Title

Expression of recombinant manganese peroxidase in Pichia pastoris: Biosynthesis, stability and kinetics analysis

Date of Award

9-2006

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical and Chemical Engineering

Advisor(s)

Christine Kelly

Keywords

Manganese peroxidase, Lignin-degrading, Fermentation

Subject Categories

Biochemical and Biomolecular Engineering

Abstract

A method for the biosynthesis and recovery of the lignin-degrading enzyme, recombinant manganese peroxidase (rMnP), was developed through the fed-batch fermentation of the 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, a logarithmic relationship was observed between heme concentration and rMnP activity, and enzyme activity increased progressively lesser with increasing heme concentrations. Scale-up from shake-flasks to 2 L fed-batch cultivations increased rMnP activities from 200 U/L to 2,500 U/L. 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 broth to 30,000 U/L in 0.1 M pH 6.0 potassium phosphate buffer. 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.

The effects of pH and temperature on rMnP production by P. pastoris αMnP1-1 and rMnP stability were investigated in shake-flask and fed-batch fermentations. Both shake-flask and fed-batch fermentation experiments demonstrated that rMnP production is highest at pH 6.0, with rMnP concentrations in the medium declining rapidly at pH less than 5.5, even though cell growth rates were similar from pH 4 to 7. Reducing the pH of the batch or fed-batch fermentation from 6 to 4.5 resulted in a fast decline in rMnP activity, and spiking of pH 4.5 fermentations with concentrated rMnP also yielded fast decline in enzyme activity. rMnP stability tests with bioreactor cultures and culture supernatants indicated that rMnP is less stable in pH 4.5 culture supernatant than in supernatant samples from pH 6 fermentations; the presence of yeast cells in the stability test increases the rate of loss of rMnP activity; samples taken later in the fermentation showed higher activity on rMnP degradation than samples taken earlier; and lowering the temperature from 30°C to 20°C decreases the rate of loss of activity. There were no benefits to altering the pH of the standard fed-batch fermentation process for rMnP production in P. pastoris αMnP1-1 from pH 6. However, the decrease of fermentation temperature from 30°C to 25°C enhanced the maximum rMnP activity by 20% (2570 U/L at 25°C vs. 2150 U/L at 30°C).

A kinetic analysis was performed on a base case fermentation, fed-batch fermentation of P. pastoris αMnP1-1 at 30°C in BSM medium; lower temperature fermentation (25°C); fermentation in modified BSM medium (25% of initial concentration); and fermentation with a doubled fed-batch feeding rate (24 mL/hr of 500 g/L glucose solution). Three distinct physiological stages were proposed to occur during the batch/fed-batch fermentations. The cell growth and enzyme production models were also developed for batch phase and fed-batch phases, based on different assumptions. The models were applied to the four fermentation cases mentioned above.

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