International Journal of Biological Macromolecules 112 (2018) 230–240

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International Journal of Biological Macromolecules

journal homepage: https://www.journals.elsevier.com/ijbiomac

Production and characterization of novel hydrocarbon degrading

enzymes from Alcanivorax borkumensis
Tayssir Kadri a, Tarek Rouissi a, Sara Magdouli a, Satinder Kaur Brar a,⁎, Krishnamoorthy Hegde a, Zied Khiari b,
Rimeh Daghrir c, Jean-Marc Lauzon d
a
INRS-ETE, Université du Québec, 490, Rue de la Couronne, Québec G1K 9A9, Canada
b
Cape Breton University, Verschuren Centre, 1250 Grand Lake Road, Sydney, Nova Scotia B1P 6L2, Canada
c
Centre des Technologies de l`Eau, 696, avenue Sainte Croix, Montréal, Québec H4L 3Y2, Canada
d
TechnoRem Inc., 4701, rue Louis-B.-Mayer, Laval, Québec H7P 6G5, Canada

a r t i c l e i n f o a b s t r a c t

Article history: This study investigates the production of alkane hydroxylase, lipase and esterase by the marine hydrocarbon
Received 2 September 2017 degrading bacteria Alcanivorax borkumensis. The focus of this study is the remediation of petroleum hydrocar-
Received in revised form 26 January 2018 bons, hexane, hexadecane and motor oil as model substrates. A. borkumensis showed an incremental growth
Accepted 27 January 2018
on these substrates with a high cell count. Growth on motor oil showed highest alkane hydroxylase and lipase
Available online 31 January 2018
production of 2.62 U/ml and 71 U/ml, respectively, while growth on hexadecane showed the highest esterase
Keywords:
production of 57.5 U/ml. The percentage of hexane, hexadecane, and motor oil degradation during A. borkumensis
Alcanivorax borkumensis growth after 72 h, was around 80%, 81.5% and 75%, respectively. Zymogram showed two different bands with a
Alkane hydroxylase molecular weight of approx. 52 and 40 kDa, respectively with lipase and esterase activity. Alkane hydroxylase
Lipase reached optimum activity at pH 8.0 and 70 ± 1 °C for hexane and hexadecane and 75 ± 1 °C for motor oil. Lipase
Esterase and esterase showed optimum activity at 35 ± 1 °C and 40 ± 1 °C, respectively and pH 7.0. The crude enzymes
Biodegradation showed higher stability in a wide range of pH, but they were not thermostable at higher temperatures.
Petroleum hydrocarbons © 2018 Elsevier B.V. All rights reserved.

1. Introduction biodegradation have demonstrated the pivotal role that A. borkumensis

play in oil bioremediation [5–7].
Petroleum hydrocarbons are an important energy resource for the As many other hydrocarbonoclastic bacteria, A. borkumensis was
industry and the population. Leaks and accidental spills occur regularly able to produce a glycolipid biosurfactant to access hydrocarbons within
during the exploration, production, refining, transport, and storage of an emulsified droplet [8]. Its genome has been completely sequenced
petroleum and petroleum products. The release of hydrocarbons into and hence leading to a better understanding of the cellular biology of
the environment accidentally or due to human activities is the principal hydrocarbons metabolism [9], and many genes encoding for enzymes
cause of water and soil contamination. The removal of petroleum-hy- initiating the degradation of these hydrocarbons have been detected
drocarbons by of mechanical and physical methods is used as a primary [10–12]. However, very few biochemical studies have been carried out
response during the oil spill. However, the efficiency by mechanical re- on the enzymatic effectiveness of this autochtonous
moval is limited and the total and ultimate oil removal completely rely hydrocarbonoclastic bacteria in oil spill remediation [13]. Therefore, un-
on bioremediation process carried out by indigenous microorganisms derstanding the biochemical pathway is a key feature in environmental
inhabiting the affected areas. Among related phylogenetic groups able bioremediation. With this aim, we investigated the production of alkane
to degrade hydrocarbon pollutants, hydrocarbonoclastic bacteria hydroxylase, lipase and esterase. The A. borkumensis alkane hydroxylase
(HCB) are key players [1–3]. Importantly, Alcanivorax borkumensis, a system is able to degrade a large range of alkanes up to C32 and
rod-shaped marine γ-proteobacterium is able to grow on a highly re- branched aliphatic, as well as isoprenoid hydrocarbons, alkylarenes
stricted spectrum of substrates, predominantly alkanes. This Gram-neg- and alkylcycloalkanes. This spectrum is much wider based on knowl-
ative, aerobic, halophilic bacteria was first to bloom in the oil-polluted edge about alkane hydroxylase complexes. This makes the choice of al-
open ocean and coastal waters, reaching 80–90% of the whole microbial kane hydroxylase of a unique importance. Furthermore, A. borkumensis
community [4,5]. Moreover, many studies on hydrocarbons genome includes 11 genes coding for different lipases/esterases of un-
known specificity. Two of these esterases were purified and functionally
⁎ Corresponding author. characterized. They show generous enzymatic activity that is up to two
E-mail address: [email protected] (S.K. Brar). orders of magnitude higher than common esterases, have a large

https://doi.org/10.1016/j.ijbiomac.2018.01.177
0141-8130/© 2018 Elsevier B.V. All rights reserved.
 T. Kadri et al. / International Journal of Biological Macromolecules 112 (2018) 230–240 231

substrate spectrum, exceptional enantioselectivity and chemical resis- Tris-HCl buffer (pH -8.0). The washed gel was immersed in 100 μM 4-
tance, which provides them a competitive advantageous over other es- methylumbelliferone butyrate (substrate) solution in the same buffer.
terases from other microorganisms and other enzymes for the A visible activity band was observed after 10 min by exposing the gel
resolution of chiral mixtures in biocatalysis [9]. Other than its extensive to UV light.
production by A. borkumensis, lipase demonstrates an important role in Zymogram for alkane hydroxylase was performed as described by
oily hydrocarbons biodegradation. In fact, lipase activity has been used Flores-Flores et al. [18]. The enzyme activity was tested using the
as a biochemical and biological parameter for testing hydrocarbon deg- crude extract and by submerging the gel in a reaction mixture com-
radation and it is an excellent indicator to monitor the decontamination posed of 10 ml of 50 mM tris buffer pH 8.5 added with 0.4 μm/ml of
of a hydrocarbon polluted site [14]. The strain was grown with hexane, NADH, 6.25 ml of o-dianisidine reagent ((20 mg 3,3′-
hexadecane or motor oil, as a unique carbon source, with a view to es- dimetoxibenzidine dissolved in 3 ml 0.025 M hydrochloride acid,
tablishing a biochemical approach adopted in response to petroleum added with agitation to 50 ml of 50 mM tris buffer pH 8.5 and brought
hydrocarbon exposure during the remediation. The choice of substrates up to 100 ml with the same buffer)) and 1 ml of substrate (hexadecane)
is not arbitrary since they are found in oil spills. Thus, a thorough char- and incubating the gel at room temperature with gentle agitation, until
acterization of these enzymes in terms of their enzymatic properties the activity bands appeared. The protein loaded was 63.5 μg per lane.
and their efficiency to degrade cited substrates has been investigated.

