Advances in Food Mycology

Ecophysiology of Fusarium culmorum and Mycotoxin Production 127
solution (40 µl) and the appropriate buffer (20 µl) were pipetted into
the wells of the microtitre plate and incubated at 37°C for 1 h along
with the appropriate controls. The reaction was stopped by the addition
of 5 µl 1M sodium carbonate solution and left for 3 min. The
enzyme activity was measured, using a MRX multiscan plate reader
(Dynex Technologies Ltd., Billinghurst, UK), by the increase in
optical density at 405 nm caused by the liberation of ρ-nitrophenol
by enzymatic hydrolysis of the substrate. Enzyme activity was calculated
from a calibration curve of absorbance at 405 nm vs ρ-nitrophenol
concentration and expressed as µmol ρ-nitrophenol
released/min.
For specific activity determinations the protein concentration was
obtained using a Bicinchoninic acid protein assay kit (Sigma-Aldrich
Ltd, Poole, Dorset, UK). This kit consisted of bicinchoninic acid
solution, copper (II) sulphate pentahydrate 4% solution and albumin
standard (containing bovine serum albumin (BSA) at a concentration
of 1.0 mg/ml). Protein reduces alkaline Cu (II) to Cu (I), which forms
a purple complex with bicinchoninic acid (a highly specific chromogenic
reagent). The resultant absorbance at 550 nm is directly proportional
to the protein concentration. The working reagent was
obtained by the addition of 1 part copper (II) sulphate solution to
50 parts bicinchoninic acid solution. The reagent is stable for one day
provided it is stored in a closed container at room temperature.
Aliquots (10 µl) of each standard or enzyme extracts were placed in
the appropriate microtitre plate wells. Potassium phosphate extraction
buffer 10 mM pH 7.2 (10 µl) was pipetted into the blank wells. The
working reagent (200 µl) was added to each well, shaken and plates
incubated at 37°C for 30 min. The plates were allowed to cool to room
temperature before measuring the absorbance at 550 nm using a MRX
multiscan plate reader. The protein concentrations in the enzyme
extracts were obtained from the calibration curve of absorbance at
550 nm against BSA concentration. These values were used to calculate
the specific activity of the enzymes in nmol ρ-nitrophenol released
per min per µg protein.
2.5. Statistical Analyses
The data were analysed using ANOVA (SigmaStat, SPSS Inc.),
with significance values of