Date of Award

7-1-1992

Degree Type

Thesis

Degree Name

M.S.

Department

Department of Biology

First Advisor

Dr. William V. Dashek

Abstract

Coriolus versicolor, a white-rot Basidiomycete, secretes lignocellulolytic enzymes and polyphenol oxidase (PPO). While the former degrade wood polymers, the latter converts diphenols to diquinones. Because C. versicolor can be ‘batch-cultured’, overproduction and enhanced secretion of agriculturally/commercially useful enzymes are feasible. Reported are attempts to: define the timed-appearances of intra- and extracellular PPO, achieve possible PPO substrate induction, ascertain its secretion route and partially purify extracellular PPO. Whereas two protein peaks (6 and 12 days in a 16 day time-course) were observed for dialyzed mycelial homogenates, PPO specific activity (spc. act.) rose between 4 and 12 days and then declined. Dialyzed growth medium protein content climbed from 6 to 15 days and the PPO spc. act. increased linearly from 4 to 15 days. When dialyzed 12 and 15 day media aliquots were added to assay mixtures containing 100 mM catechol, syringic acid or gallic acid, significant differences in PPO spc. act. between substrates were noted. Time-dependent catechol addition to medium resulted in hyphal growth inhibition at early log but stimulation at log, stationary and decline phases. Light-electron microscopies/biochemistry suggest the secretion route permitting its regulation. Scanning electron microscopy (SEM) demonstrated that the hyphal tip wall was morphologically different from that of the mature region. Transmission electron microscopy (TEM) of glutaralaldehyde pre- and 0s04 post-fixed hyphae revealed mitochondria, ER, ribosomes, vacuoles and osmiophilic bodies in the mature region’s cytoplasm but electron lucent masses appressed to the plasmalemma in the growing tip. Centrifugation of homogenates of hyphae cultured 0-16 days followed by PPO assay revealed that the enzyme was particulate. To further delineate the route, both TEM substrate localization and immunoelectron microscopies were employed. Protein G chromatography of immunized rabbits’ serum yielded two peaks; one was ELISA-positive and UV-absorbing. Antibody was tagged with colloidal gold and SEM localized PPO outside the hyphal glucan matrix. Substrate localization involved interposing dihydroxyphenylalanine between pre- and post-fixations. Although electron dense reaction product occurred in hyphae, plasmolysis was observed. As for partial purification, medium dialysis followed by 0-30% (NH4)2S04 fractionation and subsequent 12,000 xg centrifugation yielded PPO within the supernatant and a loss of one of four proteins upon SDS-PAGE. While these yielded an enhanced PPO spc. act., subsequent DEAE chromatography did not. Gel filtration of authentic PPO and medium followed by PPO assay revealed similar elution profiles suggesting an effective purification step. These results indicate that PPO secretion is organelle-mediated and that extracellular PPO can be partially purified by conventional biochemistry.

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