Transcript Butane Monooxygenase B - Oregon State University

Modifications of the Catalytic Properties of
Butane Monooxygenase in P. butanovora
Sarah Jean
Mentors: Dr. Daniel J. Arp
Dr. Luis Sayavedra-Soto
Kim Halsey
Environmental Significance
Bioremediation: Use of
 Pseudomonas butanovora
metabolizes alkanes by oxidizing
the alkanes to alcohols
 Alkane metabolism importance:
1. carbon cycling
2. bioremediation (fossil fuels
and chlorinated aliphatic
hydrocarbons such as
chloroform and trichloroethylene)
microorganisms to remove
or detoxify toxic or
unwanted chemicals from
an environment.
Chloroform: Produced
mainly by the chlorination
of methane, chloroform is
used in the production of
refrigerants and
agrochemicals. It is no
longer used as an
anesthetic.
Trichloroethylene:
(TCE) A volatile organic
compound widely used as
an industrial degreaser; as
a solvent for oils, paints,
and varnishes; and as a
dry cleaning agent.
Introduction to P. butanovora
Uses a soluble diironcontaining monooxygenase to
metabolize the alkane
Structurally similar to methane
monooxygenase (MMO), but
can’t metabolize methane
Monooxygenase: introduces one atom of dioxygen into the substrate and
reduces the other atom to water
C4H10 + O2 + AH2  C4H9OH + H2O + A
Structural genes of BMO and MMO
bmoX
bmoY
hydroxylase, a
hydroxylase, b
mmoX
mmoY
bmoB bmoZ
hydroxylase, g
mmoB mmoZ
BMO reductase
g
a
BMOB
BUTANE
b
a
b
g
bmoC
reductase
mmoD
mmoC
MMO reductase
g
BUTANOL
BMO hydroxylase
bmoD
a
METHANOL
MMO hydroxylase
MMOB
METHANE
b
b
a
g
Approach to modify BMOB
Find out how BMOB determines what
alkanes can be metabolized by BMO
Create mutations in the DNA sequence
coding BMOB
Purify the modified BMOB
Y
Y
Y
WT
BMOB
A
mutant
BMOB
Then What?
Test the effect of the BMOB mutations on
sBMO in vitro and in vivo in P. butanova
BMO reductase
BMO reductase
g
g
a
BMO hydroxylase
b
WT
BMOB
a
BMO hydroxylase METHANOL
b
Mutant
BMOB
b
METHANE
a
g
METHANE
?
a
b
g
Procedure of purification
Protein
Quantification
Clone into pET15b, propagate in
DH5-a E. Coli
dialysis
Plasmid
Prep
His-Bind
purification
Transfer
into BL21 E.
Coli
Add IPTG &
grow for 3
hours
Grow to
OD600 0.6
DNA plasmid preparation
Sequencing
Carry out the
mutagenesis
reaction
DH5a plasmid preps
Determine
concentration of
plasmid
Transformation DH5a
Methods: BMOB Expressions
IPTG
Transformation into
BL21
Expression to gel
His-Bind purification column
LB media
SDS-PAGE
Purified protein
After dialysis
BMOB Purification Progress
Mutants of
BMOB
T109A
T108S
N105G
P32H
T108A
T109Y
H8A
M114A
Y100 frame
shift
V110A
BMOB
cloned into
pET15B
pET15B
transformed
into BL21
Expression
of BMOB
Purification
of BMOB
Dialysis of
BMOB
Protein
quantification
Summary
8 BMOB mutations were purified and
protein concentrations were determined
for all of them
Future research: Growth curves and
activity assays will be carried out to see if
changes in specificity were achieved
Acknowledgements
Howard Hughes Medical Institute
Dr. Daniel J. Arp
Dr. Luis Sayavedra-Soto
Kim Halsey
Dr. Kevin Ahern
Everyone else in the Nitrogen Fixation Lab
Thank you for all the help!!