2. Materials and methods 2.4. Protein and enzymes assays

All chemical reagents of the highest purity, such as pyruvic acid, hex- Cells of each sampling were centrifuged at 4 °C for 10 min at 5000 ×g.
ane, hexadecane, Bradford reagent, NADPH (Nicotinamide adenine di- The supernatant was used for total protein estimation and enzymatic as-
nucleotide phosphate) and DMSO (Dimethyl sulfoxide) among others, says. Total protein concentration was determined according to the stan-
were procured from Sigma-Aldrich, Fisher Scientific or VWR (Missis- dard Bradford method [19].
sauga, Ontario, Canada). The strain, A. borkumensis was purchased
from Deutsche Sammlung von Mikroorganismen und Zellkulturen
GmbH, DSMZ (Braunschweig, Germany). The composition of motor oil 2.4.1. Alkane hydroxylase assay
used in this study is (in mg/l) is: 69.8 of C10–C50, 1.83 of naphthalene, For cell disruption, A. borkumensis cell pellet (1 g), was re-suspended
b44 of benzene, b30 of toluene, b44 of ethyl-benzene and b84 of xylene. in phosphate buffer (1 ml, 0.1 M, pH 8.0). The mixture was sonicated by
using two frequencies of ultrasounds (22 kHz and 30 kHz) for 6 min at
2.1. Bacterial strain 4 °C and centrifuged at 13000 ×g for 20 min. The supernatant was
used as a crude intracellular enzyme extract.
Alcanivorax borkumensis strain SK2 (DSM 11573) was used in this Alkane hydroxylase activity was measured as described by Glieder et
study. A. borkumensis was sub-cultured and streaked on agar plates, in- al. [20]. Briefly, the crude enzyme assay was carried out in sodium phos-
cubated for 72 h at 30 ± 1 °C and then preserved at 4.0 ± 1 °C for future phate buffer (0.1 M, pH 8.0) with either hexane, hexadecane or motor
use. Standard growth media consisted of (per liter of distilled water): oil as a substrate (0.5–1 mM) and dimethyl sulfoxide (DMSO; 1%, v/v).
23 g NaCl, 0.75 g KCl, 1.47 g CaCl2·2H2O, 5.08 g MgCl2·6H2O, 6.16 g The reaction was initiated by addition of NADPH (200 μM), and the ox-
MgSO4·7H2O, 0.89 g Na2HPO4·2H2O, 5.0 g NaNO3 and 0.03 g idation of NADPH was monitored at 340 nm.
FeSO4·7H2O [15]. The media was supplied with either hexane, The enzymatic assay was performed on the crude enzyme produced
hexadecane or motor oil at a concentration of 3% (v/v) as by A. borkumensis grown in three different substrates: hexane,
the carbon and energy source and the growth was monitored at 30 ± hexadecane and motor oil. One unit is defined as the amount of enzyme
1 °C, 150 rpm for 72 h. Agar plates were prepared with the same required for consumption of 1 μmol of NADPH per min.
media and 18 g/l agar was added to them. Experiments were conducted
in replicates. Cell growth was monitored by measuring the Colony
Forming Units per ml (CFU/ml). 2.4.2. Lipase assay
Extracellular lipase activity was evaluated by the titrimetric method
2.2. Inoculum preparation according to Lopes et al. [21] by using an olive oil emulsion composed of
25 ml of olive oil and 75 ml of 7% Arabic gum solution was emulsified in
For inoculum preparation, a loopful of A. borkumensis from the agar a liquefier for 2 min. About 5 ml of olive oil emulsion was then added to
plates was employed to inoculate a 250 ml Erlenmeyer flask containing 0.1 M phosphate buffer (pH 7.0) and 1 ml of the enzymatic suspension
50 ml of sterilized medium. The flask was incubated on an incubator- (10 mg/ml) and incubated at 37 °C for 30 min under shaking. Subse-
shaker at 150 rpm and 30 ± 1 °C for 24 h and the actively growing quently, the emulsion was immediately disrupted by the addition of
cells from these flasks were used as 3% (v/v) inoculum for the produc- 15 ml of a mixture of acetone-ethanol (1:1 v/v). The released fatty
tion of A. borkumensis. acids were titrated with 0.05 M NaOH. One unit of lipase activity was
defined as the amount of enzyme which liberated 1 μmol of fatty acids
2.3. Polyacrylamide gel electrophoresis (PAGE) and zymography per minute.

A native PAGE composed of 12% resolving and 4% stacking gel was

performed according to the method described by Laemmli [16] to iden- 2.4.3. Esterase assay
tify the enzymes with lipase/esterase activities by activity staining (zy- Extracellular esterase activity was measured by the titrimetric
mogram). About 50 μl of the crude enzyme produced by A. borkumensis method according to Lopes et al. [21] by using olive oil as a substrate.
was loaded on the native PAGE gel without denaturing the sample. The The reaction mixture is composed of: 5 ml of olive oil, 2 ml of 0.1 M
electrophoresis was performed at constant voltage of 85 V in Tris-gly- phosphate buffer (pH 7.0) and 1 ml of the enzymatic extract (10 mg/
cine buffer (pH -8.3) at 25 °C. ml). The mixture was incubated at 37 °C for 30 min under shaking and
Activity staining for putative lipase/esterase was performed accord- it was immediately disrupted by adding 15 ml of acetone-ethanol mix-
ing to the method described by Prim et al. [17]. In brief, the native PAGE ture (1:1 v/v). The released fatty acids were titrated with 0.05 M NaOH.
gel after electrophoresis was washed in distilled water and soaked in One unit of esterase activity was defined as the amount of enzyme
2.5% Triton X-100 at room temperature followed by a wash in 50 mM which liberated 1 μmol of fatty acids per minute.
232 T. Kadri et al. / International Journal of Biological Macromolecules 112 (2018) 230–240

2.4.4. Effect of temperature on alkane hydroxylase, lipase, esterase activity C ¼ C 0 e−Kðt−t b Þ for tNt b ð4Þ
and stability
To study the effect of temperature, the alkane hydroxylase assay was
performed between 25 ± 1 °C and 85 ± 1 °C, using hexane, hexadecane Hexane, hexadecane and motor oil half-life (T 1/2) was calculated
or motor oil as a substrate as described in section “Alkane hydroxylase using Eq. (5):
assay”.
For thermal stability, the enzyme was incubated at 50 ± 1 °C to 75 ± T 1=2 ¼ t b þ ln 2=K ð5Þ
1 °C for 5 h and the residual activity was measured every hour by alkane
hydroxylase assay. The enzyme without incubation was used as a positive In both mathematical models, C is the concentration of the different
control. substrates at a given time (t), C0 is the initial concentration of the differ-
Similarly, the optimum temperature for lipase and esterase enzymes ent substrates in the sandy soil sample, K is the biodegradation rate con-
was studied at 25–60 ± 1 °C at constant pH 7.0. For the thermal stability stant of the different substrates and tb is the breakpoint at the time at
studies, lipase and esterase were incubated at 25 ± 1 °C to 50 ± 1 °C for which rate constant changes and biodegradation starts.
5 h and the residual activity were measured.
3. Results
2.4.5. Effect of pH on alkane hydroxylase, lipase and esterase activity and
stability 3.1. Growth of A. borkumensis and enzymes production
The activity of alkane hydroxylase at different pH between 3.0 and
10 was investigated at 70 ± 1 °C for 10 min, using hexane, hexadecane The growth curves of A. borkumensis on three different culture sub-
or motor oil as a substrate. The effect of pH on enzyme stability was in- strates (hexane, hexadecane and motor oil) used as a sole source of car-
vestigated by measuring the residual activity of the enzyme after incu- bon and energy, are shown in Fig. 1 (a), 1(c) and 1(e). The best growth
bation at various pH (3.0–10) for 1 h at room temperature. was obtained between 48 h and 60 h for the three different media with
Also, the effect of pH (between 5 and 9) on the esterase and lipase 6 × 108 CFU/ml for hexane based media, 4.8 × 108 CFU/ml for
activities was studied at 37 ± 1 °C. For enzyme stability measurements, hexadecane based media and 7 × 108 CFU/ml for motor oil based
the enzymes were incubated at various pH (5.0–9.0) for 1 h at room media. The analysis of the residual concentration of the tested sub-
temperature and the respective residual enzymes were measured. strates by GC-FID as a function of incubation time is shown in Fig. 1
The following buffer system was used to maintain various pH: 100 (a), (c) and (e) and allowed calculate degradation rates of 80% for hex-
mM citrate-phosphate, pH 3.0; 100 mM glycine-HCl, pH 4.0 and 5.0; ane, 82% for hexadecane and 75% for motor oil after 72 h.
100 mM sodium acetate, pH 6.0; 100 mM phosphate buffer, pH 7.0; A. borkumensis also showed higher protein synthesis and enzymes
100 mM Tris-HCl, pH 8.0 and 100 mM glycine-NaOH, pH 9.0 and 10. production throughout the cultivation time on the three different liquid
culture media that were optimized in this study. The fermentation time
2.5. Gas chromatography-flame ionization detector (GC-FID) analysis course for alkane hydroxylase, lipase and esterase production by A.
borkumensis (Fig. 1(b), (d) and (f)) indicated that the maximum protein
GC-FID was used to test the efficiency of degradation of the different synthesis and the maximum alkane hydroxylase, lipase and esterase ac-
substrates: hexane, hexadecane and motor oil with the crude alkane hy- tivity were obtained after 72 h of cultivation on the three different sub-
droxylase produced by A. borkumensis during its growth. strates, when cells were in the stationary phase, and its production was
One drop of lube oil taken at different intervals of A. borkumensis fer- not growth associated. The concentration of crude protein produced by
mentation (6 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h) grown on hexane, A. borkumensis grown on hexane, hexadecane and motor oil presented
hexadecane and motor oil, was diluted with hexane to 2 ml and ana- no large difference using the three substrates. After 72 h of fermenta-
lyzed by a Hewlett-Packard 6890/5973 gas chromatograph coupled to tion, around 23 μg/ml was obtained for hexane, 22.5 μg/ml for
flame ionization detector. The gas chromatograph was equipped with hexadecane and 20.75 μg/ml for motor oil. Motor oil was found to be
30 m length, 0.25 mm internal diameter, and 0.11 μm film thickness the best substrate for the production of A. borkumensis crude alkane hy-
capillary column (C10–C50) (Make: Agilent). Helium was used as car- droxylase with an activity of 2.62 U/ml obtained after 72 h of
rier gas. The temperature program consisted of a heating rate of 8 °C/ fermentation.
min from 80 °C to 340 °C with a hold time of 6 min. The degradation of motor oil and other relative compounds is not
only performed by alkane hydroxylase, but also by other enzymes that
2.6. Substrates biodegradation kinetics can interfere with the entire degradation. In this regard, the determina-
tion of the lipase and esterase activities was performed.
The kinetics of different substrates was investigated using different A high lipase activity of 71 U/ml was observed when using motor oil
models. Petroleum hydrocarbon half-life (T1/2) was calculated accord- as a substrate compared to hexadecane (47 U/ml) and hexane (45.8 U/
ing to Dados et al. [22] using the following Eq. (1): ml), respectively. Also, important esterase activities were observed dur-
ing the fermentation of A. borkumensis on the three different substrates
T 1=2 ¼ ln 2=K ð1Þ with the highest activity found in motor oil (43.3 U/ml) and a lower ac-
tivity was found on hexadecane (57.5 U/ml) and hexane (39 U/ml).
where, K represents the biodegradation rate constants using the single
first-order kinetic (SFO) model given in Eq. (2): 3.2. Enzymes characterization

