Production of extracellular laccase by the white-rot fungus Pycnoporus sanguineus was examined in batch submerged cultures in shake flasks, baffled shake flasks and a stirred tank bioreactor. The biomass growth in the various culture systems closely followed a logistic growth model. The production of laccase followed a Luedeking-Piret model. A modified Luedeking-Piret model incorporating logistic growth effectively described the consumption of glucose. Biomass productivity, enzyme productivity and substrate consumption were enhanced in baffled shake flasks relative to the cases for the conventional shake flasks. This was associated with improved oxygen transfer in the presence of the baffles. The best results were obtained in the stirred tank bioreactor. At 28 °C, pH 4.5, an agitation speed of 600 rpm and a dissolved oxygen concentration of ~25 % of air saturation, the laccase productivity in the bioreactor exceeded 19 U L(-1 )days(-1), or 1.5-fold better than the best case for the baffled shake flask. The final concentration of the enzyme was about 325 U L(-1).
Drosophila melanogaster glutathione S-transferase D3 (DmGSTD3) has a shorter amino acid sequence as compared to other GSTs known in the fruit flies. This is due to the 15 amino acid N-terminal truncation in which normally active amino acid residue is located. The work has made use of homology modeling to visualize the arrangement of amino acid side chains in the glutathione (GSH) substrate cavity. The identified amino acids were then replaced with amino acids without functional groups in the side chains and the mutants were analyzed kinetically. Homology modeling revealed that the side chains of Y89 and Y97 were shown facing toward the substrate cavity proposing their possible role in catalyzing the conjugation. Y97A and Y89A GSH gave large changes in Km (twofold increase), Vmax (fivefold reduction), and Kcat /Km values for GSH suggesting their significant role in the conjugation reaction. The replacement at either positions has not affected the affinity of the enzyme toward 1-chloro-2,4-dinitrobenzene as no significant change in values of Kmax was observed. The replacement, however, had significantly reduced the catalytic efficiency of both mutants with (Kcat /Km )(GSH) and (Kcat /Km )(CDNB) of eight- and twofold reduction. The recombinant DmGSTD3 has shown no activity toward 1,2-dichloro-4-nitrobenzene, 2,4-hexadienal, 2,4-heptadienal, p-nitrobenzyl chloride, ethacrynic acid, and sulfobromophthalein. Therefore, it was evident that DmGSTD3 has made use of distal amino acids Y97 and Y89 for GSH conjugation.