to 90 mL of DI H20 and subsequently transferred to a 100 mL centrifuge tube. The

suspensions were refrigerated until use (Sinsabaugh et al., 1991).

Enzyme Analysis

 Hydrolytic enzyme activity was determined using methylumbelliferyl (MUF) and

aminomethylcoumarin (AMC) substrates. Substrate concentrations were optimized at

saturating conditions. The activities of P-glucosidase (BGL), cellobiohydrolase (CBH),

phosphatase (PHO), leucine aminopeptidase (LEU), phenol oxidase (PHE) and

peroxidase (PER) were assayed using MUF-P-D-glucoside (Sigma M3633), MUF-

cellobioside (Sigma M6018), MUF-phosphate (Sigma M8168), L-Leucine

amidomethylcoumarin (Sigma L2145), L-3,4-dihydroxyphenylalanine dopaA), and

DOPA + H202 as substrates, respectively.

 MUF and AMC substrate conversion was measured using a Cytofluor 600TM

automated spectrofluorimeter (PerSeptive Biosystems, Inc., Framingham, MA) with

KineticalcTM software at 360 nm excitation and 460 nm emission at 200 C. Assays were

performed using Corning 48-well culture plates in which 400 [iL of sample, 360 [iL of

10 mM Tris-HCI pH 8.5, and 40 [iL of substrate were added. Stock substrate

concentrations were 2000 [iM for MUF-P-D glucoside, 1000 [iM for MUF-phosphate,

1200 [iM for MUF-cellobioside, and 6000 [iM for L-Leucine aminomethylcoumarin

resulting in well concentrations of 100, 50, 60 and 300 [iM, respectively. Each sample

analysis was performed in quadruplicate. Initial and final fluorescence measurements, as

well as measurements every five minutes, were taken during the 1 hour incubation.

Graphs produced from the readings taken every five minutes were analyzed to ensure that

linear kinetics were being observed. Final apparent enzyme activities were calculated as

moless substrate released g-1 AFDM h-1.