Tai Tokuyama

Utilization of Aromatic Compounds by Trichosporon cutaneum KUY-6A

Abstract Trichosporon cutaneum KUY-6A, a cyclohexanecarboxylic acid-utilizing yeast, grew well on phenol, benzoic acid, the isomers of hydroxybenzoic acid (HBA), dihydroxybenzene and dihydroxybenzoic acid except 2.6-dihydroxybenzoic acid, but could not utilize aromatic compounds having Cl-, CH 3 - or NO 2 -groups or the isomers of phthalic acid. From the degradation behavior of all HBA isomers, it is concluded that strain KUY-6A can utilize all HBA isomers at concentrations higher than those reported previously. Furthermore, the culture conditions for o -HBA were found to differ considerably from those of m - or p -HBA.

Identification of an ice-nucleating bacterium and its ice nucleation properties

Abstract An ice-nucleating bacterium, KUIN-2, was isolated from carrot leaves. The ice-nucleating bacterium was found in the white colony group. KUIN-2 was identified as Pseudomonas viridiflava from its taxonomic characteristics. When KUIN-2 was cultured aerobically in a medium consisting of Trypticase soy broth (pH 6.0) for 24 h at 18°C, the ice-nucleating activity of KUIN-2 cells was obtained. Ice nucleation was detected at −2.8°C in cell suspensions (1.4 × 10 8 cells/ml) of KUIN-2. The nucleation frequency of KUIN-2 was greatly inhibited by the addition of urea or N -ethylmaleimide.

Purification and characterization of N,N-dimethylformamidase from Alcaligenes sp. KUFA-1

Abstract A bacterium strain capable of growing on N,N -dimethylformamide (DMF) was isolated using enrichment and isolation techniques. The isolated strain, KUFA-1, was classified as belonging to the genus Alcaligenes which has not been listed as DMF-utilizer previously. Crude soluble extracts of Alcaligenes KUFA-1 grown on DMF were found to contain dimethylformamidase (DMFase). The DMFase was purified to an apparent electrophoretic homogeneity with an overall 38.6-fold purification, a 6.7% yield and a final specific activity of 26.25 μmol DMF hydrolyzed/min/mg protein. The native DMFase had a relative molecular mass of 180 kDa and was composed of one light-chain ( M r =16,000) and one heavy-chain ( M r =74,000). The optimum pH and temperature were 6.6 and 55°C, respectively. The enzyme was highly specific for DMF. DMFase obeyed Michaelis-Menten kinetics and its K m and V max values for DMF were 1.28 mM and 83.3 U/mg, respectively, as determined using a Lineweaver-Burk plot.

Properties of cell-free ice nuclei from ice nucleation-active Pseudomonas fluorescens KUIN-1

Abstract The properties of cell-free ice nuclei (CFIN) from ice nucleation-active Pseudomonas fluorescens KUIN-1 were investigated. CFIN were found to be present in the culture supernatant liquid and were separated from P. fluorescens KUIN-1 by centrifugation, filtration (0.22 μm, pore size) and ultrafiltration. Sucrose was a good carbon source for the ice-nucleating activity of CFIN and l -sodium glutamate was a good nitrogen source. In an ice-nucleating medium in 1 l of water, maximum growth was obtained with production of 118 mg of CFIN at 18°C after 48 h with shaking. The ice-nucleating activity was greatly decreased by treating CFIN at 40°C for 30 min and was completely lost by heating at 90°C. The ice-nucleating activity of CFIN was inhibited by the addition of protease K, thermolysin or lipase. However, phospholipase A2 had no effect. The freezing difference spectra in D2O-H2O for CFIN were similar to the freezing difference spectra in D2O-H2O for the cells. Consequently, it could be inferred that there were three types of nucleating activity on CFIN. CFIN active at −3.2∼−4.3°C, −4.5 to −6.1°C or >−8.1°C were arbitrarily assigned into three groups—class A, class B or class C.

Purification and characterization of a novel cyclohexylamine oxidase from the cyclohexylamine-degrading Brevibacterium oxydans IH-35A

Cyclohexylamine oxidase (CHAO) from a cell extract of Brevibacterium grown on cyclohexylamine was purified 50.2-fold, to electrophoretic homogeneity, by serial chromatographies. The molecular mass of the native enzyme was estimated to be approximately 50 kDa by gel filtration and SDS-PAGE. The optimum pH was 7.4 and the stable pH range was 6.0 to 7.0. The enzyme was thermostable up to 30 degrees C. The enzyme was found to be highly specific for the deamination of alicyclic monoamines such as cyclopentylamine, cycloheptylamine, and N-methylcyclohexylamine and aliphatic monoamines, such as sec-butylamine. The apparent K(m) value for cyclohexylamine was 1.23 mM. The enzyme was inhibited by flavin enzyme inhibitors such as quinine and quinacrine. The N-terminal 27 amino acid residues were determined as Gly-Ser-Val-Thr-Pro-Asp-Pro-Asp-Val-Asp-Val-Ile-Ile-His-Gly-Ala-Gly-Ile-Ser-Gly-Ser-Ala-Ala-Ala-Lys-Ala-Leu-, revealing homology to conventional flavin-containing amine oxidases (EC 1.4.3.4).

