Differentiation of Salmonella typhimurium from Salmonella enteritidis and other Salmonella serotypes using random amplified polymorphic DNA analysis.

Salmonella enterica ssp. enterica serovar Typhimurium and Salmonella enterica ssp. enterica serovar Enteritidis are the major dominating serotypes of Salmonella in poultry and poultry products. Infection by Salmonella Typhimurium is an important cause of morbidity and mortality in poultry. Rapid differentiation of Salmonella Typhimurium from other Salmonella serotypes including Salmonella Enteritidis can be very crutial for public health and for epidemiologists and for the poultry industry. Ten arbitrarily designed short primers (8 to 10 bases) were used in the random amplified polymorphic DNA analysis of Salmonella Typhimurium. One of the primers, primer 3 (5'-CGT GCA CGC-3'), resulted in the amplification of a band pattern that was unique to Salmonella Typhimurium. In total, 24 strains of serotype Salmonella Typhimurium were used during the study. Eighteen of them are clinical isolates, 2 of them chicken isolates (A6, A20), 2 of them from the Pasteur Institute, 1 from Refik Saydam National Culture Collection, and 1 is a type culture strain from National Culture Type Collection. Serotype Salmonella Typhimurium strains, which were collected from several different hospitals, institutes, and culture collections, have all displayed the same amplification band by primer 3. Twenty-three strains of 16 different serotypes of salmoneallae including 11 Salmonella Enteritidis strains gave only a 300-bp amplification band or no bands, whereas an additional 700-bp amplification band was observed only in samples of Salmonella Typhimurium serotype. It is concluded that random amplified polymorphic DNA analysis with primer 3 is of potential use as a serotype-specific marker for Salmonella Typhimurium.

Production, properties and application to biocatalysis of a novel extracellular alkaline phenol oxidase from the thermophilic fungus Scytalidium thermophilum.

Scytalidium thermophilum produces an extracellular phenol oxidase on glucose-containing medium. Certain phenolic acids, specifically gallic acid and tannic acid, induce the expression of the enzyme. Production at 45 degrees C in batch cultures is growth-associated and is enhanced in the presence of 160 microM CuSO4 x 5 H2O and 3 mM gallic acid. The highest enzyme activity is observed at pH 7.5 and 65 degrees C, on catechol. When incubated for 1 h at pH 7 and pH 8, 95% and 86% of the activity is retained. Thermostability decreases gradually from 40 degrees C to 80 degrees C. Estimated molecular mass is c. 83 kDa, and pI is acidic at c. 5.4. Substrate specificity and inhibition analysis in culture supernatants suggest that the enzyme has unique properties showing activity towards catechol; 3,4-dihydroxy-L-phenylalanine (L-DOPA); 4-amino-N, N-diethylaniline (ADA); p-hydroquinone; gallic acid; tannic acid and caffeic acid, and no activity towards L-tyrosine, guaiacol, 2,2'-azino-bis(3-ethyl-benzthiazoline-6-sulphonic acid) (ABTS) and syringaldazine. Inhibition is observed in the presence of salicyl hydroxamic acid (SHAM) and p-coumaric acid. Enzyme activity is enhanced by cetyltrimethylammonium bromide (CTAB) and polyvinylpyrrolidone (PVP), and the organic solvents dimethyl sulfoxide (DMSO) and ethanol. No inhibition is observed in the presence of carbon monoxide. Benzoin, benzoyl benzoin and hydrobenzoin are converted into benzil, and stereoselective oxidation is observed on hydrobenzoin. The reported enzyme is novel due to its catalytic properties resembling mainly catechol oxidases, but displaying some features of laccases at the same time.

Submerged cultivation of scytalidium thermophilum on complex lignocellulosic biomass for endoglucanase production.

Scytalidium thermophilum endoglucanase production was analyzed on lignocellulosic biomass in submerged cultures at 45 degrees C and 155 rpm for 8 days. Endoglucanase, adsorbability of endoglucanase onto avicel, as well as exoglucanase, and filter paper activities were determined and compared with those on microcrystalline cellulose (avicel) as the main source of carbon. Lentil bran and sunflower seed bagasse yielded c. 1.5 fold more endoglucanase and avicel-adsorbable endoglucanase activity than avicel, and activities on grass clippings were similar. Grass clippings yielded the highest percentage of avicel-adsorbable endoglucanase among all lignocellulosic substrates tested. By the time when endoglucanase activities reached maximal levels, exoglucanase activities on lentil bran, sunflower seed bagasse and grass clippings were c. 1.5-3 fold lower than those on avicel, although a significant difference in filter paper activities was not observed. On lignocellulosic biomass, maximum levels of endoglucanase activity were reached within 3-4 days, and within 6-7 days on avicel.

PCR with degenerate primers amplifies a subgenomic DNA fragment from the endoglucanase gene(s) of Torula thermophila, a thermophilic fungus.

