A thermoalkaliphilic halotolerant esterase from Rhodococcus sp. LKE-028 (MTCC 5562): Enzyme purification and characterization

Article abstract of DOI:10.1016/j.procbio.2012.03.020

A newly isolated Rhodococcus sp. LKE-028 (MTCC 5562) from soil samples of Gangotri region of Uttarakhand Himalayan produced a thermostable esterase. The enzyme was purified to homogeneity with purification fold 62.8 and specific activity 861.2 U mg^-1 proteins along with 26.7% recovery. Molecular mass of the purified enzyme was 38 kDa and values of K_m and V _max were 525 nM and 1666.7 U mg^-1 proteins, respectively. The esterase was active over a broad range of temperature (40-100 °C) and pH (7.0-12.0). The esterase was most active at pH 11.0. The optimum temperature of enzyme activity was 70°C and the enzyme was completely stable after 3 h pre-incubation at 60°C. Metal ions like Ca²⁺, Mg²⁺ and Co²⁺ stimulated enzyme activities. Purified esterase remarkably retained its activity with 10 M NaCl. Enzyme activity was slightly increased in presence of non-polar detergents (Tween 20, Tween 80 and Triton X 100), and compatible with oxidizing agents (H₂O₂) and reducing agents (β-mercaptoethanol). Activities of the enzyme was stimulated in presence of organic solvents like DMSO, benzene, toluene, methanol, ethyl alcohol, acetone, isoamyl alcohol after 10 days long incubation. The enzyme retained over 75% activity in presence of proteinase K. Besides hyperthermostability and halotolerancy the novelty of this enzyme is its resistance against protease.

Full text of DOI:10.1016/j.procbio.2012.03.020

Process Biochemistry 47 (2012) 983–991
Contents lists available at SciVerse ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
A thermoalkaliphilic halotolerant esterase from Rhodococcus sp. LKE-028 (MTCC
5
562): Enzyme purification and characterization
Lokendra Kumara,b, Balvinder Singh , Dilip Kumar Adhikari , Joydeep Mukherjee , Debashish Ghosh
b
c
d
c,∗
a
Department of Biochemistry, Dolphin PG Institute of Biomedical and Natural Science, Dehradun, 248007, India
Department of Biochemistry, Sardar Bhagwan Singh PG Institute of Biomedical Sciences and Research, Balawala, Dehradun, 248161, India
Biotechnology Conversion Area (BFD), Indian Institute of Petroleum (CSIR), Dehradun, 248005, India
School of Environmental Studies, Jadavpur University, Kolkata, 700032, India
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
A newly isolated Rhodococcus sp. LKE-028 (MTCC 5562) from soil samples of Gangotri region of Uttarak-
Received 23 September 2011
Received in revised form 14 March 2012
Accepted 26 March 2012
hand Himalayan produced a thermostable esterase. The enzyme was purified to homogeneity with
−1
purification fold 62.8 and specific activity 861.2 U mg proteins along with 26.7% recovery. Molecu-
−1
lar mass of the purified enzyme was 38 kDa and values of Km and Vmax were 525 nM and 1666.7 U mg
Available online 3 April 2012
◦
proteins, respectively. The esterase was active over a broad range of temperature (40–100 C) and pH
(
7
7.0–12.0). The esterase was most active at pH 11.0. The optimum temperature of enzyme activity was
Keywords:
Esterase
Halotolerant
Thermo-alkaliphilic
Rhodococcus sp., Protease tolerant
◦
◦
2+
2+
0 C and the enzyme was completely stable after 3 h pre-incubation at 60 C. Metal ions like Ca , Mg
2
+
and Co stimulated enzyme activities. Purified esterase remarkably retained its activity with 10 M NaCl.
Enzyme activity was slightly increased in presence of non-polar detergents (Tween 20, Tween 80 and
Triton X 100), and compatible with oxidizing agents (H2O2) and reducing agents (␤-mercaptoethanol).
Activities of the enzyme was stimulated in presence of organic solvents like DMSO, benzene, toluene,
methanol, ethyl alcohol, acetone, isoamyl alcohol after 10 days long incubation. The enzyme retained
over 75% activity in presence of proteinase K. Besides hyperthermostability and halotolerancy the novelty
of this enzyme is its resistance against protease.
©
2012 Elsevier Ltd. All rights reserved.
1
. Introduction
used biocatalysts in fine chemical applications, mainly because
they could be applied efficiently in production of optically pure
compounds. Compared to other enzymes, industrial application
of esterases is relatively mature and there is an increasing inter-
est in high throughput tools for discovery and characterization of
these enzymes [8]. Despite growing interest in thermophiles and
their biocatalysts, only a limited number of esterases have been
characterized from thermophilic Archaea and Bacteria [5,6].
