Biotechnol Lett (2010) 32:497–502
DOI 10.1007/s10529-009-0177-0
ORIGINAL RESEARCH PAPER
Production of a recombinant alkane hydroxylase (AlkB2)
from Alcanivorax borkumensis
Mandana Miri • Bijan Bambai •
Fatemeh Tabandeh • Majid Sadeghizadeh •
Nasrin Kamali
Received: 21 September 2009 / Revised: 10 November 2009 / Accepted: 10 November 2009 / Published online: 2 December 2009
Ó Springer Science+Business Media B.V. 2009
Abstract Alcanivorax borkumensis is an oil-degrad-
ing marine bacterium. Its genome contains genes
coding for three cytochrome P450s and two integral
membrane alkane hydroxylases (AlkB1 & AlkB2), all
assumed to perform hydroxylation of different linear or
branched alkanes. Although, the sequence of alkB2 has
been determined, the molecular characterization and
the substrate specificity of AlkB2 require more precise
investigation. In this study, AlkB2 from A. borkumen-
sis SK2 was expressed in Escherichia coli to examine
Electronic supplementary material The online version of
this article (doi:10.1007/s10529-009-0177-0) contains
supplementary material, which is available to authorized users.
M. Miri
Science and Research Branch of Tehran, Islamic Azad
University, Tehran 1477893855, Iran
B. Bambai (&)
Faculty of Biological Sciences, Shahid Beheshti
University, GC, Tehran 19839, Iran
e-mail: B_Bambai@sbu.ac.ir
B. Bambai
F. Tabandeh
National Institute of Genetic Engineering
and Biotechnology (NIGEB), Tehran 14178, Iran
M. Sadeghizadeh
Genetics Department, Tarbiat Modares University,
Tehran 14115, Iran
N. Kamali
Malek-Ashtar University of Technology,
Tehran 83145, Iran
the functionality of AlkB2 as a hydroxylating enzyme.
Furthermore, the activity of the enzyme in the presence
of the accessory proteins, rubredoxin (RubA) and
rubredoxin reductase (RubB), produced in E. coli
BL21(DE3)plysS cells, was determined. Recombinant
AlkB2 is produced in an active form and rubredoxin is
the intermediate electron donor to AlkB2 and can
replace AlkG function, when NADH is the prime
electron donor.
Keywords Alcanivorax borkumensis

Alkane hydroxylase
Biotransformation

Rubredoxin
Introduction
Chemical hydroxylation of hydrocarbons is a
challenging reaction. The C–H bond is nonpolar and
activation of this bond in the Ziegler process requires
high energy and metal catalysts (Azapagic et al. 2003).
Production of long-chain alcohols (fatty alcohols) is
currently performed through chemical processes with
high energy demands, low yield and environmental
contamination. However, there are alternative bio-
technological systems capable of converting alkanes to
the corresponding alcohols under ambient conditions
with high specificity and negligible environmental
constrains. There is a growing list of obligatory hydro-
carbonoclastic microorganisms (van Beilen et al.
2003). These microorganisms represent a potentially
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498 Biotechnol Lett (2010) 32:497–502

valuable system for replacing chemical oxygenating Materials and methods

processes of the normal alkanes (n-alkanes). Among
these organisms, a petroleum-degrading bacterium, Bacterial strains and plasmids
Alcanivorax borkumensis has a unique position in
the global carbon cycle (Golyshin et al. 2003) as it Genomic DNA was extracted from the culture of
is a key bacterium involved in the biodegradation of Alcanivorax borkumensis SK2T strain (SK2T DSM
oil spills from marine environments (Harayama 11573T). The Escherichia coli DH5a strain (Invitrogen)
et al. 1999). and E. coli BL21(DE3)plysS strain (Promega) were
Existence of multiple hydrocarbon-degrading enzy- used for cloning and expression of the recombinant
matic systems is probably the most important reason for protein, respectively. pBluescript II KS(?) (Fermentas)
the ubiquity and competitive success of A. borkumensis and pET-21a(?) (Novagen) vectors were utilized in
(Schneiker et al. 2006). Heme-containing cytochrome cloning and expression experiments, respectively.
P450 and nonheme-diiron monooxygenases (alkane
hydroxylases) are among the most studied hydrocar-
DNA manipulation methods
bon hydroxylating systems (van Beilen et al. 2003).