C ¼ C 0 e−Kt ð2Þ ð2Þ The present study corroborates with previous studies demonstrating
that A. borkumensis is an efficient hydrocarbon-degrading microorgan-
ism. This efficiency depends on the properties of the involved key en-
The overall and specific substrate biodegradation rate constants for zymes, such as alkane hydroxylase, lipase, and esterases.
A. borkumensis were calculated using the modified Hockey–Stick
model (FOCUS 2006). The following Eqs. (3) and (4) related to this 3.2.1. Native PAGE and Zymography
method were used: Fig. 2 shows the zymogram of the crude enzyme extract of A.
borkumensis. The activity staining and Coomassie staining of the sample
C ¼ C 0 for t ≤t b ð3Þ run on the different lane of the native PAGE showed two distinct bands
 T. Kadri et al. / International Journal of Biological Macromolecules 112 (2018) 230–240 233

(b)
(a) 50 50 1,4 25
12 120 Alkane hydroxylase
Lipase
1,2
40 40 Esterase 20

Alkane hydroxylase Activity (U/ml)

10 Proteins

Protein concentration (µg/ml)

100

Residual concentration (%)

Ln((CFU/ml)t/(CFU/ml)0) 1,0
8

Esterase activity (UI)

30 30 15

Lipase Activity (UI)

80 0,8

6
20 20 0,6 10
60

4
0,4
40 10 10 5
2
0,2
Ln((CFU/ml)t/(CFU/ml)0) 20 0 0 0
0 0,0
Residual concentration (%)

0
0 20 40 60 80 0 20 40 60 80
Time (h) Time (h)

(c) (d)
12 120
70 50 1,2 25
Alkane hydroxylase
10 60 Lipase
100 1,0 Esterase

Alkane hydroxylase activity (U/ml)

40 20

Residual concentration (%)

Proteins

Protein concentration (µg/ml)

Ln((CFU/ml)t/(CFU/ml)0)

8 50
0,8

Esterase activity (UI)

80

Lipase activity (UI)

30 15
40
6 0,6
60
30 20 10
4 0,4
40 20
10 5
2 0,2
10
20
0 Ln((CFU/ml)t/(CFU/ml)0) 0 0
0 0,0
Residual concentration (%)

0
0 20 40 60 80 0 20 40 60 80
Time (h) Time (h)
(f)
(e)
50 80 3,5 25
12 120
Alkane hydroxylase
3,0 Lipase
Alkane hydroxylase activity (U/ml)

40 Esterase 20

Protein concentration (µg/ml)

10
60 Proteins
100 2,5
Residual concentration (%)
Ln((CFU/ml)t/(CFU/ml)0)

Esterase activity (UI)

Lipase activity (UI)

8 30 15
2,0
40
80
6
20 1,5 10

4 20
60 1,0
10 5
2 0,5
40 0
0 0
0 Ln((CFU/ml)t/(CFU/ml)0) 0,0
Residual concentration (%)
20
0 20 40 60 80 0 20 40 60 80

Time (h) Time (h)

Fig. 1. Colony forming units of Alkanivorax borkumensis grown on different substrates and residual concentration profile of the different substrates: hexane (a); hexadecane (c); and motor
oil (e). Kinetics of production of alkane hydroxylase, lipase and esterase and concentration of crude protein produced by A. borkumensis grown on different substrates: Hexane (b);
Hexadecane (d) and; Motor oil (f).

corresponding to the size of approx. 52 and 40 kDa, respectively with li- activity decreased slightly above 70 ± 1 °C. For motor oil, the maximum
pase/esterase activity. For alkane hydroxylase zymogram, seven trials was reached at 75 ± 1 °C, with a slight decrease above 75 ± 1 °C. The
were carried out, but the method used showed a lower resolution and relative activities at 65 ± 1 °C and 75 ± 1 °C were about 46% and 73%,
no bands were obtained. This method is usually applied for oxygenase respectively for hexane and 56% and 86.6%, respectively for hexadecane.
enzyme and no conventional method was found in the literature corre- For motor oil, the relative activities at 80 ± 1 °C and 70 ± 1 °C
sponding to the migration of alkane hydroxylase enzyme since the were about 75% and 88.4%, respectively. The thermal stability profiles
study of this enzyme is new which may suggest the modification and of alkane hydroxylase at a temperature range between 50 ± 1 °C and
the adaptation of the protocol to alkane hydroxylase properties. 75 ± 1 °C for the three different substrates hexane, hexadecane, and
motor oil are shown in Fig. 4(a), (b) and (c), respectively. Profiles
3.2.2. Effect of temperature on the activity and stability of alkane showed a high stability at temperatures below 50 ± 1 °C for the three
hydroxylase different substrates but were inactivated at higher temperatures.
The temperature profile of alkane hydroxylase activity from A. After 60 min of incubation at 75 ± 1 °C, 95.7%, 98% and 93.7% of the
borkumensis grown on hexane, hexadecane, and motor oil is presented initial activities were lost for hexane, hexadecane, and motor oil,
in Fig. 3 (a). The A. borkumensis crude enzyme extract had an optimum respectively. A. borkumensis alkane hydroxylases were stable at 50 ± 1
activity at 70 ± 1 °C in the presence of hexane and hexadecane, while and 60 ± 1 °C after 5 h of incubation for hexane, hexadecane, and
234 T. Kadri et al. / International Journal of Biological Macromolecules 112 (2018) 230–240

3.2.5. Effect of pH on the activity and stability of lipase

The pH profile of lipase activity from A. borkumensis grown on three
different substrates is shown in Fig. 3(d). For hexane, hexadecane and
motor oil, the crude enzyme was active in the pH range of 6.0–8.0,
with an optimum at pH 7.0. The relative activities at pH 6.0 and 8.0
were about 77.1% and 82.4%, respectively for hexane substrate, 59%,
and 86.2%, respectively for hexadecane substrate and around 69% to
90.1%, respectively for motor oil.
The pH stability of lipase assayed in the range of 6.0–10 on three dif-
ferent substrates is presented in Fig. 5(d). Profiles showed that the
crude extracellular lipase was highly stable in a large pH range, main-
taining N50% of its original activity in the pH range of 7.0 to 9.0 for hex-
ane, hexadecane and for motor oil.