Therefore, hunt for new microbial esterase is important for
the development of thermostable enzymes and its applications.
Enzyme resistance to denaturation in organic solvents is correlated
with their extremophilic characteristics such as high temperature
and salt tolerance (high level of toxic compounds tolerance) as
well as resistance to protease [2]. Esterases have a wide range
of applications of which few employ in combination with amy-
lase and protease. Nevertheless, there are few documents on
proteolysis-resistant lipases. Zhang et al. [9] and Dutta and Ray
[10] have produced lipases from Streptomyces fradiae and Bacillus
cereus respectively which were resistant to commercial neutral and
alkaline proteases. Lipases produced by Pseudomonas aeruginosa,
Bacillus pumilus and Bacillus licheniformis were found to be resistant
to co-produced native proteases [11–13]. Rees et al. [14] detected
esterase enzyme activities encoded by novel genes present in
Enzymes expressed by extremophiles offer new opportuni-
ties where biocatalysis and biotransformations play a pivotal role.
Major road block of industrial usage of known esterases is their
limited thermal and pH stability in various industrial processes
[
1].
Microbial esterases have attracted considerable attention
because of its current applications in food, pharmaceutical, and
chemical areas [2]. Useful reactions performed by esterases include
resolution of racemic mixtures by transesterification, initiation
and regioselective hydrolysis, synthesis of natural and non-natural
pro-drugs, non-polar detergents, polyesters, and additives [3]. Gen-
erally, most of these reactions occur in non-aqueous environments.
Esterolytic enzymes have been reported in various extremophiles
including members of the order Thermoplasmatales and Sulfolob-
ales [4–6]. In recent years, scientific and industrial significance of
these extremophiles has increased intensely due to functional and
structural stability of their proteins [7]. Esterases are most widely
∗ Corresponding author. Tel.: +91 135 2525763; fax: +91 135 2660202.
E-mail address: dghosh@iip.res.in (D. Ghosh).
1
359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.procbio.2012.03.020

9
84
L. Kumar et al. / Process Biochemistry 47 (2012) 983–991
environmental DNA libraries. Still not many reports on protease
resistance esterases have been found.
In this paper, we are reporting purification and characteriza-
tion of an organic solvent and protease stable thermoalkaliphilic
halotolerant esterase from thermophilic Rhodococcus sp. LKE-028
isolated from Gangotri region of Uttarakhand Himalaya.
2.4. Electrophoresis, molecular mass determination and activity staining
SDS-PAGE was carried out according to Laemmli [17] with 7.5% polyacrylamide.
Molecular weight markers (14–97 kD; purchased from Sigma) were run along with
samples to determine molecular mass of the target protein. Protein bands were
visualized by sensitive silver staining method.
Purified enzyme was evaluated in native PAGE for activity staining with the same
gel composition, pH and voltage like SDS-PAGE; only difference being SDS replaced
by equivalent amounts of suitable buffer in gel preparation, electrode buffer and
sample buffer. The reducing agent (␤-mercaptoethanol) was excluded in the sample
buffer and the sample was not boiled prior to application. After electrophoresis, gel
was washed with 100 mM Tris–Cl buffer (pH 7.5), followed by treatment with 3 mM
pNP acetate solution in McIlvein buffer (pH 7.5) for 30 min. Gel was finally incubated
in developing solution with 2 mM Fast Red TR (Sigma–Aldrich, USA) in 100 mM
sodium phosphate buffer (pH 7.5). Esterase activity was detected by appearance of
2. Experimental methodology
2.1. Microorganism: culture conditions and identification
Bacterial pure cultures were isolated from treated soil collected around Gan-
gotri region (average height 10,000 ft) of Uttarakhand Himalaya (India). Soil sample
◦
a deep purple colored band in gel. All PAGEs were immediately photographed with
(
physical–chemical properties during collection; temperature 6 C, pH 8.5, moisture
◦
GelDocTM XR (Bio-Rad, USA) [18].
