Catechol 2,3-oxygenase production by genetically engineered Escherichia coli and its application to catechol determination

Catechol 2,3-oxygenase was produced by Escherichia coli, harbouring the recombinant plasmid pBH100 which contained the pheB gene cloned from phenol-degrading Pseudomonas putida BH, and was applied for the determination of catechol in the liquor. E. coli JM103 (pBH100) and C600 (pBH100) showed, respectively, about 5 and 8.5 times higher activities than that of P. putida BH. Using the crude extract prepared from the culture broth of the recombinant, catechol between 0.1 and 3.0 g/ml could be determined quantitatively in phosphate buffer, synthetic sewage and in mixtures of phenol, benzoate and sallcylate, and also in sodium pyruvate solution. In addition to catechol, 3-methylcatechol, 4-methylcatechol and 4-chlorocatechol could be determined. Oxygenase activity of the crude extract was maintained completely during the 100-day storage at –20°C after being freeze-dried with 10% acelone.M. Fujita, M. Ike, Y. Kawagoshi and N. Shinohara are with the Department of Environmental Engineering, Osaka University, 2-1, Yamadaoka, Suita, Osaka 565, Japan. T. Kamiya is with the Central Research Laboratory of Mitsubishi Electric Co., Amagasaki, Hyogo 661, Japan.     

Melanin biosynthesis inhibitors from Tarragon Artemisia dracunculus

The EtOH extract of tarragon Artemisia dracunculus, a perennial herb in the family Asteraceae, was found to potently inhibit α-melanocyte-stimulating hormone (α-MSH) induced melanin production in B16 mouse melanoma cells. Bioassay-guided fractionation led to the isolation of two alkamide compounds, isobutyl (1) and piperidiyl (2) amides of undeca-2E,4E-dien-8,10-dynoic acid. The respective EC(50) values for melanin biosynthesis inhibition were 1.8 and 2.3 μg/mL for 1 and 2.     

Purification and properties of polyvinyl alcohol oxidase with broad substrate range obtained from Pseudomonas vesicularis var. povalolyticus PH

Extracellular PVA oxidase produced by Pseudomonas vesicularis var. povalolyticus PH was purified to homogeneity by ammonium sulphate fractionation followed by successive column chromatography, and a study made of its characteristics. The molecular weight of the purified enzyme was estimated to be 75,000 by gel filtration and 85,000 by SDS-PAGE, suggesting that it consists of monomeric protein. Its isoelectric point was 5.7. The purified enzyme was colourless, and contained one atom of iron per molecule. It exhibited a broad pH activity profile with maximum activity at pH 10.0, and was stable between pH 6.0 and 10.0. The optimum temperature for enzyme activity was 40°C, with stability up to 45°C. The enzyme activity was inhibited strongly by Fe2+, Hg2+ and Sn2+, and weakly by Cu2+, EDTA, thiourea and IAA. The enzyme exhibited activity toward several secondary alcohols, suggesting that it was a secondary alcohol oxidase. In particular, the enzyme exhibited strong activity towards the larger secondary alcohols such as 2-octanol and 4-decanol, and relatively strong activity towards cyclohexanol and benzyl alcohol.     

Biodegradation of a polyvinyl alcohol-starch blend plastic film

Attempts were made to elucidate the degradation mechanism of a polyvinyl alcohol (PVA)-starch blend plastic. A part of the starch fraction of this plastic was dissolved into an aqueous phase in a control test. Treatment with a PVA-degrading bacterium or enzyme gave a maximal weight loss of approximately 70% and film breakage occurred. Since this plastic contains 40% PVA, it is apparent that not only the PVA fraction but also a considerable portion of the starch fraction was lost from the film by treatment with the PVA-degrading enzyme. As the PVA-degrading bacterium and enzyme used here showed no starch-degrading activity, loss of the starch fraction seems to depend on its dissolution with degradation of the PVA fraction. These experimental results indicated that the degradation of the PVA fraction is an important requisite for complete degradation or decomposition of this plastic film.     

