Characterization of Enzymes Involved in Butane Metabolism from the Pollutant Degrading bacterium, Pseudomonas butanovora

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Characterization of Enzymes Involved in Butane
Metabolism from the Pollutant Degrading
bacterium, Pseudomonas butanovora

• John Stenberg
• Mentor Dan Arp, Ph.D.
• September 1, 2004

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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.

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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 O Cl C C Cl
Cl
H Cl C C Cl
Cl Trichloroethylene (TCE)
TCE epoxide
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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
  (C2C9), alcohols (C2C4) and organic acids as
  sources of carbon and energy
• Growth on alkanes catalyzed by a soluble butane
  monooxygenase (sBMO)

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Terminal Oxidation Pathway of Pseudomonas
butanovoraExample butane to butyric acid
(further metabolized as fatty acid)
Butane Monooxygenase (sBMO)
Butane
1-Butanol
Alcohol Dehydrogenases
Aldehyde Dehydrogenases
Butyraldehyde
Butyric Acid
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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 NADHH to the hydroxylase component
• 3. Component B (BMOB)
• - coupling protein required for substrate
  oxidation, electron transfer ??

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Proposed Catalytic Cycle of BMO
Adapted from Wallar, B.J. and J.D. Lipscomb,
1996, Chem. Rev. 96 2625-2657
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BMO Research Objectives

• Purification and characterization of BMO
  components
• Reductase
• Hydroxylase
• BMO Activity
• Methane oxidation

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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

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Enzyme Purification

• 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

Pharmacia FPLC System
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sBMO Reductase Purification
CFE
Q1
Q2
S 75
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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
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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
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Hydroxylase Purification

• 1st Q Sepharose Column Spectra

BMOH
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Hydroxylase Purification Steps
?
?
?
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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
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Methane Oxidation

• Methanol Production
• 5 picomol min-1 mg protein-1

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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

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Acknowledgements

• Howard Hughes Medical Institute
• Daniel Arp, Ph.D.
• Brad Dubbels, Ph.D.
• Arp Lab
• Kevin Ahern, Ph.D.