content 25%, w/v) were dried at 80 C for 72 h before inoculating in sterile distilled
water (5%, w/v). Soil free aqueous phase was inoculated into nutrient broth (NB) sup-
plied with glucose containing (composition in g L ): peptone, 5.0; yeast extract, 3.0;
−1
◦
3. Characterization of the enzyme
NaCl, 5.0; glucose, 10.0; pH 7.0 after proper serial dilution, and incubated at 60 C for
5
days. Randomly selected colonies were screened for esterolytic activity on trib-
utyrin agar plate containing 1.5% tributyrin in solidified nutrient agar medium at
6
3.1. Effect of pH and temperature on enzyme activity and stability
◦ ◦
0 C for 3 days. Further 4 days incubation at 40 C was continued to achieve bright
clear zone on plate [9]. On the basis of plate assay, one isolated strain designated as
LKE-028 was selected for further experiments. Master culture was preserved in 40%
◦
Effect of pH on esterase activity was determined at 70 C within
◦
pH ranges of 5.0–12.0 by various buffer solutions, viz. sodium
acetate (50 mM; pH 5.0–6.0), potassium phosphate (50 mM;
pH 6.0–8.0), Tris–Cl buffer (50 mM; pH 7.0–9.0), glycine–NaOH
(
v/v) glycerol at −80 C.
Isolated strain LKE-028 was identified by partial 16S rDNA gene sequencing.
Extraction and purificationof DNA were carried out by GenElute bacterialgenomic
DNA kit (Sigma) following the manufacturer’s instructions. Primers selected for PCR
amplification experiments were, 5 -CAGGCCTAACACATGCAAGTC (forward primer)
and 5 -GGGCGGWGTGTACAAGGC (reverse primer). Amplified DNA was purified by
TM
(
50 mM; pH 8.0–11.0) and Na HPO –NaOH (50 mM; pH 12.0)
2 4
ꢀ
ꢀ
buffer. Enzyme activity was quantified using pNP acetate as sub-
strate as mentioned earlier. To investigate pH stability, lyophilized
enzymes were dissolved in buffers of different pH mentioned above
TM
GenElute
bacterial genomic DNA kit (Sigma). 16S rDNA sequence was aligned
with submitted sequences available in NCBI database using clustal W software, and
phylogenetic position of the isolate was depicted using TREEVIEW program (3.0).
◦
and pre-incubated at 70 C for 2 h. Reaction pH was adjusted with
same buffer which has been used for pre-incubation and finally
residual activity was determined.
2
.2. Enzyme production and quantification
Effect of temperature on enzyme activity was determined
◦
◦
Isolate LKE-028 was cultured in shake flasks containing modified NB (com-
between 20 C and 120 C at pH 11.0 following standard assay
◦
position same as Section 2.1) at 60 C with constant shaking at 140 rpm. Culture
methods using pNP acetate as substrate. Thermostability was inves-
3
supernatant was collected after 24 h culture by centrifugation at 10 × 10 r.c.f. for
◦
tigated by pre-incubating enzyme solutions at 60–90 C at pH 11.0
5
min to recover extracellular enzyme. Esterase activity was determined spec-
for 150 min. After 30 min interval, residual activities were quanti-
fied and compared with non pre-incubated sample.
trophotometrically using p-nitrophenyl acetate (pNP acetate) as substrate as
described by Sana et al. [15] with some modification. 250 ␮L culture supernatant
containing enzyme was incubated with 50 ␮L of 2 mM pNP acetate and 50 mM
On the basis of these findings all characterization studies were
◦
2
00 ␮L McIIvaine buffer (pH 7.2) at 60 C for 30 min. Reaction was terminated imme-
◦
conducted in triplicate sets at 70 C and pH 11.0.
diately by adding 500 ␮L of chilled buffer and suspended particles were removed by
3
centrifuging at 10 × 10 r.c.f. for 5 min. Amount of pNP acetate released by esterase
catalyzed hydrolysis was measured at ꢀ410 nm against enzyme free blank. One unit
of esterase activity was defined as the amount of enzyme needed to liberate 1 ␮M
pNP acetate/min under this condition [15]. Total protein was measured by method
described by Lowry et al. [16] with bovine serum albumin as standard. During chro-
matographic purification steps, protein concentration was measured as a function
of its absorbance at ꢀ280 nm.
3.2. Substrate specificity of the purified enzyme
Relative enzyme activity was measured on a broad range of
substrates (procured from Sigma–Aldrich, USA) including differ-
ent fatty acid esters viz. a series of pNP esters, triglycerides and
naphthyl esters.
