Title

Heterologous expression of manganese peroxidase from Phanerochaete chrysosporium in Pichia pastoris

Date of Award

2003

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical and Chemical Engineering

Advisor(s)

Christine Kelly

Keywords

Manganese peroxidase, Phanerochaete chrysosporium, Pichia pastoris, Hyperglycosylation

Subject Categories

Biochemistry | Biochemistry, Biophysics, and Structural Biology | Chemical Engineering | Engineering | Life Sciences

Abstract

A gene encoding manganese peroxidase ( mnp1 ) was isolated from Phanerochaete chrysosporium by RT-PCR, and then cloned downstream of a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter in the methylotrophic yeast Pichia pastoris . Three different expression vectors were constructed in order to express the mnp1 gene in P. pastoris . pZBMNP contained the native P. chrysosporium fungal secretion signal and pαAMNP contained an α factor secretion signal derived from Saccharomyces cerevisiae . pZBIMNP did not have any secretion signal, and was used for intracellular expression. Both the native fungal secretion signal sequence and α-factor secretion signal sequence directed the secretion of active rMnP from P. pastoris transformants. The majority of the rMnP produced by these two P. pastoris strains had a considerably larger molecular mass (55-210 kDa) than the wild-type manganese peroxidase (wtMnP, 46 kDa). Deletion of the native fungal secretion signal yielded a molecular mass for intracellular rMnP in P. pastoris of 39 kDa. Treatment of the secreted rMnP with endoglycosidase H (Endo H) resulted in a considerable decrease in the mass of rMnP, indicating N-linked hyperglycosylation.

Recombinant P. pastoris produced more active MnP in buffered (pH 6) media than in unbuffered or low pH media. SDS-PAGE results demonstrated the degradation of rMnP in low pH medium, indicating that acidic proteases, which can be released from lysed cells, actively hydrolyzed the recombinant protein resulting in a total loss of enzyme activity. Activity increased 20-fold in the presence of 1 g/L heme. Increase in heme concentration improved the productivity of rMnP, but the amount of heme required in the medium was far more than the stoichiometric requirements of the rMnP secreted into the media, suggesting that heme permeation through the cell membrane was a limiting factor for the production of active rMnP.

Partially purified rMnP was similar to wtMnP in kinetic characteristics. Both enzymes had similar pH optima for the oxidation of Mn II and 2,6-dimethoxyphenol (2,6-DMP) and pH stability profiles. Addition of exogenous Mn II , Ca II and Fe III conferred additional thermal stability to both enzymes. However, rMnP was slightly less thermostable than wtMnP, which had a half-life 1.5-fold longer at 55°C.

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