Molecular properties of extracellular Botrytis cinerea laccase

Two molecular forms of extracellular laccase induced by different phenolics were studied in Botrytis cinerea. The enzyme induced by grapejuice had a MW of 38 000 and contained 80 % sugar while that induced by gallic acid had a MW of 36 000 and contained 70 % sugar. Both forms contained arabinose, xylose, mannose, galactose and glucose but differed markedly in the relative content of these sugars. Tunicamycin, which inhibits glycosylation of peptide chains, considerably reduced the level of laccase in both hyphae and medium. The two enzyme forms differed also in their isoelectric focusing pattern and amino acid composition, the grape juice enzyme being richer in basic amino acids and poorer in acidic ones. A third form, induced by p-coumaric acid, resembled the laccase induced by gallic acid in many of its properties but was apparently not identical to it. The possible significance of the various forms in relation to the infection process by the fungus is discussed.     

Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances

The aim of broth and agar dilution methods is to determine the lowest concentration of the assayed antimicrobial agent (minimal inhibitory concentration, MIC) that, under defined test conditions, inhibits the visible growth of the bacterium being investigated. MIC values are used to determine susceptibilities of bacteria to drugs and also to evaluate the activity of new antimicrobial agents. Agar dilution involves the incorporation of different concentrations of the antimicrobial substance into a nutrient agar medium followed by the application of a standardized number of cells to the surface of the agar plate. For broth dilution, often determined in 96-well microtiter plate format, bacteria are inoculated into a liquid growth medium in the presence of different concentrations of an antimicrobial agent. Growth is assessed after incubation for a defined period of time (16-20 h) and the MIC value is read. This protocol applies only to aerobic bacteria and can be completed in 3 d.     

Induction of laccase formation in Botrytis

Optimal conditions for laccase excretion by Botrytis cinerea were determined. Addition of gallic acid to the culture medium induced maximal enzyme production and excretion. Other inducers previously reported for other organisms were ineffective. The relationships between internal and external laccase formation were determined.