Finished Dissertation

1
DNA Sequence Analysis and Propene Oxidation using the Alkene
Monooxygenase from Rhodococcus rhodochrous B-276
by Rebecca Holmes
Research Project Unit in Partial Fulfilment of BSc (Hons) Biochemistry
Supervisor: Professor Tom Smith
Submitted May 2016
5,347 words

3
Abstract
The alkene monooxygenase (AMO) is a member of the soluble diiron
monooxygeanse family found in Rhodococcus rhodochrous. Coded by the four gene
operon amoABCD, this enzyme is able to catalyse the epoxygenation of terminal and
subterminal alkenes yielding primarily R enantiomers. Genomic DNA extraction and
DNA sequencing was used to confirm the bacterial strain. Agarose gel
electrophoresis showed that the extraction, PCR amplification and purification
methods were successful leading to samples being able to be identified using DNA
sequencing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) was run to determine the presence of proteins within the cells. No bands
were observed on the gel suggesting that the various methods of protein extraction
used were unable to break the cell membrane and therefore need to undergo further
study. Propene oxidation assays were investigated using different preparation
techniques and analysed using gas chromatography. Successful methods for
epoxypropane detection were established and were able to detect propene peaks in
the samples but epoxypropane peaks were not observed in the samples. Problems
producing epoxide products may be due to the absence of active AMO as a result of
protein degradation. Further research could be investigated in the epoxygenation of
other alkenes such as 1-Butene and 1-Hexene by using freshly cultured cells grown
on propene. Successful chiral epoxide production would be invaluable to the
pharmaceutical industry as precursor molecules and could potentially be applied to
the degradation of the hazardous pollutant trichloroethene.

5
Acknowledgements
I would like to thank my supervisor Professor Tom Smith for always making himself
available for support, despite his busy schedule. I would like to thank Dr Tim Nichol
for providing additional support with obtaining materials and developing methods. I
would also like to thank the technical team for their assistance in labs and the
analytical team for their help when using gas chromatography.

7
Contents
1. Introduction 9
1.1 Soluble Diiron Monooxygenases 9
1.2 Soluble Methane Monooxygenases 9
1.3 Alkene Monooxygenase 9
1.4 Aims and Objectives 12
2. Materials and Methods 12
2.1 Materials 12
2.2 R. rhodochrous: Genomic DNA Extraction 13
2.3 PCR of Genomic DNA 13
2.4 PCR Purification 14
2.5 SDS-PAGE Analysis 14
2.6 Epoxypropane Gas Chromatography 15
3. Results 16
3.1 DNA Sequence Analysis of R. rhodochrous B-276 16
3.2 Protein Isolation and Determination 17
3.3 Propene Oxidation and Analysis using GC 18
4. Discussion 23
4.1 DNA Sequence Analysis of R. rhodochrous B-276 23
4.2 Protein Isolation and Determination 24
4.3 Propene Oxidation and Analysis using GC 25
4.4 Further Research 25
5. Conclusion 27
6. References 28

9
1. Introduction
1.1 Soluble Diiron Monooxygenases
Soluble diiron monooxygenases are a group of multicomponent enzymes that are
involved in the oxidation of alkanes, alkenes, ethers, aromatics, carbon monoxide
and ammonia (Neilson et al. 2013; Miura et al. 1995). These enzymes use dioxygen
to initiate the hydroxylation or epoxidation step involved in the oxidation pathways for
their hydrocarbon substrates. NADPH or NADH is a cofactor that provides electrons
that are required stoichiometrically by the reaction. The following equation shows the
epoxidation step producing an epoxide with propene as the substrate:
𝑝𝑟𝑜𝑝𝑒𝑛𝑒 + 𝑂2 + 𝑁𝐴𝐷𝐻 + 𝐻+
→ 𝑒𝑝𝑜𝑥𝑦𝑝𝑟𝑜𝑝𝑎𝑛𝑒 + 𝐻2 𝑂 + 𝑁𝐴𝐷+
Three-component phenol hydroxylases and four component toluene
monooxygenases are two members that are involved in the hydroxylation of phenols
to catechols and toluene oxidation respectively (Leahy et al. 2003; Coleman et al.
2006).
1.2 Soluble Methane Monooxygenases
Extensive research has been conducted into another member, the methane
monooxygenase enzyme (sMMO) due to its potential ability in the degradation of
greenhouse gases (Leahy et al. 2003). Methane is a common greenhouse gas and
is the main substrate oxidised to methanol, as the first step in the assimilation of
biomass and energy by methanotrophic bacteria occurring at the dinuclear iron
centre (Sazinsky et al. 2015). sMMOs are a three component oxygenases
dependent on NADPH or NADH as an electron donor. The three components consist
of a dinuclear iron site-containing hydroxylase, a reductase and a gating protein. As
well as methane, sMMOs can also co-oxidise alkanes, alkenes and aromatics,
meaning that it has a wider range than other monooxygenases (Smith et al. 2001;
Coleman et al. 2006).
1.3 Alkene Monooxygenase
Another member of the soluble diiron monooxygenase family is the alkene
monooxygenase (AMO) enzyme, found in Rhodococcus rhodochrous bacteria
(formerly known as Nocardia corallina). AMOs catalyse the epoxygenation of