Two nonheme hydroxylases, AlkB1 and AlkB2, have
A. borkumensis was cultivated in synthetic sea water
been annotated in the published genomic sequence of
medium 1 (SM1) (Yakimov et al. 1998) supplemented
Alcanivorax borkumensis SK2 (Schneiker et al. 2006).
with 1 g Tween 20/l and 1 g bitumen/l as the sole
AlkB1 is located in a well-studied gene cluster, respon-
source of carbon and energy. Total genomic DNA was
sible for degrading hydrocarbons with some accessory
extracted from A. borkumensis cells according to the
proteins, e.g. AlkG, a rubredoxin with two Fe binding
bacterial genomic DNA isolation kit (Metabion) and
sites. AlkB2, on the other hand is not in this operon and
used as the template for PCR amplification of the alkB2
has different regulatory elements (Schneiker et al.
gene fragment. The DNA sequence of the gene
2006). The alkane hydroxylases require two other
encoding the AlkB2 enzyme was obtained from the
proteins to supply reducing electrons from nicotinamide
GenBank database (ABO_0122). To amplify the alkB2
adenine dinucleotide (NADH). There are two genes,
gene with an extra hexa-histidine tag at 30 -side of the
rubA and rubB, in the genome of A. borkumensis
recombinant gene, named alkB2H, PCR was carried
encoding rubredoxin, and rubredoxin reductase, respec-
out by pfu DNA polymerase (Fermentas) using gene
tively. Alkane hydroxylases in coordination with
specific primers AlkB2 F1 (50 -CAT ATG TTC GAG
electron transfer proteins constitute hydrocarbon
AAT ACG AAT CCT G-30 ) and AlkB2 R2 (50 -TTA
hydroxylation systems necessary for converting hydro-
GTG GTG GTG GTG GTG GTG AGC GAT ATT
carbons to corresponding alcohols. Alcohols are further
GTT CAC TCG C-30 ). PCR was performed as follows:
oxidized to fatty acids and finally to CO2 via beta
94°C for 5 min, followed by 30 cycles at 94°C for 40 s,
oxidation pathway to extract chemical energy buried in
59°C for 40 s, and 72°C for 1 min, followed by a final
C–H bonds for growth and reproduction (van Beilen
extension at 72°C for 10 min. Plasmids were isolated
et al. 2003). AlkB1 preferably hydroxylases C5–C12
using the plasmid miniprep kit (Metabion). Restriction
hydrocarbones, however, the substrate specificity of
endonucleases and T4 DNA ligase (Fermentas) were
AlkB2 is C8–C16 hydrocarbones (Schneiker et al. 2006).
used according to the manufacturer’s instructions.
AlkB2 enzymatic products have much higher value-
added than original saturated hydrocarbons.
There have been some attempts to express the Construction of the recombinant plasmids
recombinant alkane monooxygenase systems derived
from Gordonia sp. TF6 and Geobacillus thermoden- To create pBSK II-alkB2H, the 1167 bp PCR product
itrificans NG80-2 in Escherichia coli (Fujii et al. was ligated into the Eco321 (EcoRV) cloning site of
2004; Feng et al. 2007). The aim of this study is the digested pBluescript II KS(?). The ligation product
expression of A. borkumensis SK2 alkB2 gene in was then transformed into competent E. coli DH5a
E. coli and investigation of the functionality of the cells. Recombinant clones harboring alkB2H frag-
AlkB2 in the presence of recombinant rubredoxin and ment were selected by blue/white screening. The
rubredoxin reductase. plasmid vector containing the alkB2H insert was

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extracted from the overnight culture of a white
bacterial colony and digested with the NdeI and
HindIII restriction enzymes. The restriction digestion
product was then analyzed by 1% (w/v) agarose gel
electrophoresis and the DNA fragment corresponding
to the amplified gene (1167 bp) was purified from the
gel using the gel extraction kit (Metabion). The
purified DNA fragment was then inserted into the
NdeI/HindIII double digested expression vector,
pET-21a(?). The recombinant plasmid was named
pET-21a(?)-alkB2H. The presence of the alkB2H
fragment in the recombinant plasmid was confirmed
by the NdeI/HindIII double digestion and DNA
sequencing using the universal forward and reverse
T7-promoter and T7-terminator primers.