3.2.6. Effect of temperature on the activity and stability of esterase

The temperature profile of esterase activity from A. borkumensis
grown on hexane, hexadecane, and motor oil is presented in Fig. 3(e).
The A. borkumensis crude extract had an optimum activity at 40 ± 1 °C
for the three different substrates. The relative activities at 35 ± 1 °C
and 45 ± 1 °C were around 96.7% and 72.9%, respectively for hexane,
94.3%, and 76%, respectively for hexadecane and 86% and 79% for
motor oil.
The thermal stability profiles of esterase at a temperature range
between 25 ± 1 °C and 50 ± 1 °C for the three different substrates
namely, hexane, hexadecane, and motor oil are shown in Fig. 5(a),
(b) and (c), respectively. Like lipase, esterase was highly stable at 25 ±
1 °C with residual activities of around 70.6% for hexane, 80% for
Fig. 2. Lane 1: Zymogram of the crude enzyme as observed in UV light; Lane 2: Coomassie hexadecane and 74% for motor oil after 5 h' incubation. At 50 ± 1 °C the
staining of the crude enzyme on native PAGE; M: Molecular weight marker. enzyme lost N90% after 5 h of incubation.

3.2.7. Effect of pH on the activity and stability of esterase

motor oil. At low temperatures (−20 ± 1 and 4 ± 1 °C), the crude en- The pH profile of esterase enzymatic activity from A. borkumensis
zyme preparation retained N70% of its activity after one month. grown on three different substrates is shown in Fig. 3(f). For hexane,
hexadecane and motor oil the crude enzyme was active in the pH
range of 6.0–9.0 with a relative activity higher than 60%. The optimum
3.2.3. Effect of pH on the activity and stability of alkane hydroxylase
activity was obtained at pH 7.0. The relative activities at pH 6.0 and 8.0
The pH profile of alkane hydroxylase activity from A. borkumensis
were about 72% to 81.6% for hexane, 63 to 86% for hexadecane and fi-
grown on three different substrates: hexane, hexadecane and motor
nally 64.3 to 89% for motor oil.
oil are presented in Fig. 3(b).
The pH stability of esterase assayed in the range of 6.0–10 on the
The pH stability of A. borkumensis alkane hydroxylase assayed in the
three different substrates is presented in Fig. 6(d). Profiles showed
range of 5.0–9.0 on the three different substrates is shown in Fig. 4(d).
that the extracellular esterase was stable in a large pH range, maintain-
The crude alkane hydroxylase was highly stable over a broad pH
ing N69% of its original activity in the pH range of 7.0 to 9.0 for hexane,
range, maintaining N75% of its original activity between pH 6.0 and 9.0
hexadecane and for motor oil.
for hexane, hexadecane and also for motor oil.
The analysis of esterase activity in A. borkumensis has revealed that
For the three different substrates, the crude enzyme was active at
maximum activity is obtained at 40 ± 1 °C and pH 8.0 for the different
pH 6.0 and 8.0, with an optimum at pH 8.0. A sharp decline in activity
substrates.
was observed above pH 9.0. The relative activities at pH 6.0 and 7.0
were about 77% to 80% for hexane substrate, 70% to 75% for hexadecane
3.3. Hexane, hexadecane and motor oil degradation efficiency
substrate and around 80% to 88% for motor oil.
A. borkumensis was tested for its biodegradation potential when
3.2.4. Effect of temperature on the activity and stability of lipase growing on hexane, hexadecane and motor oil. In experiments, where
The temperature profile of lipase activity from A. borkumensis grown the media was supplemented with different hydrocarbon sources, A.
on hexane, hexadecane, and motor oil is presented in Fig. 3(c). The A. borkumensis showed higher biodegradation potential, with T1/2 values
borkumensis crude extract had an optimum at 35 ± 1 °C for the three of 40.65 h for hexane, 52.20 h for hexadecane and 40.65 h for motor
different substrates hexane, hexadecane, and motor oil. The enzymatic oil. The biodegradation pattern exhibited by this strain consisted of
activities at 30 ± 1 °C and 40 ± 1 °C were close to the optimal activity two sequential first-order curves that were characterized by drastic
with relative activities values of about 91.2% and 94.3%, respectively changes in the biodegradation rates after 6 h of experimentation (Fig.
for hexane, 81.3% and 96.7%, respectively for hexadecane and 79.10% 7(a), (b) and (c)); which could be described by the modified Hockey–
and 96% for motor oil. Stick kinetic model. The biodegradation kinetic parameters obtained
The thermal stability profiles of lipase at a temperature range be- for the three different substrates are presented in Table 1. The results
tween 25 ± 1 °C and 50 ± 1 °C for the three different substrates hex- are shown in Fig. 7(a), (b) and (c) suggested that the biodegradation
ane, hexadecane, and motor oil are shown in Fig. 4(a), (b) and (c), time for A. borkumensis grown on hexane and motor oil (40.65 h for
respectively. Lipase was highly stable at 25 ± 1 °C with residual activi- both substrates) was lesser than when the strain was grown on
ties around 80.6% for hexane, 73.3% for hexadecane and 54.5% for hexadecane (52.2 h). A. borkumensis was able to mineralize up to 80%
motor oil after 5 h incubation. In contrast, at 50 ± 1 °C, the enzyme of the initial concentration of hexane, 81.5% of the hexadecane and
was inactivated and loses the entire activity after 5 h of incubation. 75% of the motor oil.
 T. Kadri et al. / International Journal of Biological Macromolecules 112 (2018) 230–240 235

(a) (b)
120 120
Hexane Hexane
Hexadecane Hexadecane
100
Motor oil 100 Motor oil

80
Relative activity (%)

Relative activity (%)

80

60
60

40

40
20

20
0

0
20 30 40 50 60 70 80 90 2 4 6 8 10 12
Temperature (°C) pH

(c) (d)
120 120
Hexane Hexane
Hexadecane Hexadecane
100 Motor oil Motor oil
100

Relative activity (%)

Relative activity (%)

80
80

60

60
40

40
20

0 20
20 30 40 50 60 70 4 5 6 7 8 9 10
Temperature (°C) pH

(e) (f)
120 120
Hexane Hexane
Hexadecane Hexadecane
100 Motor oil Motor oil
100
Relative activity (%)

Relative activity (%)

80
80

60

60
40

40
20

0 20
20 30 40 50 60 70 4 5 6 7 8 9 10
Temperature (°C) pH

Fig. 3. Effect of temperature (a, c, e) and pH (b, d, f) on the activity of the crude alkane hydroxylase (a, b); lipase (c, d); and esterase (e, f) from A. borkumensis grown on Hexane, on
Hexadecane, and on Motor oil.

4. Discussion substances originating from crude oil had stimulatory activity. In

contrast, Kanaly et Harayama [24] have reported that petroleum hy-
4.1. Growth of A. borkumensis and enzymes production drocarbons showed toxic properties which inhibit development and
metabolic activity of microorganism in most cases. Thus, igh concen-
As shown in Fig. 1(a, c, e), growth on the different substrates (hex- trations of test substrates (around 3% v/v) correlated with their con-
ane, hexadecane and motor oil) shows typically four phases: an initial centrations detected in the contaminated soils/waters, A.
lag phase, an exponential growth phase, stationary phase (maximum borkumensis was able to grow and utilize these xenobiotics as the
growth), and a death phase, which is the result of the toxic effects of sole source of carbon and energy. In accordance with our results,
the octanol product of degradation [6]. Since A. borkumensis is a natural Bookstaver et al. [5] reported around 6.93 × 108 of counted cells of
producer of surfactants, almost all the substrates used were in the aque- A. borkumensis in the organic nitrogen free broth with octane layer.
ous phase which discarded the supposition of having a biphasic media To the best of our knowledge, this is the only work reported on A.
with cells present in the liquid-liquid interface. The high growth ob- borkumensis, most of the other reports pertain to genetics [6,25,26].
tained for the three-different media between 48 h and 60 h reflects The obtained results open the horizon to use A. borkumensis for the
the capacity of A. borkumensis to use these substrates, allowing deg- degradation of recalcitrant compounds and permit to classify this
radation at an active stage of the growth curve. Thus, hydrocarbons strain among reported xenobiotic-degrading strains, such as Pseudo-
are processed and degraded inside the cells. These results are in ac- monas, Mycobacterium, Haemophilus, Rhodococcus, Paenibacillus, and
cordance with Boopathy [23] who reported that in some cases, Ralstonia [27,28].
236 T. Kadri et al. / International Journal of Biological Macromolecules 112 (2018) 230–240

