characterization of enzymes involved in butane metabolism from the pollutant degrading bacterium,...
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Characterization of Enzymes Involved in Butane Characterization of Enzymes Involved in Butane Metabolism from the Pollutant Degrading Metabolism from the Pollutant Degrading
bacterium, bacterium, Pseudomonas butanovoraPseudomonas butanovora
John StenbergJohn Stenberg
Mentor: Dan Arp, Ph.D.Mentor: Dan Arp, Ph.D.
September 1, 2004September 1, 2004
BioremediationBioremediation
As the world population and the demands of agriculture and As the world population and the demands of agriculture and industry increase, the availability of fresh water continues to industry increase, the availability of fresh water continues to decreasedecrease
The problems associated with depleted or polluted water affect The problems associated with depleted or polluted water affect not only humans, but the plant and animal populations we not only humans, but the plant and animal populations we depend upondepend upon
The solution?The solution?
BioremediationBioremediation: The process by which living organisms act to : The process by which living organisms act to degrade hazardous organic contaminants or transform degrade hazardous organic contaminants or transform hazardous inorganic contaminants to environmentally safe hazardous inorganic contaminants to environmentally safe levels in soils, subsurface materials, water, sludges, and levels in soils, subsurface materials, water, sludges, and residues. residues.
CometabolismCometabolism Definition: the transformation of a non-growth-supporting substrate by a Definition: the transformation of a non-growth-supporting substrate by a
microorganismmicroorganism
Pseudomonas butanovoraPseudomonas butanovora contains a multi-component monooxygenase that is able to catalyze the contains a multi-component monooxygenase that is able to catalyze the degradation of many substrates including trichloroethylene, dichloroethylenes, aromatic structures, degradation of many substrates including trichloroethylene, dichloroethylenes, aromatic structures, and othersand others
Such compounds are not only environmental pollutants, but in many cases, are very stableSuch 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 Once oxidized by a monooxygenase, it is much easier for these compounds to be further degraded
H ClC C
Cl Cl
Trichloroethylene (TCE)
H O ClC C
Cl Cl
TCE epoxide
Ex. Trichloroethylene oxidation
Pseudomonas butanovoraPseudomonas butanovora
Isolated in Japan from activated sludge near an oil Isolated in Japan from activated sludge near an oil refinery refinery
Capable of growth with butane via the oxidation of Capable of growth with butane via the oxidation of butane to 1-butanol as the first step in the terminal butane to 1-butanol as the first step in the terminal oxidation pathwayoxidation pathway
CC44HH1010 + O + O22 CC44HH99OH + HOH + H22OO Also capable of growth with other alkanes (C2–C9), Also capable of growth with other alkanes (C2–C9),
alcohols (C2–C4) and organic acids as sources of alcohols (C2–C4) and organic acids as sources of carbon and energy carbon and energy
Growth on alkanes catalyzed by a soluble butane Growth on alkanes catalyzed by a soluble butane monooxygenase (sBMO)monooxygenase (sBMO)
Butane Monooxygenase(sBMO)
Butane
Terminal Oxidation Pathway of Terminal Oxidation Pathway of Pseudomonas Pseudomonas butanovorabutanovora
Example: butane to butyric acid (further metabolized as fatty acid)Example: butane to butyric acid (further metabolized as fatty acid)
CH3
CH2
CH2
CH3
CH3
CH2
CH2
CH
O
CH3
CH2
CH2
CH2
OH
1-Butanol
Alcohol Dehydrogenases
CH3
CH2
CH2
O
OH
ButyraldehydeButyric Acid
Aldehyde Dehydrogenases
Butane monooxygenaseButane monooxygenase
Responsible for oxidation of butaneResponsible for oxidation of butane CC44HH1010 + O + O22 CC44HH99OH + HOH + H22OO
Three part enzymeThree part enzyme1. Hydroxylase component (BMOH)1. Hydroxylase component (BMOH)
- - contains the substrate binding di-iron active site and is contains the substrate binding di-iron active site and is responsible for the oxidation of butane to 1-butanol responsible for the oxidation of butane to 1-butanol
2. Reductase component (BMOR)2. Reductase component (BMOR)- responsible for the transfer of electrons from NADH+H- responsible for the transfer of electrons from NADH+H++ to the to the hydroxylase component hydroxylase component
3. Component B (BMOB)3. Component B (BMOB)- coupling protein required for substrate oxidation, electron - coupling protein required for substrate oxidation, electron
transfer ??transfer ??