10
terminal and subterminal alkenes ranging from C2-C18 stereoselectively, yielding
primarily R enantiomers whereas sMMOs yield a racemic product (van Ginkel et al.
1987). As well as this, AMOs can oxidise ethane to an achiral epoxyethane. The high
stereoselectivity of this enzyme means that it can be used as a biological catalyst in
organic chemistry. Chemical routes to short-chain chiral epoxides are difficult, and
the synthesis of epoxides produces a racemic product. Epoxides can undergo a
diverse range of reactions and are optically active meaning that they are of
considerable value as versatile chiral synthons (Smith et al. 2001). They are
particularly important in the development of pharmaceuticals such as synthesis of
(S)-argylglycidylethers used in β-blockers, ferroelectric liquid crystals, pesticide
production and desulfurisation of fossil fuels (Saitoh et al. 2013) .
AMOs are also capable of degrading a range of hazardous chlorinated ethenes
including trichloroethene (TCE), 1,1-dichloroethene, 1,2-dichloroethene, 2,3-
dichloropropene and 1,3-dichloropropene. These chemical reactions mean that
AMOs have the potential to bioremediate existing pollutants that can be found in
water. Currently TCE, an industrial solvent, can be degraded to trichloroepoxyethane
at a rate of 59 nmol min-1 per mg of protein and with further investigation could be a
potential candidate in pollution reduction (Smith et al. 2009). Studies conducted by
Saeki et al. (1999), have proven that TCE can be degraded by 25% from the initial
amount within six hours. Further microbial investigation of this enzyme could lead to
faster and more effective degradation.
As a result of sequence analysis and spectroscopy, it is known that AMOs are
structurally very similar to the soluble methane monooxygenase in that it is also a
three component enzyme. The four gene operon amoABCD codes for the enzyme to
comprise of a two component epoxygenase, an NADPH-dependent reductase and a
gating protein (figure 1). Due to the 57% similarity in the primary structure of amoC
and the α-subunit of sMMO, a reasonable assumption can be made about their
tertiary structure. The alkene monooxygenase is the first enzyme in the alkene
metabolism pathway that steroselectively inserts one oxygen atom from dioxygen
across the alkene double bond yielding a chiral epoxide. The remaining oxygen is
reduced to water by electrons from NADH or NADPH (Perry et al. 2006). Alkene
epoxygenation occurs at the epoxygenase site composed of two subunits of 38 kDa
and 57 kDa encoded by amoA and amoC. EPR spectroscopy has shown that amoC

11
in the epoxygenase contains a bridged dinuclear iron centre (figure 1 (1)) (Gallagher
et al. 1997). The second component is the reductase that is encoded by amoD, a
single polypeptide of 38 kDa. This component contains a Flavin (FAD) and an Fe2S2
cluster as its prosthetic group and is responsible for NADPH or NADH oxidation
which supplies electrons to the dinuclear iron site (figure 1 (2)). Evidence suggests
that the activity of the enzyme can increase by 6.5 times when NADH is used as the
donor as opposed to NADPH. amoB codes for the 14 kDa gating protein, the
function of this component isn't fully known however it is required for AMO activity
(figure 1 (3)) (Miura et al. 1995).
Figure 1. Structure of the alkene monooxygenase.
1) The epoxygenase subunits composed of amoA (38 kDa) and amoC (57 kDa). AmoC
contains a dinuclear iron centre that stereoselectively inserts one oxygen from dioxygen
across the alkene double bond producing a chiral epoxide and water. 2) The reductase
component is encoded by amoD (38 kDa) and contains an Flavin (FAD) and Fe2S2
prosthetic group. NADH is oxidised to NAD+
which provides amoC with protons and
electrons for epoxygenation. 3) The gating protein (14 kDa) function is unknown but is
required for enzyme activity (Smith et al. 2010 with amendments).
The bacterial strain R. rhodochrous B-276 containing the AMO, grows using propene
and other alkenes as its sole source of carbon energy however it is also capable of
growing using glucose. Propene is converted to R-epoxypropane with enantiomeric
excess (e.e.) values that can be as high as 83%, which is the smallest epoxyalkane
that can be formed (Fosdike et al. 2005). Epoxide stereoselectivity varies depending
on which alkene is being oxidised by the AMO enzyme in R. rhodochrous. E.e.

