(619d) Recombinant Expression, Stability and Purification of the Lignin Oxidizing Enzyme Manganese Peroxidase

Kelly, C. J., Oregon State University
Jiang, F., Oregon State University
Lajoie, C., Oregon State University

Expression, Stability and Purification of the Lignin Oxidizing Enzyme Manganese

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
, 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 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.

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
30°C. 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 (25°C) decreased
the growth rate of yeast, but also decreased the degradation rate of rMnP in
culture, so the total rMnP activity produced at 25°C was higher than in 30°C