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