Measuring fungal cellulolytic enzyme activity in degrading wood
S M Duncan, J S Schilling
An enzyme assay which measures enzyme activity directly from wood will assist in the fundamental understanding of the enzyme components of the decay mechanism of rot fungi and any changes in the presence of wood protectants. At present, to measure fungal carbohydrate degrading enzyme activity in the presence of wood, two methods are used: either 1) the enzyme is measured in the growth media containing lignocellulosic material after the fungus has been allowed to utilize the cellulolytic substrate or 2) the fungus is grown on the lignocellulosic material and the enzymes are extracted using buffers after the growth period and the buffer is analyzed for enzyme activity. This research presents an enzyme assay where wood slivers are added to the assay mixture. The brown rot fungus Postia placenta was grown on wafers measuring 70mm by 23mm by 1.5mm for 12 days. The wafers were then sliced into 3mm by 1mm slivers and assayed using a micro assay using Azo-carbohydrates as substrates. The use of a 96-well microtiter plate allowed a large number of samples to be analyzed at one time with reduced reagent use. Using this assay method allows screening for enzyme activity along the length of the wafer. Our results showed detectable differences over the length of the wafer with a peak of endoglucanase activity closest to the colonization point of P.placenta. Small samples of decayed wood from the field were assayed for endoglucanase activity and the results indicate that this method has a potentially to be used to determine early decay. This assay will be useful in many research fields to gauge the presence and location of fungi and their carbohydrate degrading enzyme activity within a wood sample.