Title

Optimization of recombinant ligninolytic enzyme production in Pichia pastoris

Date of Award

2004

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical and Chemical Engineering

Advisor(s)

Christine Kelly

Keywords

Ligninolytic, Manganese peroxidase, Biobleaching, White-rot fungus

Subject Categories

Chemical Engineering | Engineering

Abstract

Manganese peroxidase (MnP) is a heme containing enzyme produced by white-rot fungi for the degradation of lignin in wood. This enzyme has potential applications in the biobleaching of wood pulp as an environmentally friendly alternative to chemical bleaching methods. To increase production of MnP above levels achievable with the native white-rot fungus, the mnp1 gene, encoding MnP, has been cloned into the yeast Pichia pastoris . Research is reported here on the production of recombinant MnP (rMnP) using shake flask and bench scale fermentors, and on the purification and concentration of the rMnP for use in pulp bleaching experiments.

Active recombinant MnP (rMnP) could be produced in shake-flasks provided the pH was maintained between 6 and 7. At lower pH, no active enzyme was produced, even though the yeast cells grew at the same rate down to pH 4. Use of protease inhibitors or a protease deficient strain did not appear to increase the concentration of rMnP produced below pH 6, and no increase in rMnP concentrations in fermentations at pH 6 resulted from addition of casamino acids or peptone as protease competitive inhibitors, use of pure O 2 to reduce agitation speed and release of proteases via cell lysis, and production at a lower temperature to decrease protease activity. Amendment of the medium with heme (0.1 to 1 g/L) increased the concentration of rMnP.

The rMnP concentration was increased from approximately 100 μmoles/min/L in shake flasks to greater than 3,000 moles/min/L in the fermentors using a protease deficient P. pastoris strain ( P. pastoris αMP 1-1) containing the mnp1 gene with a constitutive glyceraldehydes-3-phosphate dehydrogenase (pGAP) promoter and yeast secretion signal. A batch and fed-batch approach was employed with a basal salts medium and glucose as the carbon source. Heme was added at the beginning of fed-batch. Batch and fed-batch cultivation using glucose as the growth substrate was modeled and the kinetic parameters were determined. The model predictions agree well with the experimental data.

Due to the low aqueous solubility of heme, it formed a fine dispersion in the medium. To separate the rMnP from the medium and concentrate the enzyme, centrifugation (10,000x g , 5 minutes) was employed to remove the yeast cells and the heme, and then the enzyme was precipitated using acetone. Approximately 75% of the rMnP could be precipitated and recovered using 40% acetone, and the enzyme concentrate was found to be of adequate purity and concentration for use in pulp bleaching experiments.

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