages. As a result, the linkages had a significant effect on not only the assembly but also the binding activity. The novel and simple system described here may further facilitate the study of molecular interaction in agglutination.     

Contamination levels of PCDDs, PCDFs and Co-PCBs in commercial baby foods in Japan

To examine dioxin contamination in commercial baby foods in Japan, congener analyses of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and coplanar polychlorinated biphenyls (Co-PCBs) were performed on 102 varieties of baby foods obtained from supermarkets in 2001-2002. The toxic equivalent quantity (TEQ) levels for dioxins in samples ranged from < 0.001 to 0.135 pg-TEQ/g wet weight when undetected or trace levels of congeners were taken as zero. Among 102 samples tested, 26 samples exceeded 0.010 pg-TEQ/g. The highest TEQ value was for "sardine, vegetables" (0.135 pg-TEQ/g), followed by "Japanese radish (daikon), sardine" (0.080 pg-TEQ/g). Thus, dioxins were detected at low levels in baby foods containing animal products such as fishes and/or dairy products.     

Antioxidant Effects of Herbal Tea Leaves from Yacon (Smallanthus sonchifolius) on Multiple Free Radical and Reducing Power Assays,Especially on Different Superoxide Anion Radical Generation Systems

Yacon (Smallanthus sonchifolius), a native Andean plant, has been cultivated as a crop and locally used as a traditional folk medicine for the people suffering from diabetes and digestive/renal disorders. However, the medicinal properties of this plant and its processed foods have not been completely established. This study investigates the potent antioxidative effects of herbal tea leaves from yacon in different free radical models and a ferric reducing model. A hot‐water extract exhibited the highest yield of total polyphenol and scavenging effect on 1,1‐diphenyl‐2‐picryl hydrazyl (DPPH) radical among four extracts prepared with hot water, methanol, ethanol, and ethylacetate. In addition, a higher reducing power of the hot‐water extract was similarly demonstrated among these extracts. Varying concentrations of the hot‐water extract resulted in different scavenging activities in four synthetic free radical models: DPPH radical (EC₅₀ 28.1 μg/mL), 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) cation radical (EC₅₀ 23.7 μg/mL), galvinoxyl radical (EC₅₀ 3.06 μg/mL), and chlorpromazine cation radical (EC₅₀ 475 μg/mL). The yacon tea‐leaf extract further demonstrated superoxide anion (O₂^?) radical scavenging effects in the phenazine methosulfate‐NADH‐nitroblue tetrazolium (EC₅₀ 64.5 μg/mL) and xanthine oxidase assay systems (EC₅₀ 20.7 μg/mL). Subsequently, incubating human neutrophilic cells in the presence of the tea‐leaf extract could suppress the cellular O₂^? radical generation (IC₅₀ 65.7 μg/mL) in a phorbol 12‐myristate 13‐acetate‐activated cell model. These results support yacon tea leaves may be a good source of natural antioxidants for preventing O₂^? radical‐mediated disorders.     

Large-scale genome reorganization in Saccharomyces cerevisiae through combinatorial loss of mini-chromosomes

A highly efficient technique, termed PCR-mediated chromosome splitting (PCS), was used to create cells containing a variety of genomic constitutions in a haploid strain of Saccharomyces cerevisiae. Using PCS, we constructed two haploid strains, ZN92 and SH6484, that carry multiple mini-chromosomes. In strain ZN92, chromosomes IV and XI were split into 16 derivative chromosomes, seven of which had no known essential genes. Strain SH6484 was constructed to have 14 mini-chromosomes carrying only non-essential genes by splitting chromosomes I, II, III, VIII, XI, XIII, XIV, XV, and XVI. Both strains were cultured under defined nutrient conditions and analyzed for combinatorial loss of mini-chromosomes. During culture, cells with various combinations of mini-chromosomes arose, indicating that genomic reorganization could be achieved by splitting chromosomes to generate mini-chromosomes followed by their combinatorial loss. We found that although non-essential mini-chromosomes were lost in various combinations in ZN92, one mini-chromosome (18kb) that harbored 12 genes was not lost. This finding suggests that the loss of some combination of these 12 non-essential genes might result in synthetic lethality. We also found examples of genome-wide amplifications induced by mini-chromosome loss. In SH6484, the mitochondrial genome, as well as the copy number of genomic regions not contained in the mini-chromosomes, was specifically amplified. We conclude that PCS allows for genomic reorganization, in terms of both combinations of mini-chromosomes and gene dosage, and we suggest that PCS could be useful for the efficient production of desired compounds by generating yeast strains with optimized genomic constitutions.