Kozo Ishizaki

Isolation of clustered genes that are notably homologous to the eicosapentaenoic acid biosynthesis gene cluster from the docosahexaenoic acid-producing bacterium Vibrio marinus strain MP-1

A 40-kbp DNA fragment was isolated from the cosmid library of Vibrio marinus strain MP-1. Among the 22 putative open reading frames (ORFs) in this fragment, ORFs 8, 9, 10 and 11 had high homology with ORFs 5, 6, 7 and 8 of the eicosapentaenoic acid biosynthesis gene cluster, respectively. Then, we speculate that these ORFs are responsible for docosahexaenoic acid biosynthesis in this bacterium.

Isolation of a Pseudomonas species from fish intestine that produces a protease active at low temperature

A psychrotrophic bacterium producing a protease active at low temperatures was isolated from fish intestine and identified as a Pseudomonas species. Optimum growth and protease‐producing temperatures of this strain were 15°C and 10°C, respectively. The maximum temperature for proteolytic activity was 25°C, an unusually low temperature.

Effect of ozone on plasmid DNA of Escherichia coli in situ

Abstract The cell suspension of Escherichia coli HB101 harboring pBR322 plasmid DNA was exposed to ozone, and the damage of plasmid DNA in vivo and also cell survival were investigated. Closed circular plasmid DNA (ccDNA) converted to open circular DNA (ocDNA) by ozone treatment. The mechanism of the conversion seemed to be similar to that observed in vitro ozone treatment of plasmid DNA [Sawadaishi et al. (1985) Nucleic Acids Res. 13 , 7183; idem. (1986) ibid. 14 , 1159]. These findings indicate that ozone penetrates cell membrane and reacts with cytoplasmic substances. Therefore, chromosomal DNA may be one of targets of the ozone degradation, and its damage may be one of the factors responsible for the cell killing.

Biotechnology of enzymes from cold-adapted microorganisms

One of the main goals in enzyme research is industrial application. Nowadays, we are surrounded by enzymes as well as chemicals produced by enzymes in our daily life. Since papain (EC 3.4.22.2) was used, probably as the first exogenous enzyme, to prevent the formation of chill hazes in beer,1 many enzymes isolated from various species have been developed for industrial use. These enzymes are used as biological catalysts in various industries such as detergent, food, chemical, textile, pharmaceutical, and paper industries. For instance, proteolytic enzymes are used for detergents; pharmaceutical agents; leather bating; enzymatic conversion of peptidyl substances; and food processing such as cheese production, meat tenderizing, dough conditioning in baking, and protein recovery from waste food materials.2 Most of the industrial enzymes have been isolated from mesophiles and thermophiles, since innumerable kinds of mesophiles are easily obtained from environmental sources, and enzymes obtained from thermophiles are suitable for industrial processes due to their thermostability. Heat-stable enzymes isolated from thermophiles also have an advantage in terms of storage stability, because they can be transported and stored at the ambient temperature. On the other hand, although many enzymes have also been isolated from cold-adapted microorganisms,3 psychrophiles and psychrotrophs, there have been few reports on the industrial use of such enzymes. It is reasonable to expect that cold-adapted microorganisms produce enzymes that are active even at a low temperature, i.e., “cold-active enzymes.” They would not only be more active at a low temperature than enzymes isolated from mesophiles and thermophiles but would also presumably have distinct characteristics.

Mechanism of inactivation of tobacco mosaic virus with ozone

Abstract The inactivation mechanism of tobacco mosaic virus (TMV) in a phosphate buffer (pH 6.9) by ozone was studied. We previously reported that the damage of naked TMV-RNA occurred at the guanine moiety of RNA (Shinriki et al., Biochim. biophys. Acta655, 323, 1981). In this paper, we clarified the mode of the inactivation of TMV by using tritium-labeled TMV (TMV∗) prepared by the reconstitution of tritium-labeled TMV-RNA (TMV-RNA∗) and coat protein of TMV. It was found that the amount of extracted TMV-RNA∗ from ozone-treated TMV∗ decreased with the advance of ozonization, and that there was good correlation between the loss of infectivity and the decrease of recovery of TMV-RNA∗. When TMV lost its infectivity due to ozone, tryptophan and tyrosine of the coat protein were also degraded by ozone. Polyacrylamide gel electrophoretic analysis of the substance produced during ozonization showed that the coat protein subunits were aggregated with each other and cross-linked with TMV-RNA∗. From these results, it was concluded that the inability of uncoating is the major cause of the TMV inactivation by ozone.