Ice-nucleating activity of Pseudomonas fluorescens

Abstract Pseudomonas fluorescens KUIN-1 can cause the freezing of water at relatively warm termperatures (−2.9 to −30°C). Glucose, glycerol, and citric acid were good substrates yielding cells with high ice-nucleating activity. Ammonium salts were good nitrogen sourcfes yielding cells with high ice-nucleating activity. The ice-nucleating activity of KUIN-1 was not affected by pH between pH 5.5 to pH 8.0. The ice-nucleating activity of cells was greatly influenced by heat treatment.

Release of Cell-Free Ice Nuclei from Pseudomonas viridiflava with a Triton X-100/EDTA System and Their Ice Nucleation Properties

Abstract The release of cell-free ice nuclei from Pseudomonas viridiflava KUIN-2 cells with Triton X-100 in the presence of EDTA-2Na, NaSCN, Na 2 SO 4 , or KCI was attempted. Cell-free ice nuclei were shed from P. viridiflava KUIN-2 cells with 2% Triton X-100 in the presence of 10 mM EDTA-2Na. KUIN-2 shed cell-free ice nuclei (0.22 mg protein/ml) active at −4.9 to −5.8°C. The ice-nucleating activity was greatly decreased by treating the cell-free ice nuclei for 30 min at 40°C, and was completely lost by heating at 90°C. The ice-nucleating activity of the cell-free ice nuclei was inhibited by the addition of proteases, protein-modifying reagents and denaturants. However, phospholipase C had no such effect. It is suggested that the ice-nucleating activity sites of cell-free ice nuclei contain proteins. The ice-nucleating activity of cell-free ice nuclei containing Mg 2+ occurred within the temperature range −3.3 to −3.6°C.

Cloning of bacterial ice nucleation genes of Pseudomonas viridiflava in Escherichia coli

Abstract A cosmid library containing Pseudomonas viridiflava KUIN-2 DNA was constructed in the vector Homer III and used to transform E. coli IIB101. Five colonies (INAC-1∼-5) with ice-nucleating activity (INA) were obtained and all these transformants had in common an approximately 13.2-kb Eco R1-fragment (pNVR-1). Among them, the transformant INAC-2, which harbored a single plasmid (pCH-1) and had an excellent INA, was selected for studying the characteristics of the INA + transformant. The highest INA was obtained when INAC-2 was cultured aerobically in Luria broth (pH 7.2) for 15–20 h at 20°C. The INA in culture was not detectable until the bacterial titer reached 10 5 to 10 6 cells/ml. The heat stability of the ice-nucleating substance in the transformant was more thermostable than that in the donor strain. Further, E. coli HB101 harboring the self-circularized pNVR-1 had INA.

Expression of the ice nucleation active gene of a novel plasmid pNVR-1 from Pseudomonas viridiflava in Escherichia coli and Pseudomonas aeruginosa

A novel plasmid pNVR-1, a self-circularized DNA segment which consists of a 13.2 kb- Eco RI fragment from Pseudomonas viridiflava KUIN-2 total DNA, was introduced into both Escherichia coli JM109 and P. aeruginosa PAO 1161 by transformation. Both of the strains harboring the pNVR-1 plasmid showed good ice nucleation activity (INA) and resistance to ampicillin. In addition, the optimum growth temperatures for these strains shifted to temperatures lower than those of strains without pNVR-1. The pNVR-1 was more stable in E. coli cells than in P. aeruginosa cells. The number of copies of pNVR-1 in E. coli cells was approximately 20 per chromosome equivalent.

Construction of a new shuttle vector pNVR-1012 and the cloning of salicylate hydroxylase gene in Pseudomonas aeruginosa and Escherichia coli

Abstract A mini-plasmid, pNVR-1012 (approximately 2.7 kb), was constructed from the Pseudomonas viridiflava novel plasmid which encoded the ice-nucleation gene. This plasmid encoded an ampicillin resistance gene, and had the unique restriction sites of Eco RI, Hind III, Pst I, Sal I and Sph I. pNVR- nahG was constructed by ligation of a 3.1-kb Hind III fragment containing the salicylate hydroxylase ( nahG ) gene from the NAH plasmid with Hind III-digested pNVR-1012. The nahG gene was expressed in both Escherichia coli and Pseudomonas aeruginosa harboring this recombinant plasmid.