Proposed Catalytic Cycle of BMOProposed Catalytic Cycle of BMO
Adapted from Wallar, B.J. and J.D. Lipscomb, 1996, Chem. Rev. 96: 2625-2657
BMO Research ObjectivesBMO Research Objectives
Purification and characterization of BMO Purification and characterization of BMO componentscomponents ReductaseReductase HydroxylaseHydroxylase
BMO ActivityBMO Activity Methane oxidationMethane oxidation
Steps leading to PurificationSteps leading to Purification
1. Grow 1. Grow Pseudomonas butanovoraPseudomonas butanovora cells cells Sealed flasks, carboysSealed flasks, carboys Butane 7% overpressureButane 7% overpressure
2. Harvest cells through centrifugation2. Harvest cells through centrifugation 3. Prepare cell-free extract3. Prepare cell-free extract
Lysis by freeze/thaw and sonicationLysis by freeze/thaw and sonication Centrifuge at 46,000 x Centrifuge at 46,000 x g g
Enzyme PurificationEnzyme Purification Multiple column processMultiple column process
1. Q Sepharose resin column 1. Q Sepharose resin column (anion exchange purification)(anion exchange purification)
2. 22. 2ndnd Q Sepharose column Q Sepharose column
3. Gel filtration3. Gel filtration
Superdex 75 – reductaseSuperdex 75 – reductase
Sephacryl S-300 - Sephacryl S-300 - hydroxylasehydroxylase
What so far?What so far?-Purified reductase with activity-Purified reductase with activity
-Partially purified hydroxylase with -Partially purified hydroxylase with activityactivity
Pharmacia FPLC System
sBMO Reductase PurificationsBMO Reductase Purification
97.4
45
66.2
31
21.5
14.4
CFE Q1 Q2 S 75
Purified Reductase FractionsPurified Reductase Fractions
Reductase PropertiesReductase Properties
AA270/458270/458 ratio: 3.1 - 3.7, which is ratio: 3.1 - 3.7, which is similar to the methane similar to the methane monoxygenase reductase monoxygenase reductase and other purified and other purified oxygenase reductasesoxygenase reductases
AA458/340458/340 ratio: 1.4, also similar ratio: 1.4, also similar to the methane to the methane monoxygenase reductasemonoxygenase reductase
UV/Visible Spectra has UV/Visible Spectra has maxima at 272, 340, ~ 400, maxima at 272, 340, ~ 400, 458 nm458 nm
Reductase UV/Visible Spectra
StepStepDCPIP ReductionDCPIP Reduction
(µmol min(µmol min-1-1 mg protein mg protein-1)-1)Fold PurificationFold Purification
Cell Free ExtractCell Free Extract 5.8 ± 0.15.8 ± 0.1 11
Q1Q1 44 ± 0.844 ± 0.8 88
Q2Q2 86 ± 1.586 ± 1.5 1515
Superdex 75Superdex 75 115 ± 1.4115 ± 1.4 2020
Reductase activity and fold purificationReductase activity and fold purification
BMOH
Hydroxylase PurificationHydroxylase Purification
1st Q Sepharose Column Spectra1st Q Sepharose Column Spectra
M Q1 Q2 S-300 S-300
97.4
4566.2
31
21.5
14.4
Hydroxylase Purification StepsHydroxylase Purification Steps
StepStepEO productionEO production
(nmol min(nmol min-1-1 mg protein mg protein-1)-1)% Recovery% Recovery
Whole CellWhole Cell 300300 100100
Cell Free Cell Free ExtractExtract
106106 3535
11stst Q Q Sepharose Sepharose ColumnColumn
231231 7777
BMO Hydroxylase activity during initial BMO Hydroxylase activity during initial purification stepspurification steps
Measured by ethylene oxide (EO) production by gas Measured by ethylene oxide (EO) production by gas chromatographychromatography
Methane OxidationMethane Oxidation Methanol ProductionMethanol Production 5 picomol min5 picomol min-1-1 mg protein mg protein-1-1
0
5000
10000
15000
20000
25000
30000
35000
0 10 20 30 40 50 60 70 80
Time (min)
Peak Area
ProgressProgress
Mass culturing at 5 L/carboy is repeatable allowing for ~7-8 g Mass culturing at 5 L/carboy is repeatable allowing for ~7-8 g of cell mass/carboy with high BMO activityof cell mass/carboy with high BMO activity
Recoverable BMO hydroxylase activities in cell free extracts Recoverable BMO hydroxylase activities in cell free extracts and initial chromotography steps at high activity comparable and initial chromotography steps at high activity comparable to published sMMO purification strategy of Fox to published sMMO purification strategy of Fox et al.et al. (1989) (1989)
BMO reductase purified to homogeneity with demonstrated BMO reductase purified to homogeneity with demonstrated activity; comparable to the sMMO system reductase in activity activity; comparable to the sMMO system reductase in activity and spectral characteristicsand spectral characteristics
Possible methane oxidationPossible methane oxidation
AcknowledgementsAcknowledgements
Howard Hughes Medical InstituteHoward Hughes Medical Institute Daniel Arp, Ph.D.Daniel Arp, Ph.D.
Brad Dubbels, Ph.D.Brad Dubbels, Ph.D. Arp LabArp Lab
Kevin Ahern, Ph.D.Kevin Ahern, Ph.D.