The aim of this study was to enable the polymerase chain reaction (PCR) amplification of DNA fragments within endoglucanase gene(s) of Torula thermophila, by using degenerate primers so that the amplified fragment(s) could be used as homologous probe(s) for cloning of full-length endoglucanase gene(s). The design of the degenerate PCR primers was mainly based on the endoglucanase sequences of other fungi. The endoglucanase gene sequence of Humicola insolens was the only sequence from a thermophilic fungus publicly available in the literature. Therefore, the endoglucanase sequences of the two Trichoderma species, Trichoderma reesei and Trichoderma longibrachiatum, were used to generalize the primers. PCR amplification of T. thermophila genomic DNA with these primers resulted in a specific amplification. The specificity of the amplified fragment was shown by Southern hybridization analysis using egl3 gene of T. reesei as probe. This result suggested that the degenerate primers used in this study may be of value for studies aimed at cloning of endoglucanase genes from a range of related fungi.

Galactose oxidase of Dactylium dendroides. Gene cloning and sequence analysis.

The gaoA gene, encoding the secreted copper-containing enzyme galactose oxidase, has been isolated from the Deuteromycete fungus Dactylium dendroides. Degenerate oligonucleotide primers were designed from amino acid sequence data for use in the polymerase chain reaction. A 1.4-kilobase DNA fragment amplified from genomic DNA was used to screen a genomic library constructed in ZAP. A strongly hybridizing clone was rescued as a pBluescript derivative, pGAO9, by in vivo excision. The sequence of 3466 nucleotides of pGAO9 insert DNA was determined by progressively designing sequencing primers. The translation product of the single long open reading frame matches the available galactose oxidase peptide sequence data, which represents 42% of the residues in the protein. The mature enzyme has 639 residues, which have been assigned to a 1.7-A electron density map (Ito, N., Phillips, S. E. V., Stevens, C., Ogel, Z. B., McPherson, M. J., Keen, J. N., Yadav, K. D. S., and Knowles, P. F. (1991) Nature 350, 87-90). The gene lacks introns and encodes an mRNA of approximately 2.5 kilobases with three transcription initiation start points at least 324 nucleotides upstream of the translation start site. Multiple ATG codons are present between the transcription initiation region and the start of the mature protein; two in-frame ATGs could encode the initiating Met residue to give proteins with 89 or 41 residue N-terminal leader peptides. The shorter potential leader has N-terminal features characteristic of a secretion signal sequence and may also contain a pro-sequence processed by an enzyme specific for a monobasic (arginine) cleavage site, as proposed for other fungal genes. The codon bias of gaoA is characteristic of other filamentous fungal genes. No significant homologies exist between galactose oxidase and other protein sequences available in data bases.

Three-dimensional structure of galactose oxidase: an enzyme with a built-in secondary cofactor.

Galactose oxidase is a copper-containing enzyme, which catalyses stereospecific oxidation of primary alcohols. The three-dimensional structure of the enzyme has been determined in this study by X-ray crystallography at high resolution. The molecule is almost entirely composed of beta-structures and consists of three domains. The arrangement of 28 beta-strands in the second domain is of particular interest, having seven four-stranded antiparallel beta-sheets with pseudo-sevenfold symmetry. The copper site has square-pyramidal coordination with two histidines, one tyrosine and one exogenous ligand at the equatorial sites and another tyrosine at the axial site. The most intriguing structural feature is a covalent bond between C epsilon 1 of Tyr-272, which is one of the equatorial ligands, and S gamma of Cys-228. This unexpected thioether bond, and Trp-290 stacked above it, strongly supports the presence of a tyrosine free radical in the enzyme as a 'built-in' secondary cofactor. Calculation of the molecular surface shows a small pocket at the copper site and suggests a substrate-binding model, which can explain the substrate specificity. A model for the catalytic mechanism, involving a tyrosine free radical and basic tryptophan, is also proposed.

Novel thioether bond revealed by a 1.7 A crystal structure of galactose oxidase.

Galactose oxidase is an extracellular enzyme secreted by the fungus Dactylium dendroides. It is monomeric, with a relative molecular mass of 68,000, catalyses the stereospecific oxidation of a broad range of primary alcohol substrates and possesses a unique mononuclear copper site essential for catalysing a two-electron transfer reaction during the oxidation of primary alcohols to corresponding aldehydes. Recent evidence arguing against a Cu(III)-Cu(I) couple implies the existence of a second redox-active site proposed to involve pyrroloquinoline quinone or a tyrosine radical. We now report the crystal structure of galactose oxidase at 1.7 A resolution. This reveals a unique structural feature at the copper site with a novel thioether bond linking Cys 228 and Tyr 272 in a stacking interaction with Trp 290. We propose that these molecular components stabilize the protein free-radical species essential for catalysis and thus provide a 'built-in' secondary cofactor. This feature may represent a new mechanism for mediating electron transfer in metalloenzymes in the absence of exogenous cofactors.