12
values can vary from 5% (R-m-methylphenylglycidyl ether) to 97% (R-
pentafluorostyrene oxide), the R isomer of the epoxides are predominantly formed.
After alkene oxidation, the chiral epoxide is carboxylated by a four component
enzyme known as epoxide carboxylase (Smith et al. 2001). This yields a β-keto acid
and uses CO2, NAD+ and NADPH (Allen and Ensign, 1996). When propene is the
substrate, acetoacetate is produced and used as its energy source as shown by the
following equation:
𝑒𝑝𝑜𝑥𝑦𝑝𝑟𝑜𝑝𝑎𝑛𝑒 + 𝐶𝑂2 + 𝐻+
→ 𝑎𝑐𝑒𝑡𝑜𝑎𝑐𝑒𝑡𝑎𝑡𝑒
1.4 Aims and Objectives
The aim of this research was to develop an effective method of genomic DNA
extraction from the R. rhodochrous B-276 bacterial strain and confirm that the cells
were the correct strain as they were prepared externally. The current methods of
epoxypropane production from propene was also investigated with the aim of
analysing a range of alkenes. A genomic DNA extraction kit was used to extract
primarily bacterial genomic DNA excluding plasmid DNA and other cell components.
Polymerase chain reaction (PCR) was used to amplify the specific AMO DNA
sequence for it to be analysed by DNA sequencing. Agarose gel electrophoresis was
run throughout the DNA extraction, amplification and purification process to
determine if the techniques were successful. To determine if R. rhodochrous was
producing AMO, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) was run using protein extraction samples. Various techniques and methods
were also tested on R. rhodochrous to develop a successful approach of oxidising
propene and data was collected using gas chromatography. Successfully developing
these methods could lead future researchers to be able to investigate whether R.
rhodochrous oxidises other alkenes and with what enantiomeric excess.
2. Materials and Methods
2.1 Materials
All reagents were purchased from Sigma Aldrich (Poole, UK) unless otherwise
stated. Glucose was purchased from Acros Organics. Agarose, 16S1 and 16S2
primers were purchased from Invitrogen. Diethyl ether, ethylenediaminetetraacetic
acid (EDTA), tris(hydroxymethyl)aminomethane (Tris), sodium hydroxide, sucrose,

13
ethanol, glycerol and glycine were all purchased from Fisher Scientific. Phosphate-
buffered saline (PBS) was purchased from Gibco by Life Technologies. R.
rhodochrous was supplied in pellet form frozen using liquid nitrogen, grown
previously on propene.
Bench centrifugation was performed using a Thermo Scientific Heraeus Fresco 21
centrifuge at 4°C. DNA samples were amplified using a Techgene, Techne PCR
machine. Agarose gel electrophoresis was run on a Peqlab Biotechnologie
apparatus with a thermo EC 105 voltmeter and images were photographed using a
Quantum Vilber Lourmat. A VWR ultrasonic Cleaner B-3001 was used for sample
sonication. SDS-PAGE was run using a Biometra, An Analytik Jena Company
apparatus. The Agilent Technologies 68GON Network GC system was used with a
Stabilwax, Restek Crossband Carbowax or Grace ATTM-5 column. GC-MS was
performed using a Agilant Technologies 7890A GC system and 5975C VL MSD with
Triple-Axis Detecter fitted with a Grace ATTM-5 column.
2.2 R. rhodochrous: Genomic DNA Extraction
R. rhodochrous bacteria were supplied in pellet form, frozen with liquid nitrogen. Two
pellets were used in genomic DNA extraction, one was diluted with phosphate
buffered saline (PBS) and the other was left to thaw. Both samples underwent DNA
extraction using a GenElute™ Bacterial Genomic DNA Kit from Sigma Aldrich,
performed per the manufacturer’s protocol. 10 µL of samples were loaded onto a 1
% (w/v) agarose gel prepared in 1x TAE buffer (pH 8.0). The gel was run at 70 V for
90 mins. Photographic images of the gels were obtained immediately after
electrophoresis.
2.3 PCR of Genomic DNA
Samples obtained from the GenElute™ Bacterial Genomic DNA were amplified by
PCR using primers 16S1 (AGA GTT TGA TC TGG CTC AG) and 16S2 (TAC GGY
TAC CTT GTT ACG ACT T). PCR was carried out with a total volume of 50 µL
containing 1x PCR buffer, 50 mM MgCl2, an equimolar mixture of each dNTP with a
total concentration of 2 mM, 5 pmol of each primer and 1 unit of Taq DNA
polymerase. Amplification conditions were 95 °C for 10 min, 35 cycles of 95 °C for 1
min, 50 °C for 1 min, 72 °C for 90 s, with a final extension of 72 °C for 15 mins. Each
sample (10 µL) was loaded onto a 1 % (W/V) agarose gel as previously described.