Molecular cloning of guinea pig CYP1A1 : complete primary structure and fast mobility of expressed protein on electrophoresis

Guinea pig CYP1A1 cDNA was isolated from a liver cDNA library of guinea pig treated with 3-methylcholanthrene. The cDNA, named GPc1, was 2674 bp long and contained an entire coding region for 516 amino acids. The amino acid sequence of guinea pig CYP1A1 shared 74-78% identity with those of the other mammalian CYP1A1s. RNA blot and immunoblot analyses revealed that CYP1A1 was constitutively expressed and was induced by 3-methylcholanthrene in guinea pig liver. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, guinea pig CYP1A1 expressed in yeast had a significantly smaller apparent molecular mass than expressed mouse CYP1A1. An alignment of the amino acid sequences of mammalian CYP1A1s demonstrated that guinea pig CYP1A1 was several residues shorter than the counterparts in the N-terminal region. Thus, to clarify the contribution of the N-terminal sequence of guinea pig CYP1A1 to the fast mobility on the electrophoresis, mouse-guinea pig chimeric CYP1A1 was prepared through cDNA-directed expression in yeast. The chimeric CYP1A1 protein had an intermediate molecular mass between mouse and guinea pig CYP1A1s indicating that the anomalous mobility of guinea pig CYP1A1 is in part due to the shortened N-terminal amino acid sequence of the protein.

Mouse NADPH-cytochrome P-450 oxidoreductase: Molecular cloning and functional expression in yeast

We published isolation of a mouse NADPH-cytochrome P-450 oxidoreductase cDNA and afterward ascribed the cDNA to the guinea-pig instead of the mouse (Ohgiya, S. et al. (1992) Biochim. Biophys. Acta 1171, 103-105 and Corrigendum (1993) Biochim. Biophys. Acta 1174, 313). We report here nucleotide and deduced amino acid sequences of an NADPH-cytochrome P-450 oxidoreductase cDNA isolated from the ddY mouse. The mouse cytochrome P-450 oxidoreductase shares 98.4% identity with its rat counterpart. In particular, clusters of acidic residues that presumably participate in interaction with cytochrome P-450 are highly conserved in primary structures of mammalian cytochrome P-450 oxidoreductases. The mouse cytochrome P-450 oxidoreductase was functionally expressed in yeast using a modified cDNA clone lacking whole noncoding regions.

Lipase-mediated resolution of cis-1-amino-2-indanol, the key component of the HIV protease inhibitor indinavir

Abstract The efficient and direct resolution of cis -1-amino-2-indanol using Candida antarctica lipase B catalyzing the alcoholysis of its N , O -diacetyl derivative is reported.

Molecular cloning and sequence analysis of mouse NADPH-cytochrome P-450 oxidoreductase

Abstract A cDNA clone coding for cytochrome P -450 oxidoreductase was isolated from a mouse liver cDNA library. The cDNA, MSr2, contained a complete coding region of 678 amino acids. The amino acid sequence of the mouse cytochrome P -450 oxidoreductase showed approx. 90% identities with those of rat, human, rabbit, pig enzymes indicating conservation of primary structure of the enzyme during evolutionary divergence of species. The high conservation of acidic residues of the enzyme sustained the importance of them to maintain its function.

Upgrading of coal-derived liquids: 3. Characterization of upgraded liquids by thin-layer chromatography combined with flame-ionization detection

Abstract Four components, namely, saturates, aromatics, resins and asphaltenes, in upgraded coal-derived liquids were determined by thin-layer chromatography combined with flame-ionization detection (t.l.c./FID method). The results agree with previous results using various analytical methods. The t.l.c./FID method was found to be a simple and effective method for screening of hydroconversion catalysts and also for a follow-up investigation of the chemical changes of coal-derived liquids during the hydroconversion process.