(a) (b)
120 120
50 °C 50 °C
60 °C 60 °C
100 70 °C 100 70 °C
75 °C 75 °C

80 80

Residual activity (%)

Residual activity (%)

60 60

40 40

20 20

0 0

-20 -20
Time 0 60 120 180 240 300 0 50 100 150 200 250 300 350

Time (min) Time (min)

(c) (d)
120 100
50 °C Hexane
60 °C Hexadecane
100 70 °C 90 Motor oil
75 °C

80

Residual activity (%)

Residual activity (%)

80

60
70

40

60
20

50
0

-20 40
0 50 100 150 200 250 300 350 5 6 7 8 9 10 11
pH
Time (min)

Fig. 4. Effect of temperature (a, b, c) and pH (d) on the stability of the crude alkane hydroxylase from A. borkumensis grown on (a) Hexane; (b) Hexadecane and; (c) Motor oil.

More studies on A. borkumensis need to be done to cope with biore- studies on its alkane oxidation system. A. borkumensis SK2 is known to
mediation of petroleum contaminated sites which may lead to more possess an AlkB1 alkane hydroxylase that can oxidize medium chain

(a) (b)
120 120
25 °C 25 °C
30 °C 30 °C
100 40 °C 100 40 °C
50 °C 50 °C
Residual activity (%)

80 80
Residual activity (%)

60 60

40 40

20 20

0 0

0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350
Time (min) Time (min)

(c) (d)
120 120
25 °C Hexane
30 °C Hexadecane
100 40 °C 100 Motor oil
50 °C
80
Residual activity (%)

Residual activity (%)

80

60
60
40

40
20

20
0

-20 0
0 50 100 150 200 250 300 350 5 6 7 8 9 10 11
Time (min) pH

Fig. 5. Effect of temperature (a,b,c) and pH (d) on the stability of the crude lipase from A. borkumensis grown on (a) Hexane; (b) Hexadecane and; (c) Motor oil.
 T. Kadri et al. / International Journal of Biological Macromolecules 112 (2018) 230–240 237

(a) (b)
120 120
25 °C
30 °C
100
100 40 °C
50 °C
80
Resiadual activity (%)

Resiadual activity (%)

80

60
60
40

40
20
25 °C
20 30 °C
0 40 °C
50 °C

0
0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350
Time (min) Time (min)

(c) (d)
120 120
25 °C Hexane
30 °C Hexadecane
100 40 °C Motor oil
100
50°C
Resiadual activity (%)

80

Residual activity (%)

80
60

40 60

20
40
0

20
0 50 100 150 200 250 300 350 5 6 7 8 9 10 11

Time (min) pH

Fig. 6. Effect of temperature (a,b,c) and pH (d) on the stability of the crude esterase from A. borkumensis grown on (a) Hexane; (b) Hexadecane and; (c) Motor oil.

alkanes in the range C5 to C12 and an alkane hydroxylase AlkB2, that Glieder et al. [20] in the presence of octane, hexane, cyclohexane and
oxidizes medium-chain alkanes in the range C8 to C16. It has also pentane, major compounds present in the motor oil.
been claimed that A. borkumensis SK2 is able to degrade a large range Most of the degradation reported is a cooperative action of multi-
of alkanes up to C32 and branched aliphatic, as well as isoprenoid hy- tude enzymes as reported by Kennedy et al. [34] and Zeynalov et Nagiev
drocarbons (e.g., phytane), alkylarenes and alkylcycloalkanes [29]. Con- [35]. Besides, the lipase and esterase activity was tested as valuable tool
sequently, hexane can be degraded by the alkane hydroxylase AlkB1, to monitor oil biodegradation in freshly diesel oil-contaminated soils
hexadecane can be degraded by the alkane hydroxylase AlkB2 and the [36,37]. In this regard, important lipase activities and high esterase ac-
motor oil can be degraded by both AlkB1 and AlkB2, since it contains a tivities were observed during the growth of A. borkumensis on the
mixture of C10–C50 other that monoaromatic and polyaromatic hydro- three different substrates. Similar results were reported by Margesin
carbons. The genome also includes 11 genes coding for different lipases/ et al. [38], for induction of soil lipase activity in oil contaminated sites
esterases of unknown specificity. Two of these esterases were purified and in the presence of inorganic nutrients. Therefore, the induction of
and functionally characterized [9]. This allows the strain to grow on soil lipase activity is a valuable indicator of oil biodegradation and per-
hexane, hexadecane and motor oil at high concentrations. mits a faster and accurate assessment of the decontamination treatment
The difference of residual concentration obtained for the three sub- after an oil spill [36,37]. Moreover, Martínez-Martínez et al. [39] sug-
strates (80% for hexane, 82% for hexadecane and 75% for motor oil, gested that multifunctional esterase-like proteins from the α/β hydro-
after 72 h of growth), is attributed to the dissimilarity of chain-length lase family that can hydrolyze both C\\C and C\\O bonds may exist in
of the tested substrates as reported by [30,31]. Thus, linear alkanes are nature at much higher levels than previously reported. From an ecolog-
lipophilic substances which easily enter through the cell membrane ical perspective, such proteins may contribute to global carbon cycling
and are more easily degraded. processes for complex substrates, including recalcitrant organic
Owing to the high degradation rate, A. borkumensis can be pollutants.
considered among the potential candidates in the bioremediation,
such as for P. aeruginosa which exhibited a degradation rate of 94% in 4.2. Enzymes characterization
the presence of n-alkane [32] and Pseudomonas aeruginosa that showed
a degradation capability of 66% in the presence of diesel after 30 days 4.2.1. Native PAGE and zymography
[33]. A. borkumensis is known for degradation of petroleum-derived ali-
The results showed that motor oil is the best substrate for the phatic and aromatic hydrocarbons. However, recent studies on the ge-
production of A. borkumensis crude alkane hydroxylase (Fig. 1(b, d, f)). nome sequencing of A. borkumensis SK2 revealed that the genome has
Likewise, a higher activity of alkane hydroxylase was observed by 11 genes coding for different lipases/esterases [9]. There are also reports
238 T. Kadri et al. / International Journal of Biological Macromolecules 112 (2018) 230–240

120 weight of lipase and esterase genes are in the range of 35–52 kDa
(a) C(t) = 93.197e-0.02t (based on the amino acid length) (Alcanivorax borkumensis SK2, com-
100
R² = 0.96 plete genome, NCBI). In addition, there are few reports on other related
80 bacteria, viz. Alcanivorax dieselolei B-5(T), Marinobacter lipolyticus and
C(t) (%)

60 Pseudomonas sp., which has lipase/esterase enzymes with a molecular

weight in the range of 43–45 kDa [39-42].
40 The molecular weight of the active protein bands in our study is in
accordance with the size range of lipase/esterase reported earlier for
20
Alcanivorax sp. and other phylogenetically related bacteria. The investi-
0 gation showed that two different lipase/esterase are strongly induced
0 20 40 60 80 and produced in presence of hexadecane in the growth medium. How-
Time (h) ever, further detailed investigations, such as N-terminal sequencing and
120 MALDI-TOF are necessary to characterize these enzymes.
(b) C(t) = 101.93e-0.015t
100 4.2.2. Crude alkane hydroxylase characterization
R² = 0.79
The small difference of relative activities of alkane hydroxylase be-
80
tween substrates at different temperatures (Fig. 3(a)), may be related
C(t) (%)