14
2.4 PCR Purification
PCR amplification products were purified using a Qiagen QIAquick PCR Purification
Kit according the manufacturer’s protocol.
2.5 SDS-PAGE Analysis
A 1.5 M solution of Tris pH 8.8 was prepared by dissolving 9.09 g of Tris in water.
HCl was used to adjust the pH and the volume was made up to 50 mL. A 1 M
solution of Tris pH 6.8 was prepared by dissolving 6.06 g of Tris in water. HCl was
used to adjust the pH and the volume was made up to 50 mL. Both solutions were
stored in a cold environment. Sample Buffer (2x) (10 mL) was prepared containing
final concentrations of 100 mM Tris HCl pH 8.8, 20% glycerol, 4% SDS, 10% β-
mercaptoethanol and 0.2% bromophenol blue and kept at room temperature.
A 12% resolving gel was prepared as follows; 3.3 mL water, 2.5 mL Tris pH 8.8, 0.1
mL 10% ammonium persulfate (APS), 4 mL 30% acrylamide, 0.1 mL 10% SDS and
0.004 mL tetramethylethylenediamine (TEMED). Neat ethanol was used to remove
bubbles. A stacking gel was prepared using 0.83 mL 30% acrylamide, 3.4 mL water,
0.63 mL Tris pH 6.8, 50 µL 10% SDS, 50 µL 10 APS and 5 µL TEMED.
Samples were prepared using various methods and techniques. Thawed R.
rhodochrous was mixed with 2x sample buffer, boiled at 95°C for 5 min and
immediately put on ice. Centrifugation at 4°C, 5000 xg for 3 min was used to
separate the mixture and the supernatant was loaded onto the gel. Other samples
underwent sonication at high intensity for 5 mins with some being boiled afterwards.
The supernatant was loaded onto the gel after centrifugation. Final samples were
prepared with the addition of 5 µL protease inhibitor. After which, they were boiled;
centrifuged at 4°C, 5000 xg for 3 min and the supernatant loaded onto the gel. 10 µl
of each sample type was mixed with 10 µL of 2x sample buffer before loading. Low
range, colorPlus and EZ run markers were used to allow estimates of the molecular
masses of the protein bands.
100 mL SDS-TANK buffer (10x) was prepared with final concentrations of 250 mM
Tris, 1.92 M glycine and 1% SDS. 50 mL of 10x buffer was diluted to 1x giving a final
volume of 500 mL. The SDS-PAGE was run at 150 V for 90 mins and then the gel
was completely immersed in instant Blue and shook for 20 mins.

15
2.6 Epoxypropane Gas Chromatography
Two 10 g/L stock solutions of epoxypropane were prepared in water and in diethyl
ether and from there standards of 0.2, 0.4, 0.6, 0.8, 1, 1.5, 2, 5, 7, 8 and 9 g/L were
made. Each standard and subsequent samples were run on a gas chromatography
using a stabilwax column with GC settings with an initial temperature of 20°C and a
ramp of 1°C/min finalising at 30°C, split ratio 1:20. GC-MS was conducted using an
AT-5 column set isothermally with a temperature of 20°C.
Bacterial samples containing propene were prepared using different techniques and
methods. Some samples were prepared by adding 400 µL of thawed bacteria and
100 µL of 10% glucose. Others were made up of 250 µL bacteria 200 µL PBS and
50 µL 10% glucose solution. Both samples were pipetted into 1.5 mL crimped GC
vials and the assay was initiated by injection 1 mL of propene directly into the vials.
The vials were shaken overnight at 180 rpm at 30°C. 100 µL of diethyl ether was
then injected into each vial to extract the epoxypropane product out. The vial
contents were centrifuged at 5000 xg for 3 mins and the ether layer was analysed on
GC (Perry et al. 2006).
Other samples were prepared the same as previously in 1.5 mL crimp-seal GC vials
but 1 mL of the headspace gas was removed before propene injection and
incubation (Fosdike et al. 2005). Some samples were also prepared by sealing 250
µL bacteria and 200 µL PBS into crimp-seal GC vials and 3 mL of headspace was
removed. 3 mL of propene gas was then injected into the vials and the vials were
shook at 30°C for 30 sec. Reaction was initiated by adding 50 µL 10% (wt/vol)
glucose and vials were further shook for 3 mins. 250 µL of diethyl ether was used to
extract the epoxypropane out of the sample. Samples were centrifuged at 500 xg for
3 mins and the ether layer was analysed (Perry et al. 2006).
Other samples were prepared in 7 mL headspace vials by pipetting 450 µL thawed
bacteria into the vials. 3 mL of propene was then injected into each vial and samples
were incubated at 30°C for 1 min. The reaction was initiated by adding 50 µL of 10%
(wt/vol) glucose and samples were further incubated for 10 mins. Vials were kept in a
45°C water bath while 0.5 mL injections into the GC were performed using a gas
tight syringe (Gallagher et al. 1997)

