Concentration and Purification of Recombinant Manganese Peroxidase for Application in the Biofuel Industry

Ian GluckMentor: Dr. Christine Kelly

OSU Dept. of Chemical, Biological and Environmental Engineering

The Biofuel Industry

Cellulosic (wood-based) ethanol has several advantages

Lower greenhouse gas emissions

Biomass is cheap to produce

Majority of American ethanol is corn-based

Inefficient Conflicts with food demands

The United States is currently the world’s largest producer of ethanol

The Conversion Process

Cellulose must be converted to glucose

Performed by cellulases

Process is hindered by lignin

A large, irregular polymer

Surrounds the cellulose

Lignin

Cellulose

The Solution

Manganese

peroxidase (MnP)

Found in white-rot

fungi

Degrades lignin

The Solution

White-rot fungi cannot be effectively mass produced

DNA responsible for MnP production was cloned into a yeast (Pichia pastoris)

Yeast is cultivated to create recombinant manganese peroxidase (rMnP) Performs same lignin-degrading function

The Solution

Crude yeast broth has many other proteins, as well as rMnP

For rMnP biocatalysis pathways to be investigated, it must be:

Concentrated

Purified

The Project Purpose

To test the effectiveness of a variety

of methods in the concentration and

purification of recombinantmanganese peroxidase

The Initial Substance

White-rot fungi cultivation broth Filtered Cell-free broth submitted to:

A concentration process

A purification process

The Concentration Process

Acetone precipitation

Centrifugation/Resolubilization

Overnight freezing

Lyophilization (freeze-drying)

Dialysis

The Purification Process

Ion exchange chromatography

Uses ionic interactions to separate molecules of different charge

Diethylaminoethyl (DEAE) column-O-CH2-CH2-N+H(CH2CH3)2•Cl-

Positive charge with negative Cl- ions Cl- ions are exchanged for MnP, and

vice versa

DEAE

Cl- --

----Cl- --

----

Cl- --

----Cl- --

----

Cl- --

----

Cl- --

----

++

+

+

+

+

++

+

+

MnP

--

-

---

-

- MnP

--

-

---

-

-

Fungi Culture Protein

---

----

-

Cl- --

----Cl- --

----

Cl- --

----

Cl- --

----

Cl- --

----

Cl- --

----

Cl- --

---- Cl- --

----

Cl

MnP Measurement

Sample of subject solution is mixed with buffers, DMP and H2O2

MnP in solution reacts with peroxide and DMP, forming an orange color

Absorbance of orange solution is measured

Enzyme Activity Assay

MnP Measurement

Absorbance is used to calculate MnP concentration

Concentration * Volume Mass

Mass of MnP/ *100 = Percent Yield Previous mass of MnP

Purification Measurement

Similar process to MnP assay

Absorbance is measured and used to find protein concentration Concentration * Volume = Mass

Mass of MnP is divided by protein mass

Mass Fraction

Total Protein Assay

The Results

StepSample Volume

(L)

MnP Conc. (mg/L)

MnP Mass (mg)

Yield

(%)

Protein

Conc. (mg/L

)

Protein

Mass (mg)

Yield

(%)

Mass Fraction (*10-

5)

Crude Broth 0.1 0.139 1.39*10-2 - 1503 150.3 - 9.30

Precipitation/Centrifugatio

n0.03 0.350 1.23*10-2 88.5 2855 85.65 56.9 21.6

Lyophilization 0.004 3.00 1.19*10-2 96.7 1017 4.068 4.7 251

Dialysis 0.008 1.09 8.68*10-3 72.9 - - - -

Ion Exchange 0.015 0.433 6.50*10-3 74.9 593 8.888 - 73.1 Desired Outcomes MnP yield > 50% Dramatic increase in mass fraction

The Goal

Possible outcomes of success

Improve research on MnP transformations of lignin

Cellulosic ethanol is able to be produced more efficiently on an industrial scale

Product of lignin degradation is examined for possible applications

A model system for purifying proteins is developed

Acknowledgments

HHMI Dr. Kevin Ahern Dr. Christine Kelly Kelsey Yee OSU Department of Chemical,

Biological and Environmental Engineering