Expression of a Phanerochaete chrysosporium manganese peroxidase gene in the yeast Pichia pastoris.
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, palphaAMNP contains an alpha-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 alpha-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 degrees C.
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Lina
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L.C. Smith College of Engineering and Computer Science, Department of Chemical Engineering and Materials Science, 220 Hinds Hall, Syracuse University, Syracuse, New York 13244-1190, USA.
Lajoie
Curtis
C
Kelly
Christine
C
D004789:Enzyme Activation; D004795:Enzyme Stability; D005240:Feasibility Studies; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010544:Peroxidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020075:Phanerochaete / Q000201:enzymology* / Q000235:genetics*; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013696:Temperature; D014170:Transformation, Genetic / Q000235:genetics
D011994:Recombinant Proteins; D010544:Peroxidases; C051129:manganese peroxidase