16
3. Results
3.1 DNA Sequence Analysis of R. rhodochrous B-276
Genomic DNA was extracted from R. rhodochrous using the method described
previously and an agarose gel electrophoresis was run. DNA preparations 2-3 were
made by thawing the bacteria in PBS and samples 4-5 were pure thawed bacteria.
Bands for all four samples were compared with the BstEII marker and appeared at
8,454 base pairs (bp) in length (figure 2A). All samples that were run on the first gel
underwent PCR amplification and loaded onto another agarose gel. DNA samples
prepared in PBS (figure 2B) showed bands at 100 bp and only well 4 showed bands
at 100 bp and 1,517 bp in length. Samples containing pure thawed bacteria after
PCR and produced prominent bands at 1,517 bp. A negative control was used
containing no DNA presenting no bands (figure 2B). Samples that were in wells 4, 5
and 6 from the PCR agarose gel (figure 2B), were used for PCR purification and then
loaded onto an agarose gel as show in figure 2C. One well contained purified R.
rhodochrous in PBS and the others contained purified thawed bacteria. All three
sample wells exhibited bands at 1,517 bp in length (figure 2C).
Figure 2. Agarose gel electrophoresis of R. rhodochrous genomic DNA.
A) Agarose gel electrophoresis of thawed bacteria and bacteria in PBS after genomic DNA
extraction. All four wells exhibited bands at 8,454 bp with wells 2-3 containing bacteria in
PBS and wells 4-5 containing the thawed bacteria. B) Agarose gel of samples after PCR

17
amplification. Wells 2-4 contained R. rhodochrous in PBS producing bands at 100 bp and
1,517 bp. The thawed R. rhodochrous samples were loaded onto wells 5-7 and showed
bands at 1,517bp. Well 8 was used as a negative control and contained no DNA. C)
Samples 4, 5 and 6 from the previous gel underwent purification and loaded onto a gel in
order. All three wells displayed bands at 1,517 bp. All gels were loaded with a 100 bp marker
and a BstEII marker for base pair identification.
3.2 Protein Isolation and Determination
Liquid nitrogen frozen R. rhodochrous was thawed and prepared using techniques
described in the materials and methods section and loaded onto an SDS-PAGE.
ColorPlus, EZ and Low range were all used as markers to determine the weight in
kDa. Wells 3 and 4 contained bacteria that had undergone the sonication method of
extraction. The bacteria that were boiled with sample buffer and immediately put in
ice, was loaded onto wells 5 and 6. The bacterial samples prepared using protease
inhibitors were loaded onto wells 7 and 8. The yellow line along the bottom is the dye
front showing that the proteins were not run off the bottom of the gel (figure 3).
Figure 3. SDS-PAGE of R. rhodochrous after protein extraction.
Wells 1-2 contained a ColorPlus and an EZ marker resulting in bands with the most
prominent being at 80 kDa and 25 kDa. Sonicated samples were loaded onto wells 3 and 4.

18
Samples that were boiled at 95°C and immediately put on ice, were loaded onto wells 5 and
6. The samples prepared with protease inhibitors were loaded onto wells 7 and 8. All
methods of protein extraction resulted in no bands other than the dye front at the bottom of
the gel. A Low Range marker was loaded onto well 9 creating a smear.
3.3 Propene Oxidation and Analysis using GC
Standards prepared with various concentrations as described is the materials and
methods section were run on GC and plotted against the average of the
corresponding peak area. The lowest detection limit for epoxypropane was identified
as being 0.2 mg/mL with the highest being 9 mg/mL. All standards were run with a
minimum of two replicates and standard deviations and standard errors were
calculated. Standard errors are represented by the error bars on each point showing
an increase as epoxypropane increases. The equation of the line was determined to
be; 𝑦 = 433077𝑥 which could then be used to calculate the epoxypropane
concentration that would be obtained later. A coefficient of determination value was
calculated to be 𝑅2
= 0.9876 to determine how well the regression line fit the data
points.
Figure 4. Epoxypropane standard curve.
Different epoxypropane concentrations were analysed using GC in increasing concentration.
Their corresponding peak areas were averaged and plotted against the concentration. The
lowest detection limit was identified as being 0.2 mg/mL and the highest as being 9 mg/mL.
y = 433077x
R² = 0.9876
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
0 2 4 6 8 10
PeakArea(μV*sec)
Epoxypropane Concentration (mg/mL)

19
Standard deviations and standard error from the standard deviations was calculated for each
concentration and represented by the error bars. Concentration at 0.4 mg/mL was measured
twice. Concentrations 0.6 and 5 mg/mL were measured four times. Concentration 0.8 mg/mL
was measured five times. All other concentrations were measured three times. The equation
of the line was calculated as 𝒚 = 𝟒𝟑𝟑𝟎𝟕𝟕𝒙 with the R2
value being 0.9876.
The retention time of the expected product epoxypropane, was established through
analysis of the epoxypropane standards in diethyl ether. The diethyl ether solvent
peak appeared at 4.47 min and the epoxypropane peak at 7.08 with the
concentration of 8 mg/mL giving an area of 152473.88 μV*sec (figure 5A). Propene
gas was also analysed using GC to establish its peak retention time (3.87 min) to
determine if the enzymatic reaction had taken place (figure 5B).

