Simple and Rapid Detection of ESBL blaSHV gene from an Urban River in Tokyo by Loop-Mediated Isothermal Amplification.

Extended-spectrum ß-lactamases (ESBLs) are produced mainly by gram-negative bacteria in Enterobacteriaceae. One of the major types of ESBLs is sulfhydryl variable (SHV) -type ESBL. Herein, we attempted to develop a simple and rapid method for the detection of the ESBL blaSHV gene by loop-mediated isothermal amplification (LAMP) . The five-primer set designed could amplify blaSHV gene at an isothermal temperature of 65℃. The detection limit of the LAMP method with the LF loop primer was 1 copy/tube, which was 10,000-fold more sensitive than that of the conventional PCR. The LAMP assay could also detect the direct amplification of blaSHV gene from a single river water sample in Tokyo. The LAMP method has great potential for applications in hospital, soil and water environment, food, and livestock.

Comparison of Benzoï¼»aï¼½pyrene-Degrading Activities between Olleya sp. ITB9 Isolated from Tokyo Bay and Other Type Strains of the Genus Olleya.

Benzoï¼»aï¼½pyrene (BaP) is one of the strongest carcinogenic compounds among polycyclicaromatic hydrocarbons (PAHs) .We previously identified the ITB9 strain of Olleya species, which shows BaP-degrading activity; our report was the first about BaP degradation by the genus Olleya. In this study, BaP-degradation efficiency by ITB9 was about 50% when the strain was suspended in 20 ml of L9 liquid medium with 100 µg/ml BaP and 0.2 M NaCl, with pH 8.0, and incubated at 25℃ for 5 days. Under the same conditions, all four type strains (O. marilimosa CIP108537, O. aquimaris KCTC22661, O. namhaensis KCTC23673, and O. algicola KCTC22024) also showed BaP-degrading activities, at efficiencies ranging from 49% to 63%. Olleya sp. ITB9 and O. aquimaris KCTC22661 were found to be in the same clade in the phylogenetic tree of the genus Olleya, given that the homology of 16S rRNA gene sequences between ITB9 and KCTC22661 was 99.77%.

Citeromyces matritensis M37 is a salt-tolerant yeast that produces ethanol from salted algae.

Algae are referred to as a third-generation biomass for ethanol production. However, salinity treatment is a problem that needs to be solved, because algal hydrolysates often contain high salt. Here, we isolated the salt-tolerant ethanol-producing yeast Citeromyces matritensis M37 from the east coast of Miura Peninsula in Japan. This yeast grew under osmotic stress conditions (20% NaCl or 60% glucose). It produced 6.55 g/L ethanol from YPD medium containing 15% NaCl after 48 h, and the ethanol accumulation was observed even at 20% NaCl. Using salted Undaria pinnatifida (wakame), we obtained 6.33 g/L glucose from approx. 150 g/L of the salted wakame powder with acidic and heat pretreatment followed by enzymatic saccharification, and the ethanol production reached 2.58 g/L for C. matritensis M37. The ethanol concentration was 1.4 times higher compared with that using the salt-tolerant ethanol-producing yeast Zygosaccharomyces rouxii S11.

Characterization of 9-fatty acid hydroperoxide lyase-like activity in germinating barley seeds that transforms 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid into 2(E)-nonenal.

Previously, we reported that 2(E)-nonenal, having a low flavor threshold (0.1 ppb) and known as the major contributor to a cardboard flavor (stale flavor) in stored beer, is produced by lipoxygenase-1 and a newly found factor named 9-fatty acid hydroperoxide lyase-like (9-HPL-like) activity in malt. To assess the involvement of 9-HPL-like activity in beer staling, we compared the values of the wort nonenal potential, an index for predicting the staleness of beer, with the lipoxygenase and 9-HPL-like activity of 20 commercial malts. There was a significant correlation between the malt 9-HPL-like activity and the values of wort nonenal potential (r=0.53, P<0.05), while the correlation between malt lipoxygenase activity and the wort nonenal potential was statistically insignificant. Analysis of the partially purified 9-HPL-like activity from embryos of germinating barley seeds indicated that 9-HPL-like activity consisted of fatty acid hydroperoxide lyase and 3Z:2E isomerase.

Identification and functional analyses of two cDNAs that encode fatty acid 9-/13-hydroperoxide lyase (CYP74C) in rice.

