Shitsuw Shikata
Kao Corporation
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Microbiology | 1990
Katsuya Ozaki; Shitsuw Shikata; Shuji Kawai; Susumu Ito; Kikuhiko Okamoto
A gene for alkaline cellulase from the alkalophilic Bacillus sp. KSM-635 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. Although the recombinant plasmid contained two HindIII inserts of 2.6 kb and 4.0 kb, the inserts were found to be contiguous in the Bacillus genome by hybridization analysis. Nucleotide sequences of a 2.4 kb region which was indispensable for the production of cellulase, and the flanking, 1.1 kb region, were determined. There was an open reading frame (ORF) of 2823 bp in the 3498 bp sequence determined, which encoded 941 amino acid residues. Two putative ribosome-binding sites and a sigma 43-type, promoter-like sequence were found upstream from an initiation codon in the ORF. The deduced amino-terminal sequence resembles the signal peptide of extracellular proteins. A region of amino acids, 249 to 568, of the deduced amino acid sequence of the cellulase from this organism is homologous with those of alkaline and neutral enzymes of other micro-organisms, but nine amino acid residues were found to be conserved only in the alkaline enzymes.
Microbiology | 1990
Tadashi Yoshimatsu; Katsuya Ozaki; Shitsuw Shikata; Yu-ichi Ohta; Kenzo Koike; Shuji Kawai; Susumu Ito
Summary: Two carboxymethylcellulases (CMCase, 1,4-1,4-β-d-glucan glucanohydrolase, EC 3.2.1.4), designated E-H and E-L, were purified to homogeneity from a culture filtrate of the alkalophilic Bacillus sp. KSM-635, by chromatography on DEAE-Toyopearl 650S and gel filtration on Bio-Gel A-0.5m. The purified CMCases both contained approximately 2–3% (w/w) glucosamine. Molecular masses deduced from SDS-PAGE were 130 kDa for E-H and 103 kDa for E-L. The pH optima of the enzymes were both about 9.5, and their optimum temperatures were around 40°C. Activities of both enzymes were inhibited by Hg2+, Cu2+, Fe2+ and Fe3+, but sulphydryl inhibitors, such as N-ethylmaleimide, monoiodoacetate and 4-chloromercuribenzoate, had either no effect or a slightly inhibitory effect. N-Bromosuccinimide was strongly inhibitory, suggesting that a tryptophan residue is essential for the activity of the CMCases from Bacillus. In addition, the activities of both E-H and E-L were stimulated by Co2+, and they required Mg2+, Ca2+, Mn2+ or Co2+ for stabilization. Both enzymes efficiently hydrolysed carboxymethylcellulose (β-1,4-linkage) and lichenan (β-1,3; 1,4-linkage), but crystalline cellulosic substrates, curdlan (β-1,3-linkage), laminarin (β-1,3; 1,6-linkage) and 4-nitrophenyl-β-d-glucopyranoside were hydrolysed very little, if at all. 4-Nitrophenyl-β-d-cellobioside was hydrolysed by both enzymes to liberate 4-nitrophenol, and their hydrolysis rates were higher at neutral pH than at alkaline pH.
Biochimica et Biophysica Acta | 1988
Shitsuw Shikata; Katsuya Ozaki; Shuji Kawai; Susumu Ito; Kikuhiko Okamoto
We have succeeded in purifying to homogeneity a very labile NADP+-linked isocitrate dehydrogenase (isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) from a strain of alkalophilic Bacillus, by a simple method, with an overall yield over 76% of the original activity. The molecular weight on Sephadex G-200 was around 90,000; and that by electrophoresis on SDS-polyacrylamide gels was about 44,000. The sedimentation coefficient (s020,w) and isoelectric point of the enzyme were determined to be 3.22 S and pH 4.7, respectively. The enzyme required Mn2+ for the reaction and for stability. The optimum pH for the reaction was in the range 7.8-8.4 at 30 degrees C; the optimum temperature at pH 8.0 was 75 degrees C; the activation energy of the reaction was 6.2 kcal/mol. The Km values for threo-Ds-isocitrate, DL-isocitrate, and NADP+ were 5.4 microM, 9.9 microM, and 7.3 microM, respectively. This enzyme was inhibited by NADPH, glyceraldehyde 3-phosphate, 3-phosphoglycerate, phosphoenol pyruvate, cis-aconitate, alpha-ketoglutarate, and oxaloacetate. In addition, it was subject to a concerted inhibition by a combination of glyoxylate and oxaloacetate, and also to a cumulative inhibition by nucleoside triphosphates.
Agricultural and biological chemistry | 1989
Susumu Ito; Shitsuw Shikata; Katsuya Ozaki; Shuji Kawai; Kikuhiko Okamoto; Shigeo Inoue; Akira Takei; Yu-ichi Ohta; Tomokazu Satoh
Agricultural and biological chemistry | 1990
Shitsuw Shikata; Katsuhisa Saeki; Hiromi Okoshi; Tadashi Yoshimatsu; Katsuya Ozaki; Shuji Kawai; Susumu Ito
Archive | 1987
Susumu Ito; Tomokazu Sato; Katsuya Ozaki; Shitsuw Shikata; Kikuhiko Okamoto; Shigeo Inoue; Kenzo Koike; Yuichi Ota; Akira Takei
Archive | 1987
Shuji Kawai; Kazushi Oshino; Hiromi Okoshi; Hajime Mori; Katsuya Ozaki; Shitsuw Shikata; Susumu Ito; Kikuhiko Okamoto
Archive | 1989
Susumu Ito; Shuji Kawai; Shitsuw Shikata; Katsuya Ozaki; Tadashi Yoshimatsu
Agricultural and biological chemistry | 1990
Hiromi Okoshi; Katsuya Ozaki; Shitsuw Shikata; Kazushi Oshino; Shuji Kawai; Susumu Ito
Bioscience, Biotechnology, and Biochemistry | 1992
Decorosa D. Lusterio; Franklin G. Suizo; Nellie M. Labunos; Marietta N. Valledor; Shinta Ueda; Shuji Kawai; Kenzo Koike; Shitsuw Shikata; Tadashi Yoshimatsu; Susumu Ito