20
Figure 5. Gas chromatogram of epoxypropane in diethyl ether and propene.
A) The epoxypropane standard 8 mg/mL in diethyl ether representing their corresponding peaks. A solvent diethyl ether peak is present at 4.47
min and epoxypropane at 7.08 min with a peak are of 152473.88 μV*sec. B) Using a gas tight syringe, 1 mL of propene was analysed using
GC giving a retention time of 3.87 min.

21
The majority of samples run on the GC wax column presented a peak at approx.
3.93 min with an area of 632924.06 μV*sec and the solvent diethyl ether peak at
4.40 (figure 6). One sample was then run on GC-MS with an AT-5 column to
determine the 3.93 min peak as being propene.
Figure 6. GC chromatogram of R. rhodochrous reaction sample.
The sample using the overnight incubation method along with the other methods presented
a peak at 3.93 min and a solvent diethyl ether peak at 4.40 min. The peak at 3.93 min had a
peak area of 632924.06 μV*sec and was further analysed on GC-MS to determine
compound. GC-MS analysis showed the peak to be propene.
Some samples of both PBS thawed and purely thawed R. rhodochrous displayed
unknown peaks at approx. 11.21 min with an approx. peak area of 993538.92
μV*sec. The diethyl ether peak remained at a similar retention time of 5.10 min
(figure 7).

22
Figure 7. GC chromatogram of thawed R. rhodochrous reaction sample.
The reaction sample containing thawed R. rhodochrous in overnight incubation displaying a diethyl ether peak at 5.10 min and an unknown
peak at 10.70 min with a peak area of 1358716.3 μV*sec.

23
4. Discussion
The work carried out was designed to develop an effective method of bacterial
genomic DNA extraction and to confirm that the strain given was R. rhodochrous B-
276 by using DNA sequencing. The various protein extraction methods were tested
to confirm that the cells could be broken up and protein extracted to conclude if there
was any protein still present within the cells.
The results obtained through agarose gel electrophoresis analysis confirmed that
genomic DNA extraction could be used successfully and the PCR method used is
effective for 16s RNA gene amplification. Work was conducted on developing a
method of propene oxidation to then apply those methods to other alkene substrates.
Results confirmed an effective epoxide identification method with GC, which could
then be used to retest propene oxidation samples and other substrates.
4.1 DNA Sequence Analysis of R. rhodochrous B-276
The method used for extraction of genomic DNA out of R. rhodochrous was
confirmed successful by analysing samples with agarose gel electrophoresis. The
bands present in figure 2A suggest that the kit used to isolate the genomic DNA was
effective and can therefore be continually used in future research. Similar work
conducted by Saitoh et al. (2013) also showed success in the isolation of genomic
DNA through the use of a specialist kit allowing them to further analyse the genomic
structure.
PCR was conducted to amplify the specific DNA sequence coding for the 16s RNA
gene to confirm the bacterial strain given was R. rhodochrous B-276. The strong
bands exhibited in the purely thawed samples compared to the PBS thawed samples
in figure 2B, suggest that this method of sample preparation is the most effective.
The samples prepared in PBS created mixed results as one well showed a faint
band at 1,517 bp whereas the other two showed faint bands at 100 bp suggesting
that this method is less effective and alternatives should be considered. The kit used
to purify the DNA after PCR did prove to be successful as bands were present at the
1,517 bp mark (figure 2C) which corresponds to the bands on gel B. The bands on
gel C were faint suggesting that a higher concentration of PCR product would need
to be used in the purification process in order for DNA sequence analysis to be
successful.

24
4.2 Protein Isolation and Determination
The results shown from the SDS-PAGE (figure 3) suggest that protein isolation from
R. rhodochrous needed further investigation as proteins could not be visualised.
However, the presence of the marker bands shows that the method for gel
preparation was a success. The liquid nitrogen frozen bacteria provided was
previously grown on propene suggesting that there would have been active AMO
produced at one time, however it may have been degraded during the time it had
been frozen and be present within the dye front. R. rhodochrous grown on glucose is
an alternative method of cell preparation but produces fewer AMO and other proteins
that aren’t necessary for the cells. Another problem may have been that the methods
used for protein extraction were not effective enough to break the cell membrane.
Various methods of cell lysis were investigated such as sonication and high
temperature boiling to troubleshoot the problem but a more vigorous method may
need to be applied. In order to successfully extract proteins from R. rhodochrous,
fresh cells should be used and other methods of extraction should be investigated.
Miura et al. (1995) prepared cell extracts by passing the thawed cells through a cell
distributer, centrifuging and then using DEAE cellulose column chromatography to
prepare samples for further phenyl sepharose column chromatography or separation
by other means. The outcome was successful in isolating the four different active
AMO components with the addition of glycerol and SDS-PAGE exhibited relevant
bands relating to the proteins.
Use of a magnesium sulphate buffer followed by French pressure cell press, dialysis
and centrifugation, is another alternative that can be for the preparation of cell
extracts. This method uses high pressure that disrupts the cell membrane and the
dialysis then removes cell debris from the sample. A biuret assay could be used as
an alternative to SDS-PAGE to determine the presence and concentration of
proteins within the sample. These methods that were performed by Allen et al.
(1996), showed them to be successful when applied to Xanthobacter strain Py2. As
this bacterial strain also produces AMO, it suggests that this method could be
applied to R. rhodochrous.

