Production and separation of manganese peroxidase from heme amended yeast cultures
Autor: | Puapong Kongsaeree, Rose Charron, Haowen Xu, Christine Kelly, Curtis A. Lajoie, Gary Scott, Fei Jiang |
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Rok vydání: | 2007 |
Předmět: |
Cell Culture Techniques
Bioengineering Heme Phanerochaete Protein Engineering Applied Microbiology and Biotechnology Cofactor Pichia Pichia pastoris chemistry.chemical_compound Manganese peroxidase Enzyme Stability Cell Proliferation biology biology.organism_classification Yeast Enzyme assay Recombinant Proteins Enzyme Activation Biochemistry chemistry Peroxidases biology.protein Biotechnology Peroxidase |
Zdroj: | Biotechnology and bioengineering. 99(3) |
ISSN: | 1097-0290 |
Popis: | A method for the production and concentration of the lignin-degrading enzyme, manganese peroxidase (rMnP), was developed using the yeast Pichia pastoris in high cell density, fed-batch cultivations. A gene encoding manganese peroxidase (mnp1) from the white-rot fungus Phanerochaete chrysosporium was cloned into a protease deficient (pep4-) strain of the methylotrophic yeast P. pastoris. Heme is an important cofactor for active rMnP production, and amendment of yeast cultures with heme increased active rMnP concentrations. In both shake-flasks and fed-batch bioreactors, the relationship between heme concentration and rMnP activity was logarithmic, with increasing heme concentrations resulting in progressively lesser increases in enzyme activity. Scale-up from shake-flasks to 2 L fed-batch cultivations increased rMnP activities from 200 U/L to 2,500 U/L, with addition of 0.1 g/L heme (added heme per liquid volume) at the beginning of the fed-batch phase resulting in higher enzyme activities than addition at the beginning of the batch phase. A combination of centrifugation, acetone precipitation, dialysis, and freeze drying was found to be effective for concentrating the rMnP from 2,500 U/L in the P. pastoris bioreactor culture to 30,000 U/L in 0.1 M potassium phosphate buffer pH 6. The rMnP recovery yield was 60% and the purity was 1-4%. By using 0.1 g/L heme during the fed-batch cultivation, the heme content of the final enzyme preparation could be reduced by 97%, and had sufficiently high rMnP activity and low enough color to be suitable for pulp bleaching experiments. |
Databáze: | OpenAIRE |
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