Optimization of culture conditions for Aspergillus sojae expressing an Aspergillus fumigatus alpha-galactosidase

Using Response Surface Methodology, carbon and nitrogen sources and agitation speed for cultivation of Aspergillus sojae expressing the alpha-galactosidase gene, aglB of Aspergillus fumigatus IMI 385708 were optimized. Compared to cultivation in modified YpSs medium, cultivation in 250-mL Erlenmeyer flasks agitated at 276 rpm and containing 100 mL of optimized medium consisting of 10.5% molasses (w/v) and 1.3% NH4NO3 (w/v), 0.1% K2HPO4, and 0.005% MgSO4 center dot 7H(2)0 achieved a 4-fold increase in alpha-galactosidase production (10.4 U/mL). These results suggest the feasibility of industrial large scale production of an alpha-galactosidase known to be valuable in galactomannan modification

Oxidation of phenolic compounds by the bifunctional catalase-phenol oxidase (CATPO) from Scytalidium thermophilum

The thermophilic fungus Scytalidium thermophilum produces a novel bifunctional catalase with an additional phenol oxidase activity (CATPO); however, its phenol oxidation spectrum is not known. Here, 14 phenolic compounds were selected as substrates, among which (+)-catechin, catechol, caffeic acid, and chlorogenic acid yielded distinct oxidation products examined by reversed-phase HPLC chromatography method. Characterization of the products by LC-ESI/MS and UV-vis spectroscopy suggests the formation of dimers of dehydrocatechin type B (hydrophilic) and type A (hydrophobic), as well as oligomers, namely, a trimer and tetramer from (+)-catechin, the formation of a dimer and oligomer of catechol, a dimer from caffeic acid with a caffeicin-like structure, as well as trimeric and tetrameric derivatives, and a single major product from chlorogenic acid suggested to be a dimer. Based on the results, CATPO oxidizes phenolic compounds ranging from simple phenols to polyphenols but all having an ortho-diphenolic structure in common. The enzyme also appears to have stereoselectivity due to the oxidation of (+)-catechin, but not that of epicatechin. It is suggested that CATPO may contribute to the antioxidant mechanism of the fungus and may be of value for future food and biotechnology applications where such a bifunctional activity would be desirable

Cloning, expression and characterization of endo-β-1,4-mannanase from aspergillus fumigatus in aspergillus sojae and pichia pastoris

To be utilized in biomass conversion, including ethanol production and galactosylated oligosaccharide synthesis, namely prebiotics, the gene of extracellular endo-β-1,4-mannanase (EC 32.1.78) of Aspergillus fumigatus IMI 385708 (formerly known as Thermomyces lanuginosus IMI 158749) was expressed first in Aspergillus sojae and then in Pichia pastoris under the control of the glyceraldehyde triphosphate dehydrogenase (gpdA) and the alcohol oxidase (AOX1) promoters, respectively. The highest production of mannanase (352 U mL -1)in A. sojae was observed after 6 days of cultivation. In P. pastoris, the highest mannanase production was observed 10 h after induction with methanol (61 U mL-1). The fold increase in mannanase production was estimated as ∼12-fold and ∼2-fold in A. sojae and P. pastoris, respectively, when compared with A. fumigatus. Both recombinant enzymes showed molecular mass of about 60 kDa and similar specific activities (∼350 U mg-1 protein). Temperature optima were at 60°C and 45°C, and maximum activity was at pH 4.5 and 5.2 for A. sojae and P. pastoris, respectively. The enzyme from P. pastoris was more stable retaining most of the activity up to 50°C, whereas the enzyme from A. sojae rapidly lost activity above 40°C

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

Towards Active Applications: the Virtual Virtual Machine Approach.

With the wide acceptance of distributed computing a rapidly growing number of application domains are emerging, leading to a growing number of ad hoc solutions that are rigid and poorly interoperable. Our response to this situation is a platform for building flexible and interoperable execution environments called the Virtual Virtual Machine. This article presents our approach, the architecture of the VVM and some of its primary applications