25
4.3 Propene Oxidation and Analysis using GC
The epoxypropane standards used to prepare the standard curve can be considered
reliable due to the R2 value being high. If the samples were to produce
epoxypropane peaks, the peak area could be entered into the standard curve
equation and the concentration calculated.
The majority of samples made produced peaks at around 3.93 min suggesting that
the AMO did not oxidise the propene injected into the vials (figure 6). The presence
of the propene gas peak also indicates that the crimped sealed vials used for
sampling are suitable for gas injection. If the method was to be repeated, R.
rhodochrous that has been freshly grown on propene and frozen using liquid
nitrogen would be used and the propene assay would expect to show an
epoxypropane peak and no propene peak on GC. The absence of results in the GC
analysis suggests that the thawed bacteria did not contain the active AMO enzyme in
order for propene oxidation to occur, possibly due to protein degradation over time.
The GC-MS analysis of the propene peak at 3.93 also confirms that propene had not
being oxidised. The mass spectrum was compared to other mass spectra on a
computer database which identified the peak as being propene.
The samples that produced an unknown peak at approx. 11.21 min need further
investigation in order to identify it using GC-MS. It is unknown whether the enzyme
actively produced this compound as a by-product or if it was a result of low-level
contaminants in the diethyl ether or coextractants from the assay. The presence of
the peak in both the PBS samples and the non PBS samples indicates that it is not a
contaminant in the PBS. All the techniques described in the materials and methods
section used by researchers Perry et al. (2006), Fosdike et al. (2005) and Gallagher
et al. (1997) showed success in their investigations. Perry et al. (2006) further
investigated the chirality of the epoxypropane product and concluded that a
genetically modified version of the AMO is able to produce a mean e.e. of 73%
epoxypropane compared to the wild-type that can produce 79%.
4.4 Further Research
The peroxide shunt reaction is another method to be considered which could
increase the probability of epoxypropane production; however the enzymes have
shown to be 10% less active. The peroxide shunt reaction allows terminal alkenes to

26
be epoxygenated in the absence of the reductase component (figure 1) and NADPH
or NADH. Hydrogen peroxide can be used as an alternative to dioxygen by providing
oxygen for the epoxygenation and water production. In sMMO analysis of the
peroxide shunt reaction, O2-mediated turnover was bypassed and was capable of at
least 30 turnovers with propene with a rate of ~1 s-1 (Bailey et al. 2009). Due to the
absence of information on the epoxide shunt reaction with the AMO of R.
rhodochrous, this could be an interesting area to investigate and could lead to
increased epoxide production.
If future study leads to successful epoxidation with the various alkenes by the AMO
of R. rhodochrous, this could lead into the possibility of it being used as an anti-
pollutant technique by degrading chlorinated compounds. Testing with chlorinated
alkanes would need to be done using an alternative GC method due to their flame
retardant properties; the FID connected to the GC would be unable to detect the
compounds. MS in Electron Capture Negative Ionisation mode is the current method
that has been well established, however a new technique called carbon skeleton gas
chromatography is currently being investigated. This method catalytically
hydrodechlorinates chlorinated paraffin’s to the corresponding n-alkanes and is
proving to be a positive technique by providing a better performance in
reproducibility and being less time-consuming than current techniques (Pellizzato et
al. 2008). Analysis of TCE would be an interesting area of investigation and if
effective, would be very significant in future strategies to remove the pollutant.
Studies by Saeki et al. (1999) found that TCE degradation by the AMO in R.
rhodochrous was not affected by the growth strategy used. Both glucose and
propene grown cells showed the same efficiency of degradation.
Developments in this research could also be beneficial to the pharmaceutical
industry by providing chiral epoxides that can be used as precursors. An example of
new substrates that could be investigated would include 1-octene, 1-butene, 1-
hexene, trans-2-butene and 1-tetradecene. Production of corresponding chiral
epoxides would be invaluable to organic synthesis of pharmaceuticals due to the
difficulties of the chemical synthesis pathway.

