Date of Award:

5-1991

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Chemistry and Biochemistry

Department name when degree awarded

Biochemistry/Molecular Biology

Committee Chair(s)

Joseph K. -K. Li

Committee

Joseph K. -K. Li

Committee

Steven D. Aust

Committee

Thomas F. Emery

Committee

Thomas Grover

Committee

Dennis L. Welker

Abstract

In a collaborative effort with Dr. Steven D. Aust's group at Utah State University, this research is the first to describe the heterologous expression of active peroxidases from the filamentous fungus Panerochaete chrysosporium. The cDNA clones λML-1, λML-6, λML-5 and λMP-1, which had been tentatively identified as fungal peroxidase clones, were subcloned into baculovirus transfer vectors and recombinant Autographa californica nuclear polyhedrosis viruses were constructed which contained copies of these potential peroxidase genes. The recombinant baculovirus clones were used to infect insect cells for the heterologous production of active recombinant enzymes. Three recombinant lignin peroxidases (H2, H6 and H8) and one recombinant manganese (II)-dependent peroxidase (H4) were identified, using monoclonal and polyclonal antibodies raised against the native fungal isozymes, within the extracellular fluid of the infected insect cells. All of the recombinant fungal peroxidases were shown to be hemoproteins containing N-linked oligosaccharide(s). The recombinant lignin peroxidases H2 and H8 were purified to near homogeneity and characterized. These two enzymes had apparent molecular weights, anion-exchange chromatographic profiles and substrate specificities which were similar to those of the native fungal isozymes H2 and H8. The recombinant Mn(II)-dependent peroxidase H4 was partially purified and also characterized. This enzyme had an apparent molecular weight, an anion-exchange chromatographic profile and substrate specificities which paralleled those of the native fungal isozyme H4. Since the recombinant proteins were similar in most respects to the native isozymes, this heterologous system appears to be amenable for detailed structure/function analysis of the fungal peroxidases and probably horseradish peroxidase as well.

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