Effects of pH and Temperature on Recombinant Manganese Peroxidase Production and Stability |
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Authors: | Fei Jiang Puapong Kongsaeree Karl Schilke Curtis Lajoie Christine Kelly |
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Institution: | (1) Department of Biomedical and Chemical Engineering, Syracuse University, 121 Link Hall, Syracuse, NY 13244-1240, USA;(2) School of Chemical, Biological, and Environmental Engineering, Oregon State University, 102 Gleeson Hall, Corvallis, OR 97331-2701, USA;(3) Cell Genesys, Inc., 500 Forbes Boulevard, South San Francisco, CA 94080, USA;(4) G C Hanford MFG Company, 304 Oneida St., Syracuse, NY 13201, USA |
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Abstract: | The enzyme manganese peroxidase (MnP) is produced by numerous white-rot fungi to overcome biomass recalcitrance caused by
lignin. MnP acts directly on lignin and increases access of the woody structure to synergistic wood-degrading enzymes such
as cellulases and xylanases. Recombinant MnP (rMnP) can be produced in the yeast Pichia pastoris αMnP1-1 in fed-batch fermentations. The effects of pH and temperature on recombinant manganese peroxidase (rMnP) production
by P. pastoris αMnP1-1 were investigated in shake flask and fed-batch fermentations. The optimum pH and temperature for a standardized fed-batch
fermentation process for rMnP production in P. pastoris αMnP1-1 were determined to be pH 6 and 30 °C, respectively. P. pastoris αMnP1-1 constitutively expresses the manganese peroxidase (mnp1) complementary DNA from Phanerochaete chrysosporium, and the rMnP has similar kinetic characteristics and pH activity and stability ranges as the wild-type MnP (wtMnP). Cultivation
of P. chrysosporium mycelia in stationary flasks for production of heme peroxidases is commonly conducted at low pH (pH 4.2). However, shake
flask and fed-batch fermentation experiments with P. pastoris αMnP1-1 demonstrated that rMnP production is highest at pH 6, with rMnP concentrations in the medium declining rapidly at
pH less than 5.5, although cell growth rates were similar from pH 4–7. Investigations of the cause of low rMnP production
at low pH were consistent with the hypothesis that intracellular proteases are released from dead and lysed yeast cells during
the fermentation that are active against rMnP at pH less than 5.5. |
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Keywords: | Manganese peroxidase Yeast Pichia Biofuels Proteases |
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