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Expression of a Phanerochaete chrysosporium Manganese Peroxidase Gene in the Yeast Pichia pastoris
Author(s) -
Gu Lina,
Lajoie Curtis,
Kelly Christine
Publication year - 2003
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp025781h
Subject(s) - pichia pastoris , phanerochaete , signal peptide , manganese peroxidase , biochemistry , microbiology and biotechnology , secretion , peroxidase , biology , yeast , molecular mass , recombinant dna , enzyme , gene
A gene encoding manganese peroxidase ( mnp1 ) from Phanerochaete chrysosporium was cloned downstream of a constitutive glyceraldehyde‐3‐phosphate dehydrogenase promoter in the methylotrophic yeast Pichia pastoris . Three different expression vectors were constructed: pZBMNP contains the native P. chrysosporium fungal secretion signal, pαAMNP contains an α‐factor secretion signal derived from Saccharomyces cerevisiae , and pZBIMNP has no 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 recombinant manganese peroxidase (rMnP) from P. pastoris transformants. The majority of the rMnP produced by P. pastoris exhibited a molecular mass (55–100 kDa) considerably larger than that of the wild‐type manganese peroxidase (wtMnP, 46 kDa). Deletion of the native fungal secretion signal yielded a molecular mass of 39 kDa for intracellular rMnP in P. pastoris . Treatment of the secreted rMnP with endoglycosidase H (Endo H) resulted in a considerable decrease in the mass of rMnP, indicating N‐linked hyperglycosylation. Partially purified rMnP showed kinetic characteristics similar to those of wtMnP. Both enzymes also had similar 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 demonstrated an extended half‐life at 55 °C.