Transcript Ian Gluck Mentor: Dr. Christine Kelly OSU Dept. of Chemical, Biological and Environmental Engineering.

Ian Gluck
Mentor: Dr. Christine Kelly
OSU Dept. of Chemical, Biological and Environmental Engineering
The United States is currently the world’s largest producer of
ethanol
Majority of American ethanol is corn-based
Inefficient
Conflicts with food demands
Cellulosic (wood-based) ethanol has several advantages
Lower greenhouse gas emissions
Biomass is cheap to produce

Cellulose must be converted to glucose
 Performed by cellulases

Process is hindered by lignin
 A large, irregular polymer
 Surrounds the cellulose
Lignin
Cellulose
Manganese
peroxidase
(MnP)
Found in
white-rot
fungi
Degrades
lignin

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
Crude yeast broth has many other proteins, as well as rMnP
For rMnP biocatalysis pathways to be investigated, it must be:
Concentrated
Purified
To test the effectiveness of a variety
of methods in the concentration and
purification of recombinant
manganese peroxidase

White-rot fungi cultivation broth

Filtered

Cell-free broth submitted to:
 A concentration process
 A purification process
Acetone precipitation
Centrifugation/Resolubilization
Overnight freezing
Lyophilization (freeze-drying)
Dialysis

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
- Cl - Cl - - - - - - - Cl - - - Cl
- -
-
-
-
- Cl - -
Cl
-
-
MnP
-
- Cl - - - Cl
- - - - -- Cl - -
- Cl - +
- Cl - +
+
- Cl - +
DEAE
+
- Cl - -
+
+
- Cl - -
MnP
-
+
+
+
- Cl - -
-
-
-
Fungi Culture
- Protein
-
Enzyme Activity Assay
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
Absorbance is used to calculate MnP
concentration
Concentration * Volume
Mass
Mass of MnP/ *100 = Percent Yield
Previous mass of MnP
Total Protein Assay
Mass of MnP is divided by protein mass
Absorbance is
measured and used
to find protein
concentration
Concentration * Volume = Mass
Similar process to
MnP assay
Mass
Fraction
Step
Sample
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/
Centrifugation
0.03
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

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

HHMI

Dr. Kevin Ahern

Dr. Christine Kelly

Kelsey Yee

OSU Department of Chemical, Biological and
Environmental Engineering