60 to the structures. For the thermal stability profiles of alkane hydroxylase

produced from A. borkumensis, Fig. 3(a), (b) and (c), high stabilities
40 were shown at temperatures below 50 ± 1 °C, but were inactivated at
20 higher temperatures. Similarly, Li et al. [43] studied the alkane
monooxygenase produced by the bacterium Pusillimonas sp. and
0 found that the optimal reaction condition for this enzyme was pH 7.5
0 20 40 60 80 at 30 °C. Also, this monooxygenase system showed better cold toler-
Time (h) ance, with activity retained at temperatures as low as 0 °C.
As shown in Fig. 3(b), alkane hydroxylase was active within pH 6.0
120
and 8.0, with an optimum at pH 8.0 for the three different substrates.
(c) C(t)= 92.265e-0.02t A similar study done by Lu et al. [44] reported a pH of 8.0 in laccase-
100
R² = 0.91 like multicopper oxidase produced by Streptomyces sp. C1 in the pres-
80 ence of ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic
acid)) and guaiacol as substrates. However, Salvachúa et al. [45] have
C(t) (%)

60 found that acidic pH was required to reach high activities for other oxi-
dative enzymes, such as peroxidase (DyP)-Type form.
40
4.2.3. Crude lipase characterization
20
The temperature profile of lipase activity and stability (Figs. 3(c), 4
0 (a), (b) and (c)) shows that the crude enzyme is highly active at 35 ±
1 °C and has enhanced stability at 25 ± 1 °C, for the three different sub-
0 20 40 60 80
Time (h) strates. This is related to the cells growth temperature range [46], which
is defined to be 30 °C. In this regard, Margesin et al. [37] reported that
Fig. 7. Biodegradation of hexane (a); hexadecane (b); and motor oil (c) by Alkanivorax lipase enzyme was active at pH 7.25 and 30 °C. This activity was consid-
borkumensis. Lines represent exponential equation estimates of the first-order kinetics ered as a valuable tool to monitor oil biodegradation in freshly diesel oil-
model of the samples where R2 is the correlation coefficient, C(t) is the residual contaminated soils, most probably due to a high content of available al-
concentration in (%) of the different substrates at a given time (t).
iphatic compounds. On the other side, at 50 ± 1 °C, the enzyme loses
the entire activity after 5 h of incubation. This is probably due to the
that lipases and esterases are actively involved in the degradation of pe- thermal denaturation of the enzyme. The temperature stability of lipo-
troleum hydrocarbons [38,39]. Thus, as shown in Fig. 2, zymogram lytic is of high importance, particularly for applications in industry
showed two distinct bands of approx. 52 and 40 kDa, respectively [47]. Although some studies have investigated the effects of high tem-
with lipase/esterase activity. The fact that both lipase and esterase can peratures on the activity of esterase and lipase isolated from
hydrolyze the substrate, 4-methylumbelliferone butyrate, it is difficult Acenitobacter, yet no study was reported on the effects of temperatures
to distinguish the active enzyme between lipase and esterase. Further, on the activity of lipolytic enzymes of A. borkumensis.
scarce literature is available on the molecular characterization of lipase This same enzyme, was highly active at pH 7.0 for hexane,
and esterase from Alcanivorax family. However, from the available se- hexadecane and motor oil (Fig. 3(d)) and stable in a wide range of pH
quence data in the NCBS database for A. borkumensis, the molecular from 7.0 to 9.0 (Fig. 5(d)). Similarly, Bisht et al. [48] have identified an
extracellular alkaline lipase from a mutant strain of P. aeruginosa with
Table 1
a maximum activity at pH 8.0 with a considerable stability in pH range
Biodegradation kinetic parameters obtained by the Hockey–Stick modified method in me- 7.0–11.0. Moreover, lipase from P. aeruginosa SRT9 and Burkholderia
dia enriched with hexane, hexadecane and motor oil. sp. had shown maximum lipase activity at pH 6.9 and 8.5, respectively
[49,50]. These characteristics provided a clear indication for their indus-
Sample K (h−1) R2a tbb (h) T1/2 (h)
trial use as effective agents to degrade hydrocarbons even at a high
Hexane 0.02 0.96 6 40.65
range of pH.
Hexadecane 0.015 0.79 6 52.20
Motor oil 0.02 0.91 6 40.65
4.2.4. Crude esterase characterization
tb: breakpoint at the time that biodegradation starts; K, overall biodegradation rate of the
substrate; T1/2: substrates half-life.
Esterases were reported to degrade alkanes and aromatic rings in
a
Coefficient of determination of the modified Hockey–Stick model. different bacterial and fungal isolates [51,52]. In our study, the analysis
b
Constant rate from T = tb. of esterase activity in A. borkumensis has revealed that maximum
 T. Kadri et al. / International Journal of Biological Macromolecules 112 (2018) 230–240 239

