MUF and AMC substrate enzymatic analysis 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 Coming 48-well culture plates in which 400 giL of sample, 360

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

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

and 6000 [iM for L-Leucine amidomethylcoumarin resulting in well concentrations of

100 giM, 50 giM, and 300 giM, respectively. Each sample run 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 was being

observed. Concentrations of MUF and AMC released were calculated by the application

of standard curves to the initial and final fluoresences. The difference in concentrations

yielded the substrate released during the incubation period.

 The effects of quenching on MUF and AMC substrates were determined in order to

account for fluorescence blocking or absorption effects caused by coloring, particle

suspension, humic matter, self-quenching, or other inhibitions in the suspensions. MUF

and AMC standards were placed in each sample suspension to determine the quench

percentage of each matrix. The final and initial fluoresences were then converted using

the appropriate quench percentage.

 Phenol oxidase and peroxidase analyses were performed per Sinsabaugh (personal

communication). 2.0 mL soil suspension was added to 2.0 mL 10 mM L-DOPA

dissolved in 10 mM Tris-HCL pH 8.5 in 10 mL EppendorfFM centrifuge tubes. The