Seasonal, Locality, and Habitat Variation in Assemblages of Carrion-Associated Diptera in Gauteng Province, South Africa.

Seasonal, spatial, and habitat responses of carrion-associated Diptera assemblages can provide valuable information about the presence or absence of species and their relative abundance, and thereby enhance understanding of their responses to environmental variables and how this may have an impact on forensic investigations. Three different nature reserves (localities) within the Municipality of Tshwane, South Africa, were selected to determine whether species assemblages of carrion-feeding flies differ between seasons, localities, and habitat types. A total of 59,511 adult Diptera, identified to 35 species in eight different families, were collected using modified Redtop hanging traps, baited with liver and fish, during four seasons in three different habitat types. Species assemblages differed temporally, with season being the main factor determining species diversity and not locality or habitat. However, savanna and human-disturbed habitats supported a higher abundance and species richness than grassland habitats. Areas adjacent to the localities, such as large urban expanses in Dinokeng or agricultural holdings in Rietvlei, led to an increase in the abundance and mean species richness of carrion-associated Diptera, and in increased numbers of pest or invasive species such as Chrysomya megacephala (F.). Despite this, the overall species assemblages present in human-disturbed areas were very similar to those recorded in natural habitats.

Biochemical characterization and mode of action of a thermostable endoglucanase purified from Thermoascus aurantiacus.

A major extracellular endoglucanase purified to homogeneity from Thermoascus aurantiacus had a M(r) of 34 kDa and a pI of 3.7 and was optimally active at 70-80 degrees C and pH 4.0-4.4. It was stable at pH 2.8-6.8 at 50 degrees C for 48 h and maintained its secondary structure and folded conformation up to 70 degrees C at pH 5.0 and 2.8, respectively. A 33-amino acid sequence at the N terminus showed considerable homology with 14 microbial endoglucanases having highly conserved 8 amino acids (positions 10-17) and Gly, Pro, Gly, and Pro at positions 8, 22, 23, and 32, respectively. The enzyme is rich in Asp (15%) and Glu (10%) with a carbohydrate content of 2.7%. Polyclonal antibodies of endoglucanase cross-reacted with their own antigen and with other purified cellulases from T. aurantiacus. The endoglucanase was specific for polymeric substrates with highest activity toward carboxymethyl cellulose followed by barley beta-glucan and lichenan. It preferentially cleaved the internal glycosidic bonds of Glc(n) and MeUmbGlc(n) and possessed an extended substrate-binding site with five subsites. The data indicate that the endoglucanase from T. aurantiacus is a member of glycoside hydrolase family 5.

Biochemical characterization and mechanism of action of a thermostable beta-glucosidase purified from Thermoascus aurantiacus.

An extracellular beta-glucosidase from Thermoascus aurantiacus was purified to homogeneity by DEAE-Sepharose, Ultrogel AcA 44 and Mono-P column chromatography. The enzyme was a homotrimer, with a monomer molecular mass of 120 kDa; only the trimer was optimally active at 80 degrees C and at pH 4.5. At 90 degrees C, the enzyme showed 70% of its optimal activity. It was stable at pH 5.2 and at temperatures up to 70 degrees C for 48 h, but stability decreased above 70 degrees C and at pH values above and below 5.0. The enzyme hydrolysed aryl and alkyl beta-d-glucosides and cello-oligosaccharides, and was specific for substrates with a beta-glycosidic linkage. The hydroxy groups at positions 2, 4 and 6 of a glucose residue at the non-reducing end of a disaccharide appeared to be essential for catalysis. The enzyme had the lowest K(m) towards p-nitrophenyl beta-d-glucoside (0.1137 mM) and the highest k(cat) towards cellobiose and beta,beta-trehalose (17052 min(-1)). It released one glucose unit at a time from the non-reducing end of cello-oligosaccharides, and the rate of hydrolysis decreased with an increase in chain length. Glucose and d-delta-gluconolactone inhibited the beta-glucosidase competitively, with K(i) values of 0.29 mM and 8.3 nM respectively, while methanol, ethanol and propan-2-ol activated the enzyme. The enzyme catalysed the synthesis of methyl, ethyl and propyl beta-d-glucosides in the presence of methanol, ethanol and propan-2-ol respectively with either glucose or cellobiose, although cellobiose was preferred. An acidic pH favoured hydrolysis and transglycosylation, but high concentrations of alcohols favoured the latter reaction. The stereochemistry of cellobiose hydrolysis revealed that beta-glucosidase from T. aurantiacus is a retaining glycosidase, while N-terminal amino acid sequence alignment indicated that it is a member of glycoside hydrolase family 3.

Crystallization and preliminary X-ray analysis of the major endoglucanase from Thermoascus aurantiacus.

The major endoglucanase (35 kDa) from the thermophilic fungus Thermoascus aurantiacus has been purified from culture filtrates using an affinity method and the sequence for 35 N-terminal amino acids determined. This has allowed assignment of the enzyme to subtype A6 of family 5 endoglucanases. The enzyme has been crystallized as thick plates by the hanging-drop method using ammonium sulfate as precipitant. The crystals belong to space group P2(1)2(1)2(1) with cell edges a = 76.4, b = 85.7 and c = 89.5 A, with two molecules in the asymmetric unit, and diffract to 1.62 A resolution using synchrotron radiation. The structure will be solved by isomorphous replacement.