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Yokoi K.-J.,Toyama prefectural Food Research Institute | Taketo A.,Fukui University of Technology | Kodaira K.-I.,University of Toyama
Food Science and Technology Research | Year: 2013

The traditional fermented food ika-kurozukuri, salted squid with ink and liver, isa noted product of Toyama prefecture. Compared with traditional ika-kurozukuri, the salt content of the modern type of product is considerably lower. The microflora of threecommercial ika-kurozukuri was investigated using the 16S rDNA clone library method. Staphylococcus saprophyticus was dominant in one product, while Weissella paramesenteroides-dominated in the other two products. During ripening at 10°C, the microflora of the modern ika-kurozukuri (6% NaCl content) was analyzed periodically. At the beginning of ripening, Sphingomonas sp. was in the majority. After 10 days of ripening, S. saprophyticus was dominant, and the microflora was stable up to 35 days. The S. saprophyticusstrain isolated from ika-kurozukuri could grow at 10°C. These results suggested thatthe dominant bacterial species of modern ika-kurozukuri products are S. saprophyticus and W. paramesenteroides. Source

Yokoi K.-J.,Toyama prefectural Food Research Institute | Kuzuwa S.,University of Toyama | Iwasaki S.-I.,White Food Industry Co. | Yamakawa A.,Wakayama National College of Technology | And 2 more authors.
Bioscience, Biotechnology and Biochemistry | Year: 2016

The aureolysin (Aur) gene of S. warneri M (aurWM) was cloned and sequenced. Analyses of the aurWM-inactivated mutant (S. warneri Mau) suggested that AurWM was probably associated with efficient processing of the PROM protease (homolog of V8/SspA serine protease), whereas considerable amount of mature-PROC protease (homolog of SspB cysteine protease) accumulated without AurWM. Additionally, AurWM appeared to affect biofilm formation in an uncertain suppressive way. © 2016 Japan Society for Bioscience, Biotechnology, and Agrochemistry. Source

Yokoi K.-J.,Toyama prefectural Food Research Institute | Kuzuwa S.,University of Toyama | Kondo M.,University of Toyama | Yamakawa A.,Wakayama National College of Technology | And 2 more authors.
Gene | Year: 2013

Unlike other members of coagulase negative staphylococci (CNS), strain warneri has proMCD operon, a homologue of sspABC proteinase operon of S. aureus. The proM and proC encode serine glutamyl endopeptidase and cysteine protease respectively, whereas proD directs homologue of SspC, putative cytoplasmic inhibitor which protects the host bacterium from premature activation of SspB. We determined whole nucleotide sequence of proMCD operon of S. warneri M, succeeded in expression of these genes, and investigated their functions by gene inactivation and complementation experiments. In gelatin zymography of the culture supernatant, a 20-kDa band corresponding to PROC cysteine protease was detected. By Western blotting, PROD was also confirmed in the cytoplasmic protein fraction. PROC and PROD showed significant similarity to SspB and SspC of S. aureus (73% and 58%, respectively). Inactivation mutants of proMCD, proCD and proD genes were established, separately. In the proMCD mutant, degradation/processing of extracellular proteins was drastically reduced, suggesting that PROM was responsible for the cleavage of extracellular proteins. By the proD mutation, the growth profile was not affected, and secretion of PROC was retained. Extracellular protein profiles of the proCD and proD mutants were not so different each other, but autolysin profiles were slightly dissimilar, around 39-48. kDa and 20. kDa bands in zymogram. Experiments in buffer systems showed that autolysis was significantly diminished in proMCD mutant, and was promoted by addition of purified PROM. The proC gene was cloned into a multicopy plasmid, and introduced into the proMCD mutant. Compared with the wild type, autolysis of the proC-complemented strain was definitely enhanced by addition of purified PROM. These results suggested that PROM and PROC affected the coccal autolysis, through processing of the autolysin. © 2012 Elsevier B.V. Source

Kuzuwa S.,University of Toyama | Yokoi K.-J.,Toyama prefectural Food Research Institute | Kondo M.,University of Toyama | Kimoto H.,Fukui Prefectural University | And 3 more authors.
Gene | Year: 2012

Though some genetic features of lactobacillar fructan hydrolases were elucidated, information about their enzymology or mutational analyses were scarce. Lactobacillus casei IAM1045 exhibits extracellular activity degrading inulin. After partial purification of the inulin-degrading protein from the spent culture medium, several fragments were obtained by protease digestion. Based on their partial amino-acid sequences, oligonucleotide primers were designed, and its structural gene (levH1) was determined using the gene library constructed in the E. coli system. The levH1 gene encoded a protein (designated as LevH1), of which calculated molecular mass and p. I were 138.8-kDa and 4.66, respectively. LevH1 (1296 amino-acids long) was predicted to have a four-domain structure, containing (i) an N-terminal secretion signal of 40 amino-acids, (ii) variable domain of about 140 residues whose function is unclear, (iii) a catalytic domain of about 630 residues with glycoside-hydrolase activity consisting of two modules, a five-blade β-propeller module linked to a β-sandwich module, (iv) a C-terminal domain of about 490 residues comprising five nearly perfect repeat sequences of 80 residues homologous to equivalents of other hypothetical cell surface proteins, followed by 37-residues rich in Ser/Thr/Pro/Gly, a pentad LPQAG (the LPXTG homologue). When overproduced in E. coli, the putative variable-catalytic domain region of about 770 residues exhibited exo-inulinase activity. Deletion analyses demonstrated that the variable-catalytic domain region containing two modules is important for enzymatic activity. Presence of eight conserved motifs (I-VIII) was suggested in the catalytic domain by comparative analysis, among which motif VIII was newly identified in the β-sandwich module in this study. Site-directed mutagenesis of conserved amino-acids in these motifs revealed that D198, R388, D389 and E440, were crucial for inulinase activity. Moreover, mutations of D502A and D683A in motif VI and VIII respectively caused significant decrease in the activity. These results suggested that the variable domain and β-sandwich module, besides the β-propeller module, are important for inulin-degrading activity of LevH1. © 2011 Elsevier B.V. Source

Yokoi K.-J.,Toyama prefectural Food Research Institute | Yokoi K.-J.,University of Toyama | Harada Y.,Toyama prefectural Food Research Institute | Shozen K.-I.,Toyama prefectural Food Research Institute | And 3 more authors.
Gene | Year: 2011

Histamine production from histidine in fermented food results in food spoilage, and is harmful to consumers. From fish-miso, we have isolated a new bacterial strain Staphylococcus epidermidis TYH1, which produced histamine under acidic condition in the medium supplemented with glucose. Using oligonucleotides deduced from the histidine decarboxylase gene (hdcA) of Lactobacillus hilgardii, about 14-kbp DNA region of the TYH1 genome was cloned and sequenced. This region contained two putative genes hdcA TYH1 and hdcP TYH1 encoding proteins HdcA TYH1 (310 amino acid residues) and HdcP TYH1 (495 residues), respectively. Nucleotide sequence around this hdc cluster showed similarity to SCCpbp4 region of S. epidermidis ATCC 12228. Downstream of the cluster, ccrA, ccrB (Type II, respectively) and pbp4 were located. The CcrA and CcrB proteins catalyzed excision of the hdc cluster from the TYH1 chromosome, upon introduction into the TYH1 strain via multicopy plasmid. When hdcA TYH1 was expressed in Staphylococcus warneri M, histamine was extracellularly accumulated in dependence on exogenous histidine. These results indicate that the gene encoding a histidine decarboxylase resides in a movable genetic element, SCC. This new element is designated as SCChdc. © 2011 Elsevier B.V. Source

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