Production and separation of manganese peroxidase from heme amended yeast cultures.
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.
Cell Genesys, Inc., South San Francisco, California, USA.
Fei
F
Jiang
Puapong
P
Kongsaeree
Rose
R
Charron
Curtis
C
Lajoie
Haowen
H
Xu
Gary
G
Scott
Christine
C
Kelly
D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D004789:Enzyme Activation; D004795:Enzyme Stability; D006418:Heme / Q000378:metabolism*; D010544:Peroxidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D020075:Phanerochaete / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000502:physiology*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D006418:Heme; D010544:Peroxidases; C051129:manganese peroxidase