activity was obtained at 40 ± 1 °C and pH 8.0 for the different sub- hydrocarbons, but faster biodegradation rate was observed in hexane
strates (Fig. 3(f) and (e)) and the highest stability was achieved at 25 and motor oil than in hexadecane.
± 1 °C and in a wide range pH from 7.0 to 9.0 (Figs. 5(a), (b), (c) and
6(d)). As mentioned previously for lipase, esterase activity is also used
as a biological indicator to monitor total petroleum hydrocarbons bio- Acknowledgement(s)
degradation and both hydrolases (lipases and esterase) were induced
in the presence of hydrocarbons [38]. Our results were in agreement The authors are sincerely thankful to the Natural Sciences and
with Lubna Tahir [52] who found that temperature of a 45 ± 1 °C en- Engineering Research Council of Canada (Discovery Grant 355254,
hanced the high esterase activity in Lentinus tigrinus. Besides, a basic CRD Grant and Strategic Grant 447075) and Techno-Rem Inc. (CRDPJ
pH was required to reach a high activity [51,52]. Thus, the role of pH is 476649-14) for financial support. The views or opinions expressed in
highly important and may be related to the stability of the enzyme this article are those of the authors.
[51]. All these observations proved the role of assay conditions (e.g.
pH and temperature) in maintaining higher enzymatic activities [53]. References
These insights could have larger implications for the future of [1] M.M. Yakimov, K.N. Timmis, P.N. Golyshin, Obligate oil-degrading marine bacteria,
bioinspired oil spill remediation. Thus, this study can be further Curr. Opin. Biotechnol. 18 (2007) 257–266.
exploited by applying A. borkumensis enzymes for the bioremediation [2] L. Wang, W. Wang, Q. Lai, Z. Shao, Gene diversity of CYP153A and AlkB alkane hy-
droxylases in oil-degrading bacteria isolated from the Atlantic Ocean, Environ.
of the contaminated sites, which is being explored at laboratory scale. Microbiol. 12 (2010) 1230–1242.
Moreover, in all the previous results, we noticed that the profile of [3] W. Wang, L. Wang, Z. Shao, Diversity and abundance of oil-degrading bacteria and
lipase enzyme and esterase showed same kinetics and behavior during alkane hydroxylase (alkB) genes in the subtropical seawater of Xiamen Island,
Microb. Ecol. 60 (2010) 429–439.
incubation time under different pH and temperatures. This proved that [4] S.-H. Naing, S. Parvez, M. Pender-Cudlip, J.T. Groves, R.N. Austin, Substrate specificity
both enzymes exhibited a synergistic action [54]. and reaction mechanism of purified alkane hydroxylase from the
hydrocarbonoclastic bacterium Alcanivorax borkumensis (AbAlkB), J. Inorg. Biochem.
121 (2013) 46–52.
4.3. Hexane, hexadecane and motor oil degradation efficiency [5] M. Bookstaver, M.P. Godfrin, A. Bose, A. Tripathi, An insight into the growth of
Alcanivorax borkumensis under different inoculation conditions, J. Pet. Sci. Eng. 129
(2015) 153–158, https://doi.org/10.1016/j.petrol.2015.02.038.
The substrate degradation capacity of A. borkumensis was evaluated [6] D.J. Naether, S. Slawtschew, S. Stasik, M. Engel, M. Olzog, L.Y. Wick, K.N. Timmis, H.J.
by calculating the different degradation rates and kinetic constants. Dif- Heipieper, Adaptation of the hydrocarbonoclastic bacterium Alcanivorax
borkumensis SK2 to alkanes and toxic organic compounds: a physiological and
ferences between biodegradation rates may be due to the type and the
transcriptomic approach, Appl. Environ. Microbiol. 79 (2013) 4282–4293, https://
bioavailability of the hydrocarbons. In fact, it has been demonstrated doi.org/10.1128/AEM.00694-13.
that different bacterial species have different dissipation potentials de- [7] M. Hassanshahian, G. Emtiazi, G. Caruso, S. Cappello, Bioremediation (bioaugmenta-
tion/biostimulation) trials of oil polluted seawater: a mesocosm simulation study,
pending on those two factors [55].
Mar. Environ. Res. 95 (2014) 28–38.
The Hockey–Stick model is commonly used to describe dissipation [8] N. Qiao, Z. Shao, Isolation and characterization of a novel biosurfactant produced by
patterns with a lag-phase where the concentration of the pollutant is hydrocarbon-degrading bacterium Alcanivorax dieselolei B-5, J. Appl. Microbiol. 108
not constant but declines very slowly up to a point where the biodegra- (2010) 1207–1216, https://doi.org/10.1111/j.1365-2672.2009.04513.x.
[9] S. Schneiker, V.A.M. dos Santos, D. Bartels, T. Bekel, M. Brecht, J. Buhrmester, T.N.
dation process starts (FOCUS 2006). In this study, A. borkumensis exhib- Chernikova, R. Denaro, M. Ferrer, C. Gertler, A. Goesmann, O.V. Golyshina, F.
ited this pattern when grown on hexane, hexadecane and motor oil Kaminski, A.N. Khachane, S. Lang, B. Linke, A.C. McHardy, F. Meyer, T. Nechitaylo, A.
with a high correlation (R2 = 0.96, 0.79 and 0.91, respectively; Fig. 7 Pühler, D. Regenhardt, O. Rupp, J.S. Sabirova, W. Selbitschka, M.M. Yakimov, K.N.
Timmis, F.-J. Vorhölter, S. Weidner, O. Kaiser, P.N. Golyshin, Genome sequence of the
(a), (b) and (c)). ubiquitous hydrocarbon-degrading marine bacterium Alcanivorax borkumensis, Nat.
These results are similar to those found for the remediation of soil Biotechnol. 24 (2006) 997–1004, https://doi.org/10.1038/nbt1232.
heavily contaminated with hydrocarbons by Pseudomonas sp. [22] or [10] C. Liu, W. Wang, Y. Wu, Z. Zhou, Q. Lai, Z. Shao, Multiple alkane hydroxylase systems
in a marine alkane degrader, Alcanivorax dieselolei B-5, Environ. Microbiol. 13
other microbial consortia [56,57]. (2011) 1168–1178.
[11] W. Wang, Z. Shao, Diversity of flavin-binding monooxygenase genes (almA) in ma-
rine bacteria capable of degradation long-chain alkanes, FEMS Microbiol. Ecol. 80
5. Conclusions (2012) 523–533.
[12] W. Wang, Z. Shao, The long-chain alkane metabolism network of Alcanivorax
dieselolei, Nat. Commun. 5 (2014) http://www.nature.com/ncomms/2014/141212/
The growth of Alcanivorax borkumensis was investigated on various
ncomms6755/full/ncomms6755.html (accessed February 13, 2017).
substrates, hexane, hexadecane and motor oil that could exist in the ma- [13] R.N. Austin, J.T. Groves, Alkane-oxidizing metalloenzymes in the carbon cycle,
rine environment during an oil spill situation. Alcanivorax borkumensis Metallomics 3 (2011) 775–787.
showed excellent growth on the three different substrates with the pro- [14] G.A.-E. Mahmoud, M.M. Koutb, F.M. Morsy, M.M. Bagy, Characterization of lipase en-
zyme produced by hydrocarbons utilizing fungus Aspergillus terreus, Eur. J. Biol. Res.
duction of high activities of alkane hydroxylase, lipase, and esterase en- 5 (2015) 70–77.
zymes. A higher percentage removal of hexane, hexadecane and motor [15] M.M. Yakimov, P.N. Golyshin, S. Lang, E.R. Moore, W.-R. Abraham, H. Lünsdorf, K.N.
oil during Alkanivorax bokumensis growth was obtained (80%, 81.5%, Timmis, Alcanivorax borkumensis gen. nov., sp. nov., a new, hydrocarbon-degrading
and surfactant-producing marine bacterium, Int. J. Syst. Evol. Microbiol. 48 (1998)
and 75%, respectively). 339–348.
The best production of alkane hydroxylase and lipase was found when [16] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of
using motor oil as a substrate while the best esterase production was bacteriophage T4, Nature 227 (1970) 680–685.
[17] N. Prim, M. Sánchez, C. Ruiz, F.J. Pastor, P. Diaz, Use of methylumbeliferyl-derivative
reached when using hexadecane as a substrate. Zymogram of the crude substrates for lipase activity characterization, J. Mol. Catal. B Enzym. 22 (2003)
enzyme extract of the studied strain showed two distinct bands corre- 339–346.
sponding to the size of approx. 52 and 40 kDa, respectively with lipase/es- [18] T.C. Flores-Flores, M. Gutiérrez-Rojas, S. Revah, E. Favela-Torres, Comparative study
for oxygenases produced by Aspergillus niger, ATCC 9642, in solid-state and sub-
terase activity. Crude alkane hydroxylase was shown to have optimum merged fermentation, rev. Mex, Ing. Quím. 10 (2011) 189–207.
activity at pH 8.8 and temperature 70 ± 1 °C for hexane and hexadecane, [19] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram
and temperature 75 ± 1 °C for motor oil. Characterization of lipase and es- quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem.
72 (1976) 248–254.
terase showed optimum activity pH 7.0 and temperatures 35 ± 1 °C and
[20] A. Glieder, E.T. Farinas, F.H. Arnold, Laboratory evolution of a soluble, self-sufficient,
40 ± 1 °C, respectively. All the enzymes possessed stability in a wide highly active alkane hydroxylase, Nat. Biotechnol. 20 (2002) 1135–1139.
range of pH, but they were not thermostable at high temperatures. [21] D.B. Lopes, L.P. Fraga, L.F. Fleuri, G.A. Macedo, Lipase and esterase: to what extent
Kinetics of Alcanivorax borkumensis showed higher biodegradation can this classification be applied accurately? Food Sci. Technol. Camp. 31 (2011)
603–613.
efficiency in terms of hydrocarbon removal. Moreover, A. borkumensis [22] A. Dados, M. Omirou, K. Demetriou, C. Papastephanou, I.M. Ioannides, Rapid remedi-
responded to the same kinetic models when growing on different ation of soil heavily contaminated with hydrocarbons: a comparison of different
240 T. Kadri et al. / International Journal of Biological Macromolecules 112 (2018) 230–240