Production of the recombinant AlkB2
To obtain the recombinant AlkB2 with a hexa-histidine
tag at the C-terminal, AlkB2H, E. coli BL21(DE3)-
plysS cells harboring the pET-21a(?)-alkB2H plasmid
was grown in a specific medium containing 10 g
peptone/l, 10 g glucose/l, 19.95 g KH2PO4/l, 6 g (NH4)2
HPO4/l, 1.2 g MgSO4
7H2O/l, 14.1 mg EDTA/l, 2.5 mg
CoCl2
6H2O/l, 15 mg MgCl2
4H2O/l, 1.5 mg CuCl2

2H2O/l, 3 mg H3BO3/l, 2.1 mg Na2MoO4
2H2O/l,
33.8 mg zinc acetate/l and 100.8 mg ferric citrate/l
supplemented with 100 lg ampicillin/ml and 30 lg
chloramphenicol/ml at 37°C with shaking at 200 rpm
to reach an OD600 of 0.6–0.8. The expression of the
recombinant AlkB2H was then induced by addition of
0.5 mM IPTG (Roche).The cells were incubated at
30°C with shaking at 150 rpm for an additional 6 h.
Purification of the recombinant AlkB2
The induced cells from 1 l culture (about 1 g dry wt
cells) were collected by centrifugation and the result-
ing pellet was resuspended in 10 ml binding buffer A
(50 mM sodium dihydrogen phosphate, 500 mM
NaCl, 10 mM imidazole, 0.1% Triton X-100, 0.05%
Tween 20, pH 8.0). After adding 1 mg lysozyme/ml,
the cells suspension was incubated for 30 min at 4°C
and sonicated for 59 10 s bursts at 200–300 W with a
10 s cooling intervals. After the treatment with 10 lg
RNase/ml and 5 lg DNase/ml for 15 min on ice, the
insoluble debris were removed by centrifugation at
120009g at 4°C for 20 min. The supernatant (cleared
lysate) was transferred to two milliliters of 50% slurry
499
Ni2?-nitrilotriacetic acid (NTA) agarose beads (Qia-
gen), pre-equilibrated with buffer A and stirred gently
for 1 h at 4°C. The Ni-NTA resin was sequentially
washed four times with 5 ml of each of the B, C, D
and E buffers (the same composition as buffer A but
containing 20, 30, 50 and 60 mM imidazole, respec-
tively). The recombinant AlkB2 was eluted with buffer
F (buffer A containing 400 mM imidazole) and dia-
lyzed against PBS. The protein concentration of the
purified AlkB2 fraction was determined by Bradford
method and the purified sample was analyzed on 12%
SDS-PAGE.
Western blotting analysis
Western blotting analysis was performed using the
monoclonal anti-his antibody to visualize of the purified
recombinant protein. The purified enzyme was trans-
ferred onto polyvinylidene difluoride (PVDF) mem-
brane (Hi-bond Amersham Biosciences) following
separation on a 12% (v/v) SDS-PAGE under 86 mA
current for 16 h in the transfer buffer (CAPS). The
membrane was then blocked overnight with the block-
ing buffer containing phosphate buffered saline, 0.05%
Tween 20 (PBS-T) and 5% (w/v) skimmed milk, and
washed five times in PBS-T subsequently. After 1 h
incubation with the monoclonal mouse anti-his antibody
(Amersham) used at 1/3000 dilution, the membrane was
washed with PBS-T and then incubated for 30 min at the
room temperature with the horseradish peroxidase
(HRP)-conjugated anti-mouse IgG (Ray BioTech)
diluted 1/2000. Finally, the band was revealed by the
peroxidase activity on 4-chloro-1-naphthol (Sigma) as
the chromogenic substrate.
Enzyme assay
The alkane hydroxylase activity of the recombinant
AlkB2 was determined by monitoring the decrease in
absorbance at 340 nm for 300 s. The reaction mixtures
contained 50 mM Tris/HCl (pH 7.5), 1 mM oleic acid,
1 mM each of NADH and MgSO4, 0.1 mM FAD and
10% (v/v) each of the cell crude extracts of BL21
(DE3)plysS expressing the recombinant alkane hydrox-
ylase (alkB2) and BL21(DE3)plysS co-expressing
rubredoxin (rubA) and rubredoxin reductase (rubB) of
A. borkumensis (The 5th Annual Biotechnology and
Bioinformatics Symposium-BIOT-2008, University of
Texas at Arlington). Mixtures without the cell crude
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extracts (either the cell crude extract containing AlkB2
or containing RubA and RubB) were used as controls.
To prepare the cell crude extracts, all cells were
collected following induction from 2 ml culture by
centrifugation and the resulting pellets were resus-
pended in one ml lysis buffer (50 mM Tris/HCl, 0.1%
Triton X-100, 0.05% Tween 20, pH 7.5). The cells
suspensions were ultra sonicated with 59 10 s bursts at
200–300 W with a 10 s cooling intervals. This assess-
ment was carried out at the room temperature. Each unit
of the enzyme activity was defined as 1 lmol oxidized
NADH per min/mg protein.
