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.     

Removal of carboxymethyl cellulase activity from the culture filtrate of Termitomyces clypeatus producing xylanase

Termitomyces clypeatus produced 450 IU xylanase ml^–1 in a medium containing starch-free wheat bran powder as the carbon source. Carboxymethyl cellulase (CMCase) activity in the culture filtrate was removed by keeping the filtrate at pH 10 for 60 min followed by a change to pH 6. Treatment of Kraft pulp (bamboo) with the filtrate at pH 7 decreased the kappa number from 10.5 to 5 with release of reducing groups equivalent to 0.15 mg glucose g^–1 pulp.