27
5. Conclusion
A working method for gene amplification of the 16s RNA gene for R. rhodochrous
was developed through use of a genomic DNA extraction kit, followed by PCR using
specific primers and agarose gel electrophoresis. The concentration of pure PCR
product obtained would be enough for DNA sequence analysis to confirm the base
pair sequence. The GC method developed for propene oxidation analysis was
effective in detection of epoxypropane standards and propene. Liquid nitrogen frozen
cells that have been freshly grown on propene would be used in future research as
they may contain active AMO enzyme for epoxygenation and protein degradation
may not have had an impact yet. SDS-PAGE confirmed that the cells either
contained degraded proteins or that the methods of protein extraction were not
vigorous enough the break the cell membrane. Further analysis should be conducted
using the freshly grown cells and the specific AMO extraction method tested to fully
determine the enzymes presence on SDS-PAGE. The GC method developed in this
research can be used in the future along with the propene oxidation assays which
should be effective with the fresh bacteria. Other alkene substrates such as 1-
hexene, 1-butene and 1-octene could also be investigated to determine the enzymes
activity and percentage chiral production.

28
6. References
ALLEN, J. and ENSIGN, S. (1996). Carboxylation of Epoxides to Beta-Keto Acids in
Cell Extracts of Xanthobacter strain Py2. J Bacteriol, 178 (5), 1469-72.
BAILEY, L. and FOX, B. (2009). Crystallographic and Catalytic Studies of the
Peroxide-Shunt Reaction in a Diiron Hydroxylase. Biochemistry, 48 (38), 8932-8939.
COLEMAN, N.V., BUI, N.B. and HOLMES, A.J. (2006). Soluble Di-Iron
Monooxygenase Gene Diversity in Soils, Sediments and Ethene Enrichments.
Environmental Microbiology, 8 (7), 1228-1239
FOSDIKE, W., SMITH, T. and DALTON, H. (2005). Adventitious Reactions of Alkene
Monooxygenase Reveal Common Reaction Pathways and Component Interactions
Among Bacterial Hydrocarbon Oxygenases. FEBS Journal, 272 (11), 2661-2669.
GALLAGHER, S., CAMMACK, R. and DALTON, H. (1997). Alkene Monooxygenase
from Nocardia Corallina B-276 is a Member of the Class of Dinuclear Iron Proteins
Capable of Stereospecific Epoxygenation Reactions. Eur J Biochem, 247 (2), 635-
641.
LEAHY, J., BATCHELOR, P. and MORCOMB, S. (2003). Evolution of the Soluble
Diiron Monooxygenases. FEMS Microbiology Reviews, 27 (4), 449-479.
MIURA, A. and DALTON, H. (1995). Purification and Characterization of the Alkene
Monooxygenase from Nocardia corallina B-276. Bioscience, Biotechnology and
Biochemistry, 59 (5), 853-859.
NEILSON, A. and ALLARD, A. (2013). Organic Chemicals in the Environment. Boca
Raton, FL: CRC Press.
PELLIZZATO, F., RICCI, M., HELD, A. and EMONS, H. (2008). Determination of
Short-Chain Chlorinated Paraffins by Carbon Skeleton Gas Chromatography.
Organohalogen Compounds, 70 (1), 000776.
PERRY, A. (2006). Protocol for Mutagenesis of Alkene Monooxygenase and
Screening for Modified Enantiocomposition of the Epoxypropane Product. Journal of
Biomolecular Screening, 11 (5), 553-556.

29
SAEKI, H., AKIRA, M., FURUHASHI, K., AVERHOFF, B. and GOTTSCHALK, G.
(1999). Degradation of Trichloroethene by a Linear-Plasmid-Encoded Alkene
Monooxygenase in Rhodococcus corallinus (Nocardia corallina) B-276. Microbiology,
145 (7), 1721-1730.
SAITOH, S., AOYAMA, H., AKUTSU, M., NAKANO, K., SHINZATO, N. and
MATSUI, T. (2013). Genomic Sequencing-Based Detection of Large Deletions in
Rhodococcus rhodochrous Strain B-276. Journal of Bioscience and Bioengineering,
116 (3), 309-312.
SAZINSKY, M. and LIPPARD, S. (2015). Methane Monooxygenase: Functionalizing
Methane at Iron and Copper. Metal Ions in Life Sciences, 15 (1), 205-256.
SMITH, T., LLOYD, J., GALLAGHER, S., FOSDIKE, W., MURRELL, J. and
DALTON, H. (2001). Heterologous Expression of Alkene Monooxygenase from
Rhodococcus rhodochrous B-276. European Journal of Biochemistry, 260 (2), 446-
452.
SMITH, T.J. 2010. Oxidation of alkenes by bacterial monooxygenases.
Encyclopaedia of Industrial Biotechnology. 1-13. Ed. Flickenger, M. C. Published by
Wiley
VAN GINKEL, C.G., WELTEN, H.G.J., and DE BONT, J.A.M. (1987). Oxidation of
Gaseous and Volatile Hydrocarbons by Selected Alkene-Utilizing Bacteria.
Environmental Microbiology, 53 (12), 2903-7

Finished Dissertation

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Viewers also liked

Viewers also liked (11)

Similar to Finished Dissertation

Similar to Finished Dissertation (20)

Finished Dissertation