Characterization of Enzymes Involved in Butane
Metabolism from the Pollutant Degrading
bacterium, Pseudomonas butanovora
John Stenberg
Mentor: Dan Arp, Ph.D.
September 1, 2004
Bioremediation

As the world population and the demands of agriculture and
industry increase, the availability of fresh water continues to
decrease

The problems associated with depleted or polluted water affect
not only humans, but the plant and animal populations we
depend upon

The solution?

Bioremediation: The process by which living organisms act to
degrade hazardous organic contaminants or transform
hazardous inorganic contaminants to environmentally safe
levels in soils, subsurface materials, water, sludges, and
residues.
Cometabolism

Definition: the transformation of a non-growth-supporting substrate by a
microorganism

Pseudomonas butanovora contains a multi-component monooxygenase that is able
to catalyze the degradation of many substrates including trichloroethylene,
dichloroethylenes, aromatic structures, and others

Such compounds are not only environmental pollutants, but in many cases, are very
stable

Once oxidized by a monooxygenase, it is much easier for these compounds to be
further degraded
Ex. Trichloroethylene oxidation
H
Cl
C
Cl
H
C
O
C
Cl
Trichloroethylene (TCE)
Cl
Cl
C
Cl
TCE epoxide
Pseudomonas butanovora




Isolated in Japan from activated sludge near an oil
refinery
Capable of growth with butane via the oxidation of
butane to 1-butanol as the first step in the terminal
oxidation pathway
C4H10 + O2 C4H9OH + H2O
Also capable of growth with other alkanes (C2–C9),
alcohols (C2–C4) and organic acids as sources of
carbon and energy
Growth on alkanes catalyzed by a soluble butane
monooxygenase (sBMO)
Terminal Oxidation Pathway of Pseudomonas
butanovora
Example: butane to butyric acid (further metabolized as fatty acid)
H3C
H2
C
C
H2
CH3
Butane Monooxygenase
(sBMO)
H3C
Butane
H2
C
OH
C
H2
CH2
1-Butanol
Alcohol Dehydrogenases
H3C
H2
C
Aldehyde Dehydrogenases
O
C
H2
OH
Butyric Acid
H3C
H2
C
O
C
H2
CH
Butyraldehyde
Butane monooxygenase

Responsible for oxidation of butane
C4H10 + O2

C4H9OH + H2O
Three part enzyme
1. Hydroxylase component (BMOH)
- contains the substrate binding di-iron active site and is
responsible for the oxidation of butane to 1-butanol
2. Reductase component (BMOR)
- responsible for the transfer of electrons from NADH+H+ to the
hydroxylase component
3. Component B (BMOB)
- coupling protein required for substrate oxidation, electron
transfer ??
Proposed Catalytic Cycle of BMO
Adapted from Wallar, B.J. and J.D. Lipscomb, 1996, Chem. Rev. 96: 2625-2657
BMO Research Objectives

Purification and characterization of BMO
components



Reductase
Hydroxylase
BMO Activity

Methane oxidation
Steps leading to Purification
 1.


Grow Pseudomonas butanovora cells
Sealed flasks, carboys
Butane 7% overpressure
 2.
Harvest cells through centrifugation
 3. Prepare cell-free extract


Lysis by freeze/thaw and sonication
Centrifuge at 46,000 x g
Enzyme Purification
Pharmacia FPLC System

Multiple column process
1. Q Sepharose resin column
(anion exchange purification)
2. 2nd Q Sepharose column
3. Gel filtration
Superdex 75 – reductase
Sephacryl S-300 - hydroxylase

What so far?
-Purified reductase with activity
-Partially purified hydroxylase with
activity
sBMO Reductase Purification
CFE
97.4
66.2
45
31
21.5
14.4
Q1
Q2
S 75
Purified Reductase Fractions
Reductase Properties
A270/458 ratio: 3.1 - 3.7, which
is similar to the methane
monoxygenase reductase
and other purified
oxygenase reductases
A458/340 ratio: 1.4, also similar
to the methane
monoxygenase reductase
UV/Visible Spectra has
maxima at 272, 340, ~ 400,
458 nm
Reductase UV/Visible Spectra
Reductase activity and fold purification
Step
DCPIP Reduction
(µmol min-1 mg protein-1)
Fold Purification
Cell Free Extract
5.8 ± 0.1
1
Q1
44 ± 0.8
8
Q2
86 ± 1.5
15
Superdex 75
115 ± 1.4
20
Hydroxylase Purification
1st Q Sepharose Column Spectra
BMOH
Hydroxylase Purification Steps
M Q1
Q2
S-300 S-300



97.4
66.2
45
31
21.5
14.4
BMO Hydroxylase activity during initial
purification steps

Measured by ethylene oxide (EO) production by gas
chromatography
Step
EO production
(nmol min-1 mg protein-1)
% Recovery
Whole Cell
300
100
Cell Free
Extract
106
35
1st Q
Sepharose
Column
231
77
Methane Oxidation

Methanol Production
5 picomol min-1 mg protein-1
35000
30000
25000
Peak Area

20000
15000
10000
5000
0
0
10
20
30
40
Time (min)
50
60
70
80
Progress

Mass culturing at 5 L/carboy is repeatable allowing for ~7-8 g
of cell mass/carboy with high BMO activity
 Recoverable BMO hydroxylase activities in cell free extracts
and initial chromotography steps at high activity comparable
to published sMMO purification strategy of Fox et al. (1989)
 BMO reductase purified to homogeneity with demonstrated
activity; comparable to the sMMO system reductase in activity
and spectral characteristics
 Possible methane oxidation
Acknowledgements

Howard Hughes Medical Institute
 Daniel Arp, Ph.D.
 Brad Dubbels, Ph.D.
 Arp Lab
 Kevin Ahern, Ph.D.