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619d

Recombinant Expression, Stability and Purification of the Lignin Oxidizing Enzyme Manganese Peroxidase

Christine J. Kelly1, Fei Jiang1, and Curtis Lajoie2. (1) Chemical Engineering, Oregon State University, 103 Gleeson Hall, Corvallis, OR 97331, (2) Civil, Structural, and Environmental Engineering, Oregon State University, 103 Gleeson Hall, Corvallis, OR 97331

Recombinant Expression, Stability and Purification of the Lignin Oxidizing Enzyme Manganese Peroxidase Lignocellulosic materials (e.g., wood, crop residues) will likely play an increasingly important role as raw materials for manufacturing and energy production in a sustainable environment. One of the major impediments to using these feedstocks is the presence of lignin. Sustainable biomanufacturing will require suitable methods for the removal of lignin in the process of separation, modification and conversion of lignocellulosic materials into manufacturing feedstocks and biofuels. This approach is currently employed in the pulp and paper manufacture, where cellulose is separated from lignin and hemicellulose using mechanical or chemical pulping methods. Enzymes (xylanases) and microorganisms (white-rot fungi) are finding significant applications in decreasing chemical and energy usage in existing pulp and paper plants. Pulp has been treated using white-rot fungi such as Phanerochaete chrysosporium, Trametes versicolor, and strain IZU-154, resulting in lignin depolymerization, and consequently, a reduction in chemical usage during bleaching. It appears that among the array of wood-degrading enzymes secreted by white-rot fungi, manganese peroxidase (MnP; EC 1.11.1.7) is quantitatively the most important lignin-degrading enzyme in biobleaching. MnP is a heme-peroxidase that employs H2O2 as a substrate to catalyze the oxidation of MnII to MnIII. The MnIII-organic acid chelator formed by MnP can oxidize lignin, and has potentially valuable applications in the pulp and paper industry.

 

White-rot fungi typically grow as mycelia, and do not perform well under the high agitation conditions of industrial scale stirred-tank bioreactors. Only limited amounts (5 mg/L) of multiple isozymes are produced by these naturally-occurring strains, and are only produced under nutrient limiting conditions. An array of wood-degrading enzymes other than MnP are typically produced and secreted into the culture supernatant, some of which could damage the essential cellulose fibers. Advances have been made by screening large numbers of white-rot fungus isolates to find strains with more favorable production characteristics, and new cultivation methods have increased the yield of MnP. An alternative approach is to clone the white-rot fungus mnp cDNA downstream of a constitutive promoter in a host more suitable for industrial cultivations. Because these hosts do not produce native wood-degrading enzymes, negative effects of cellulases or other related enzymes are avoided. The MnP-encoding gene mnp1 from P. chrysosporium has been into successfully expressed in the filamentous fungi Aspergillus niger and Aspergillus oryzae and in the yeast Pichia pastoris. Unlike the native host, these strains require high concentrations of exogenous heme in the medium (0.5 g/L heme or 5 g/L hemoglobin) and produce from 5 ~ 100 mg/L rMnP. The rMnP produced by P. pastoris is hyperglycosylated, and ranges in molecular weight from 55 to 100 kDa, but otherwise has similar kinetic and stability properties to the native MnP. In this paper, research is reported on the construction of a protease deficient rMnP producing P. pastoris. 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 Pichia 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. The maximum rMnP activity was observed at about 0.1 g/L heme, which is a 100-fold stoichiometric molar heme excess. 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 heme at the beginning of the fed-batch phase resulted 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 4 %. The heme content was reduced by 97%, resulting in an enzyme preparation of sufficiently high rMnP activity and low enough color to be suitable for pulp bleaching experiments. In addition to experiments examining the effect of heme on rMnP expression, production and stability of rMnP was studied at different temperatures and pH. rMnP activity was not observed with cultivation at less than pH 5.5, and rMnP not stable under any conditions examined at pH 4.5 and 30C. The lack of rMnP activity in pH 4.5 culitvations can be accounted for quantitatively by the increased degradation rate at the lower pH in independent studies. Degradation of rMnP in culture broths and supernatants seemed to be a result of two processes: (1) a fast, but transient inactivation, and (2) a constant slow thermal inactivation. The presence of the yeast correlated with increased degradation rate of added rMnP. Reduced temperature (25C) decreased the growth rate of yeast, but also decreased the degradation rate of rMnP in culture, so the total rMnP activity produced at 25C was higher than in 30C cultivation.