The Staurotypus Turtles and Aves Share the Same Origin of Sex Chromosomes but Evolved Different Types of Heterogametic Sex Determination

Reptiles have a wide diversity of sex-determining mechanisms and types of sex chromosomes. Turtles exhibit temperature-dependent sex determination and genotypic sex determination, with male heterogametic (XX/XY) and female heterogametic (ZZ/ZW) sex chromosomes. Identification of sex chromosomes in many turtle species and their comparative genomic analysis are of great significance to understand the evolutionary processes of sex determination and sex chromosome differentiation in Testudines. The Mexican giant musk turtle (Staurotypus triporcatus, Kinosternidae, Testudines) and the giant musk turtle (Staurotypus salvinii) have heteromorphic XY sex chromosomes with a low degree of morphological differentiation; however, their origin and linkage group are still unknown. Cross-species chromosome painting with chromosome-specific DNA from Chinese soft-shelled turtle (Pelodiscus sinensis) revealed that the X and Y chromosomes of S. triporcatus have homology with P. sinensis chromosome 6, which corresponds to the chicken Z chromosome. We cloned cDNA fragments of S. triporcatus homologs of 16 chicken Z-linked genes and mapped them to S. triporcatus and S. salvinii chromosomes using fluorescence in situ hybridization. Sixteen genes were localized to the X and Y long arms in the same order in both species. The orders were also almost the same as those of the ostrich (Struthio camelus) Z chromosome, which retains the primitive state of the avian ancestral Z chromosome. These results strongly suggest that the X and Y chromosomes of Staurotypus turtles are at a very early stage of sex chromosome differentiation, and that these chromosomes and the avian ZW chromosomes share the same origin. Nonetheless, the turtles and birds acquired different systems of heterogametic sex determination during their evolution.     

Production and recovery of an enzyme fromPseudomonas vesicularis var.povalolyticus PH that degrades polyvinyl alcohol

A PVA-degrading enzyme was produced byPseudomonas vesicularis var.povalolyticus PH and accumulated intracellularly when grown in nutrient medium including tryptone and yeast extract without PVA. The internal enzyme activity increased with cell growth and was maximal when growth was maximal, whereas, external activity continued to increase. It was presumed that the enzyme secretion was induced by the presence of PVA in the culture medium. It was established that crude enzyme can be effectively recovered from the cell by osmotic shock treatment with sucrose or NaCl.     

Ribonuclease activity of rat liver perchloric acid-soluble protein, a potent inhibitor of protein synthesis.

Rat liver perchloric acid-soluble protein (L-PSP) is a potent inhibitor of cell-free protein synthesis; however, its mechanism of action is not known. Here we show that the protein is a unique ribonuclease and that this activity is responsible for the inhibition of translation. The addition of perchloric acid-soluble protein to a rabbit reticulocyte cell-free system at a concentration of 6.2 microM led to an almost complete inhibition of protein synthesis. The kinetics are unlike those of hemin-controlled inhibitor, a protein that acts at the initiation step. The inhibition appears to be due to an endoribonucleolytic activity of perchloric acid-soluble protein because L-PSP directly affects mRNA template activity and induces disaggregation of the reticulocyte polysomes into 80 S ribosomes, even in the presence of cycloheximide. These effects were observed with authentic as well as recombinant L-PSP. Analysis by thin-layer chromatography of [alpha-32P]UTP-labeled mRNA incubated with the protein showed production of the ribonucleoside 3'-monophosphates Ap, Gp, Up, and Cp, providing direct evidence that the protein is an endoribonuclease. When either 5'- or 3'-32P-labeled 5 S rRNA was the substrate, L-PSP cleaved phosphodiester bonds only in the single-stranded regions of the molecule.     

Purification and properties of the polyvinyl alcohol-degrading enzyme 2,4-pentanedione hydrolase obtained from Pseudomonas vesicularis var. povalolyticus PH

World Journal of Microbiology and Biotechnology -