Results
Cloning of the alkB2 gene
The alkB2H fragment was PCR amplified from the
genome of A. borkumensis strain SK2. The PCR ampli-
fication yielded a single specific product of 1167 bp
(Supplementary Figure 1). The PCR product was then
inserted into the lacZ site of pBluescript II KS. The
alkBH2 fragment was digested by the NdeI/HindIII
restriction enzymes from pBSK II KS(?)-alkB2H
plasmid and sub-cloned into the pET-21a(?) expression
vector. The colonies harboring the recombinant pET-
21a(?)-alkB2H plasmid were screened by the NdeI/
HindIII double digestion (Supplementary Figure 2).
DNA sequencing analysis of the correct clone resem-
bled 100% similarity between the cloned gene and the
published sequence of the alkB2 sequence. Further-
more, the sequencing results revealed the appropriate
position of a hexa-histidine tag added at the carboxylic
terminal of the recombinant protein to facilitate the
subsequent purification of the recombinant protein.
Production and purification of the recombinant
AlkB2
Under optimized expression conditions, the highest
protein yield of the recombinant AlkB2 was obtained.
The SDS-PAGE of the recombinant enzyme exhib-
ited an apparent molecular mass of 44 kDa. This
band agreed with the expected molecular weight of
AlkB2H (Fig. 1, lane AI). The recombinant AlkB2
was purified using Ni-NTA affinity chromatography
(Fig. 1, lane P). Lower and higher bands observed on
the SDS-PAGE might be proteins that co-purified
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Biotechnol Lett (2010) 32:497–502
with AlkB2 or resulted from the degradation of the
protein by bacterial proteases. 3.75 mg purified
recombinant AlkB2 was obtained from 1 l culture.
The purified enzyme was recognized by western
blotting analysis using the monoclonal anti-his anti-
body (Fig. 2). No specific band, however, was
observed on the blot of the non-induced bacteria as
the negative control.
Assessing the activity of the recombinant AlkB2
Supplementary Figure 3 depicts the average changes
of the reaction mixture absorbance at 340 nm from
the triplicate assays after the addition of the cell crude
extracts expressing AlkB2. The activity was 177 ±
31 units (lmol NADH produced min-1 mg-1). Assay
mixtures lacking AlkB2, or the mixtures lacking
RubA and RubB proteins, failed to demonstrate any
measurable activity.
Discussion
Biological hydroxylation of alkanes is of great interest.
Alkanols derived from this reaction are intermediates
as plasticizers and solvents in chemical and pharma-
ceutical industries while their corresponding alkanes
are relatively low priced. The first and the most critical
step in biological utilization of alkanes in hydrocar-
bonoclastic bacteria is hydroxylation of one of the two
terminal carbons by inserting an oxygen atom into
relatively stable C–H bonds. This reaction leads to the
production of the corresponding alcohols (van Beilen
et al. 2003). There are six known enzymatic systems
capable of hydroxylation C–H bonds (Rozhkova-
Novosad et al. 2007). A. borkumensis, a key marine
oil-degrading bacterium (Yakimov et al. 1998), pos-
sesses three P450s and two alkane hydroxylases,
namely AlkB1 and AlkB2. AlkB1 is located in a
well-characterized operon with other genes coding for
accessory proteins such as AlkSB1GHJ. However,
AlkB2 is located in another region of the genome that is
not far from RubB and RubA (ABO_122 to ABO_162
-163; Schneiker et al. 2006). RubA is a rubredoxin
peptide similar to AlkG and RubB encodes a rubre-
doxin reductase. Rubredoxin is the simplest known
metalloprotein responsible for supplying electrons to
alkane hydroxylases and possibly other oxygenases.
Rubredoxin reductase regenerates reduced rubredoxin
Biotechnol Lett (2010) 32:497–502
Fig. 1 Analysis of the expressed and purified recombinant
AlkB2 by SDS-PAGE. SDS-PAGE analysis showed the
expression of an approximately 44 kDa protein band upon
induction of 0.5 mM IPTG which was expected according to
the calculated molecular mass of AlkB2. This protein band,
however was absent in uninduced bacteria. The gel (12%) was
stained with Coomassie Blue G-250. Lane M Protein molecular
weight marker (Fermentas # SM0431). Lane BI Total proteins
of E. coli BL21(DE3)plysS harbouring pET-21a(?)-alkB2H
before induction. Lane AI Total proteins after induction with
IPTG. Lane P Purified enzyme AlkB2 with metal affinity
chromatography
by transferring electrons from NAD(P)H to rubre-
doxin. Interestingly, the sequence homology between
AlkB1 and AlkB2 is below 30% in A. borkumensis, and
AlkB1 was not present even in the top 100 similar
sequences in a BLASTP search as a quest for AlkB2.