Fatty acid hydroperoxide lyase (HPL), a member of cytochrome P450 (CYP74), produces aldehydes and oxo-acids involved in plant defensive reactions. In monocots, HPL that cleaves 13-hydroperoxides of fatty acids has been reported, but HPL that cleaves 9-hydroperoxides is still unknown. To find this type of HPL, in silico screening of candidate cDNA clones and subsequent functional analyses of recombinant proteins were performed. We found that AK105964 and AK107161 (Genbank accession numbers), cDNAs previously annotated as allene oxide synthase (AOS) in rice, are distinctively grouped from AOS and 13-HPL. Recombinant proteins of these cDNAs produced in Escherichia. coli cleaved both 9- and 13-hydroperoxide of linoleic and linolenic into aldehydes, while having only a trace level of AOS activity and no divinyl ether synthase activity. Hence we designated AK105964 and AK107161 OsHPL1 and OsHPL2 respectively. They are the first CYP74C family cDNAs to be found in monocots.

Characterization of factors involved in the production of 2(E)-nonenal during mashing.

To characterize the factors involved in the production of volatile aldehydes during mashing, a model mashing experiment was done. After we inactivated the endogenous lipoxygenase (LOX) activity in the mash by mashing at 70 degrees C for 30 min, further incubation with recombinant barley LOX-1 stimulated the accumulation of 2(E)-nonenal; however, this effect was significantly reduced by boiling the mash sample. The result suggests that both LOX-1 and a heat-stable enzymatic factor are involved in the production of 2(E)-nonenal during mashing. Malt contained fatty acid hydroperoxide lyase-like activity (HPL-like activity) that transformed 9-hydroperoxy-10(E), 12(Z)-octadecadienoic and 13-hydroperoxy-9(Z), 11(E)-octadecadienoic acid into 2(E)-nonenal and hexanal, respectively. Proteinase K sensitivity tests showed that they are distinct factors. 9-HPL-like activity survived through the mashing at 70 degrees C for 30 min but was inactivated by boiling, suggesting it will be the heat-stable enzymatic factor found in the model mashing experiment.

Characterization of factors that transform linoleic acid into di- and trihydroxyoctadecenoic acids in mash.

The qualities of beer are deteriorated by the presence of either di- or trihydroxyoctadecenoic acids, which reduce the beer 'head' and produce an astringent flavor. In this study we found that native extracts of malt mash transformed linoleic acid into di- and trihydroxyoctadecenoic acids, but this transforming activity and lipoxygenase activity were inactivated by heating the mash at 70 degrees C for 30 min. Recombinant barley lipoxygenase 1 was not able to transform linoleic acid into di- and trihydroxyoctadecenoic acids. The transforming activity of mash extract heated at 70 degrees C for 30 min could be restored by the addition of recombinant barley lipoxygenase 1; in contrast, the activity of boiled mash extract was not substantially restored by the recombinant enzyme. These results indicate that di- and trihydroxyoctadecenoic acids are generated from linoleic acid by both lipoxygenase and a heat-stable enzymatic factor present in the mash.

Analysis of an inactivated Lg-FLO1 gene present in bottom-fermenting yeast.

During the course of investigating a flocculation-related gene of a bottom-fermenting yeast, we identified a new Lg-FLO1 homologue which contains the N-terminal domain of the Lg-FLO1 gene. The results of the partial DNA sequence analysis of the amplified product obtained by inverse-PCR suggested that the homologue contains a sequence present in the YIL169c (chr. IX of Saccharomyces cerevisiae). Southern blot analyses using the VTH1, HXT12, SDL1 and UBP7 genes as probes for chr. IX strongly indicated that an approximately 20-kb region from the YIL169c ORF to the left telomere in chr. IX translocated to the Lg-FLO1 ORF region in chr. VIII of bottom-fermenting yeast. This translocation might convert a flocculent cell to a non-flocculent one.

Breeding of brewer's yeast by hybridization between a top-fermenting yeast Saccharomyces cerevisiae and a cryophilic yeast Saccharomyces bayanus.

To improve the fermentability of a top-fermenting yeast at low-temperature, we performed hybridization trials between four top-fermenting Saccharomyces cerevisiae strains and a cryophilic yeast Saccharomyces bayanus YM84 with good fermentability at low-temperature. The hybrids selected using 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside were checked with pulsed-field gel electrophoresis and their brewing performance at the low-temperature of 10.5 degrees C was observed using small-scale (2 l) fermentation trials.