approaches, Ann. Microbiol. 65 (2014) 241–251, https://doi.org/10.1007/s13213- terase that is most catalytically active with polyaromatic esters, Microb. Biotechnol.
014-0856-5. 7 (2014) 184–191.
[23] R. Boopathy, Factors limiting bioremediation technologies, Bioresour. Technol. 74 [40] L.T. Izrael-Zivkovic, G.D. Gojgic-Cvijovic, K.R. Gopcevic, M.M. Vrvic, I.M. Karadzic, En-
(2000) 63–67, https://doi.org/10.1016/S0960-8524(99)00144-3. zymatic characterization of 30 kDa lipase from Pseudomonas aeruginosa ATCC
[24] R.A. Kanaly, S. Harayama, Biodegradation of high-molecular-weight polycyclic aro- 27853, J. Basic Microbiol. 49 (2009) 452–462.
matic hydrocarbons by bacteria, J. Bacteriol. 182 (2000) 2059–2067, https://doi. [41] M. de Lourdes Moreno, D. Pérez, M.T. García, E. Mellado, Halophilic bacteria as a
org/10.1128/JB.182.8.2059-2067.2000. source of novel hydrolytic enzymes, Life 3 (2013) 38–51.
[25] P.N. Golyshin, V.A.P. Martins Dos, O. Kaiser Santos, M. Ferrer, Y.S. Sabirova, H. [42] S. Zhang, G. Wu, Z. Liu, Z. Shao, Z. Liu, Characterization of EstB, a novel cold-active
Lünsdorf, T.N. Chernikova, O.V. Golyshina, M.M. Yakimov, A. Pühler, K.N. Timmis, and organic solvent-tolerant esterase from marine microorganism Alcanivorax
Genome sequence completed of Alcanivorax borkumensis, a hydrocarbon-degrading dieselolei B-5 (T), Extremophiles 18 (2014) 251–259.
bacterium that plays a global role in oil removal from marine systems, J. Biotechnol. [43] P. Li, L. Wang, L. Feng, Characterization of a novel Rieske-type alkane monooxygenase
106 (2003) 215–220, https://doi.org/10.1016/j.jbiotec.2003.07.013. system in Pusillimonas sp. strain T7-7, J. Bacteriol. 195 (2013) 1892–1901.
[26] J.B. Van Beilen, M.M. Marín, T.H.M. Smits, M. Röthlisberger, A.G. Franchini, B. [44] L. Lu, G. Zeng, C. Fan, X. Ren, C. Wang, Q. Zhao, J. Zhang, M. Chen, A. Chen, M. Jiang,
Witholt, F. Rojo, Characterization of two alkane hydroxylase genes from the marine Characterization of a laccase-like multicopper oxidase from newly isolated Strepto-
hydrocarbonoclastic bacterium Alcanivorax borkumensis, Environ. Microbiol. 6 myces sp. C1 in agricultural waste compost and enzymatic decolorization of azo
(2004) 264–273, https://doi.org/10.1111/j.1462-2920.2004.00567.x. dyes, Biochem. Eng. J. 72 (2013) 70–76.
[27] C.C. De Carvalho, B. Parreño-Marchante, G. Neumann, M.M.R. Da Fonseca, H.J. [45] D. Salvachúa, A. Prieto, Á.T. Martínez, M.J. Martínez, Characterization of a novel dye-de-
Heipieper, Adaptation of Rhodococcus erythropolis DCL14 to growth on n-alkanes, colorizing peroxidase (DyP)-type enzyme from Irpex lacteus and its application in en-
alcohols and terpenes, Appl. Microbiol. Biotechnol. 67 (2005) 383–388. zymatic hydrolysis of wheat straw, Appl. Environ. Microbiol. 79 (2013) 4316–4324.
[28] A.K. Haritash, C.P. Kaushik, Biodegradation aspects of polycyclic aromatic hydrocar- [46] G. Feller, M. Thiry, J.-L. Arpigny, M. Mergeay, C. Gerday, Lipases from psychrotropic
bons (PAHs): a review, J. Hazard. Mater. 169 (2009) 1–15, https://doi.org/10.1016/j. Antarctic bacteria, FEMS Microbiol. Lett. 66 (1990) 239–243.
jhazmat.2009.03.137. [47] D.-W. Choo, T. Kurihara, T. Suzuki, K. Soda, N. Esaki, A cold-adapted lipase of an Alas-
[29] T.K. Dutta, S. Harayama, Biodegradation ofn-alkylcycloalkanes and n-alkylbenzenes kan psychrotroph, Pseudomonas sp. strain B11-1: gene cloning and enzyme purifica-
via new pathways in Alcanivorax sp. strain MBIC 4326, Appl. Environ. Microbiol. 67 tion and characterization, Appl. Environ. Microbiol. 64 (1998) 486–491.
(2001) 1970–1974. [48] D. Bisht, S.K. Yadav, N.S. Darmwal, An oxidant and organic solvent tolerant alkaline
[30] J.B. Van Beilen, E.G. Funhoff, Alkane hydroxylases involved in microbial alkane deg- lipase by P. aeruginosa mutant: downstream processing and biochemical character-
radation, Appl. Microbiol. Biotechnol. 74 (2007) 13–21. ization, Braz. J. Microbiol. 44 (2013) 1305–1314.
[31] F. Rojo, Degradation of alkanes by bacteria, Environ. Microbiol. 11 (2009) [49] D. Park, H. Oh, S. Heo, W. Jeong, D.H. Shin, K.S. Bae, H. Park, et al., Characterization of
2477–2490, https://doi.org/10.1111/j.1462-2920.2009.01948.x. an extracellular lipase in Burkholderia sp. HY-10 isolated from a longicorn beetle, J.
[32] A.K. Karamalidis, A.C. Evangelou, E. Karabika, A.I. Koukkou, C. Drainas, E.A. Voudrias, Microbiol.-Seoul 45 (2007) 409.
Laboratory scale bioremediation of petroleum-contaminated soil by indigenous mi- [50] P.S. Borkar, R.G. Bodade, S.R. Rao, C.N. Khobragade, Purification and characterization
croorganisms and added Pseudomonas aeruginosa strain Spet, Bioresour. Technol. of extracellular lipase from a new strain: Pseudomonas aeruginosa SRT 9, Braz. J.
101 (2010) 6545–6552. Microbiol. 40 (2009) 358–366.
[33] A. Sharma, P. Kumar, M.B. Rehman, Biodegradation of diesel hydrocarbon in soil by [51] S. Ueda, Y. Fujio, J.Y. Lim, Production and some properties of pectic enzymes from
bioaugmentation of Pseudomonas aeruginosa: a laboratory scale study, Int. J. Environ. Aspergillus oryzae A-3, J. Appl. Biochem. 4 (1982) 524–532.
Bioremediation Biodegrad. 2 (2014) 202–212. [52] M.I.A. Lubna Tahir, Production and characterization of esterase in Lantinus tigrinus
[34] J. Kennedy, N.D. O'leary, G.S. Kiran, J.P. Morrissey, F. O'Gara, J. Selvin, A.D.W. Dobson, for degradation of polystyrene, Pol. J. Microbiol. Pol. Tow. Mikrobiol. Pol. Soc.
Functional metagenomic strategies for the discovery of novel enzymes and Microbiol. 62 (2013) 101–108.
biosurfactants with biotechnological applications from marine ecosystems, J. Appl. [53] E. Topakas, C. Vafiadi, P. Christakopoulos, Microbial production, characterization and
Microbiol. 111 (2011) 787–799. applications of feruloyl esterases, Process Biochem. 42 (2007) 497–509.
[35] E. Zeynalov, T. Nagiev, Enzymatic catalysis of hydrocarbons oxidation “in vitro” [54] C. Breuil, D.B. Shindler, J.S. Sijher, D.J. Kushner, Stimulation of lipase production dur-
(review), http://old.lp.edu.ua/fileadmin/ICCT/journal/Vol.9/No_2/05.pdf 2015 ing bacterial growth on alkanes, J. Bacteriol. 133 (1978) 601–606.
(accessed November 20, 2015). [55] P. Cyplik, M. Schmidt, A. Szulc, R. Marecik, P. Lisiecki, H.J. Heipieper, M. Owsianiak,
[36] R. Margesin, G. Feller, M. Hämmerle, U. Stegner, F. Schinner, A colorimetric method M. Vainshtein, \Lukasz Chrzanowski, Relative quantitative PCR to assess bacterial
for the determination of lipase activity in soil, Biotechnol. Lett. 24 (2002) 27–33. community dynamics during biodegradation of diesel and biodiesel fuels under var-
[37] R. Margesin, D. Labbe, F. Schinner, C.W. Greer, L.G. Whyte, Characterization of hydro- ious aeration conditions, Bioresour. Technol. 102 (2011) 4347–4352.
carbon-degrading microbial populations in contaminated and pristine alpine soils, [56] W. Liu, Y. Luo, Y. Teng, Z. Li, L.Q. Ma, Bioremediation of oily sludge-contaminated soil
Appl. Environ. Microbiol. 69 (2003) 3085–3092. by stimulating indigenous microbes, Environ. Geochem. Health 32 (2010) 23–29.
[38] R. Margesin, M. Hämmerle, D. Tscherko, Microbial activity and community compo- [57] M. Nikolopoulou, N. Kalogerakis, Biostimulation strategies for fresh and chronically
sition during bioremediation of diesel-oil-contaminated soil: effects of hydrocarbon polluted marine environments with petroleum hydrocarbons, J. Chem. Technol.
concentration, fertilizers, and incubation time, Microb. Ecol. 53 (2007) 259–269. Biotechnol. 84 (2009) 802–807, https://doi.org/10.1002/jctb.2182.
[39] M. Martínez-Martínez, I. Lores, C. Peña-García, R. Bargiela, D. Reyes-Duarte, M.-E.
Guazzaroni, A.I. Peláez, J. Sánchez, M. Ferrer, Biochemical studies on a versatile es-