The differences between AlkB2 and AlkB1 or even
among the AlkB2 and other known alkane hydroxy-
lases in primary structure can be indicative of the
different mechanical/kinetics properties and the sub-
strate range of A. borkumensis AlkB2.
Heterologous expression of the A. borkumensis
alkB1 and alkB2 genes in Pseudomonas revealed the
function of alkane hydroxylase enzymes (Smits et al.
2002; van Beilen et al. 2004; Hara et al. 2004). The
alkB2 gene product enabled a functional alkB gene
deficient strain, Pseudomonas putida GPo12 to grow
on C10–C16 n-alkanes (van Beilen et al. 2004).
Proteomic analysis of A. borkumensis revealed the
metabolic effects of hydrocarbon on gene expression
in A. borkumensis (Sabirova et al. 2006). There has
501
Fig. 2 Western blotting analysis of purified AlkB2 using
monoclonal anti-his antibody. Lane M Prestained Protein
Molecular weight marker (Fermentas # SM0441). Lane 1
Non-induced cell extract of BL21(DE3)plysS cells containing
pET-21a(?)-alkB2H as a negative control. Lane 2 Assessment
of purified AlkB2 enzyme probed with anti-his mAb. Western
blotting confirmed the identity of AlkB2 and no degradation
fragment was detected
been no report on the recombinant expression of
Alcanivorax alkane hydroxylases in any model organ-
ism such as E. coli. Our results presented here reveal
the feasibility of production of active hexa-histidine
tagged AlkB2 from A. borkumensis SK2 in E. coli
cells. Furthermore, presence of a hexa-histidine tag
enables us to raise anti-hydroxylase serum in the future
studies with purified samples obtained in this study.
This facilitates the identification of the native form in
A. borkumensis or the hexa-histidine tag free enzyme
in the recombinant form in E. coli.
To construct an expression system for functional
studies of the recombinant AlkB2, the PCR product
obtained by the gene specific primers with coding
sequence of a hexa-histidine residues at 50 -end of the
reverse primer, were cloned into pET vectors. Western
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blotting of the recombinant protein tagged with a
hexa-histidine peptide confirmed the successful
expression of a 44 kDa band as expected for AlkB2
from Alcanivorax in E. coli cells. Moreover, integra-
tion of a hexa-histidine residue at C-terminal of the
hydroxylase enhanced the probing of the cell extract
with the monoclonal anti-his antibody as the visual-
ization of the hydroxylase band was not possible in the
absence of the histidine tag. The activity of the hexa-
histidine tagged version of the enzyme demonstrates
the proper folding and membrane localization of
AlkB2.
To evaluate functional properties of the recombi-
nant AlkB2 in vitro, we designed an assay based on
the oxidation of NADH in the presence of the cell
crude extracts containing all three components of
AlkB2 hydroxylation system (RubA, RubB and
AlkB2). To increase the solubility of the membrane
bound enzyme, Triton X-100 and Tween 20 were
added to the cells harvested following induction by
IPTG (Bambai et al. 2004).
An average hydroxylase activity of 177 lmol min-1
mg-1 was obtained corresponding to a theoretical
value of 18.6 mg h-1 mg-1 of octanol production from
octane. Economical factors favor using NADH over
NADPH in industrial or semi-industrial applications.
Therefore, we assayed NADH solely as an electron
donor in the hydroxylase activity measurement. There is
no homologue of RubA and AlkB2 reported in the
genome of E. coli. However, a NADH:flavorubredoxin
oxidoreductase enzyme (accession # AP_003278) is
presented in genome of E. coli K12 with 34% identity
and 50% similarity of amino acid residues to RubB. The
oxidation of NADH in the presence of the cell crude
extract and substrate revealed the functional activity of
the expressed enzyme on the oleic acid substrate. These
results show that the substrate range of AlkB2 is not
limited to hydrocarbons and may also react with fatty
acids.
Application of alkane hydroxylases as a biocatalyst
is a major drive in the enzymology of this class of
enzymes. Our results demonstrated that AlkB2 from
A. borkumensis has the potential as the biocatalyst in
the biotransformation reactions of fatty acids as well as
alkanes.
Acknowledgments Authors thank Dr, Kenneth N. Timmis at
German Research Center for Biotechnology in Braunschweig,
Germany for providing Alcanivorax borkumensis.
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Biotechnol Lett (2010) 32:497–502
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