2.3. Purification of esterase
3.2.1. p-Nitrophenyl esters
LKE-028 esterase was purified to homogeneity in four steps. Crude enzyme solu-
Assay with different pNP ester substrates was performed with
standard assay technique. pNP acetate was replaced by correspond-
ing substrates [19].
tion (cell free supernatant) was cooled and ammonium sulfate crystals were slowly
added with constant stirring up to 30% saturation. The solution was allowed to stand
◦
3
overnight at 4 C. Precipitate was collected after centrifugation at 12 × 10 r.c.f. at
◦
4
C for 15 min and dissolved in 20 mM Tris–Cl buffer (pH 8.0) to achieve a vol-
ume of 1/10th of the original crude sample. Undissolved substance was removed
3.2.1.1. ˛-Naphthyl esters. Reaction mixture of 250 ␮L enzyme
solution, 50 ␮L ␣-naphthyl acetate and 200 ␮L glycine–NaOH
buffer was incubated with 50 ␮L 10 mM Fast Red TR. Activity
was quantified at ꢀ560 nm by measuring the degree of substrate
hydrolysis. Spontaneous substrate hydrolysis without enzyme was
considered as blank. One unit of enzyme activity was defined as
amount of enzyme essential to release 1 ␮M ␣-naphthol in 1 h.
Methods were adopted from the work done by Sana et al. [15] with
slight modifications.
3
◦
by centrifuging at 12 × 10 r.c.f. at 4 C for 15 min, precipitated protein were esti-
mated and dialyzed against 20 mM Tris–Cl buffer (pH 8.0) to remove excess salts.
Concentrated protein solution was loaded in a DEAE cellulose (Himedia, India) col-
umn (2.2 cm × 20 cm) pre-equilibrated with 20 mM Tris–Cl buffer (pH 8.0). Column
was washed with same buffer until ꢀ280 nm of effluent become zero and bound
−
1
proteins were eluted by NaCl gradient (0–0.5 M, flow rate 0.2 mL min ). Each frac-
tion of 1 mL volume was collected and those with high specific activity were pooled
and lyophilized. Lyophilized powder was dissolved in minimum volume of 20 mM
Tris–Cl buffer (pH 8.0) and was passed through pre-equilibrated carboxy methyl cel-
lulose (CM-cellulose) (Sigma–Aldrich, USA) column (2.2 cm × 25 cm). Column was
then washed until ꢀ280 nm of effluent reached zero. Bound proteins were eluted by
NaCl gradient (0–0.5 M) in same buffer. Active fractions were pooled and loaded to a
sephadex-G-100 (Fluka chemicals, Switzerland) column (2.25 cm × 35 cm) and frac-
3.2.1.2. ˇ-Naphthyl esters. Assay mixture containing 50 ␮L ␤-
−1
naphthyl acetate, 200 ␮L of glycine–NaOH buffer, and 250 ␮L
enzyme was incubated for 30 min and reaction was terminated by
chilling on ice. Esterolytic activity was measured at ꢀ320 nm. One
tions of 1 mL each were collected at a flow rate of 5 mL h . Each purification step
was monitored by SDS-PAGE. High specific activity fractions of purified enzyme
were pooled and lyophilized for further characterization.

L. Kumar et al. / Process Biochemistry 47 (2012) 983–991
985
Fig. 1. Phylogenetic dendrogram, indicating the position and distance matrix based comparison of the strain Rhodococcus sp. LKE-028 (GenBank accession number:
GU944774).
unit of enzyme activity was defined as amount of enzyme prepara-
3.6. Effect of organic solvents on enzyme activity and stability
tion capable of releasing 1 ␮M ␤-naphthol in 1 h [20].
Stability of purified esterase was determined against various
organic solvents. In different screw cap test tubes, enzyme solution
in 50 mM glycine–NaOH buffer was mixed with different organic
3
.2.2. Fatty acid esters
Enzymatic hydrolysis of fatty acid esters was determined by
◦
solvents (enzyme:solvent::2:1) and incubated at 70 C with con-
titration of liberated fatty acid. Reaction mixture containing 400 ␮L
of 10 mM fatty acid ester, 300 ␮L glycine–NaOH buffer and 300 ␮L
enzyme solution was incubated for 30 min and then stopped by
addition of terminating agent (10 ␮L 1:1 (v/v) mixture of acetone
and ethanol and 10 ␮L of 1% (w/v) phenolphthalein in a 9:1 (v/v)
stant moderate shaking for 10 days. Residual activity of each aliquot
was measured by standard assay procedure at different interval
of days. Loss of solvents due to evaporation was replenished with
fresh solvent. Relative activities were calculated on the basis of
enzyme activity, considering solvent at 0 day without incubation is
mixture of ethanol and H O). Final mixture was titrated with 0.05 M
2
1
00%.
NaOH until mixture turned red. Amount of liberated fatty acid was
estimated from the volume of NaOH required to reach end point.
One unit of enzyme activity was defined as the amount of enzyme
required to produce 1 ␮M fatty acid in 1 h [21].
3.7. Stability of enzyme against protease
Enzyme stability was also checked against proteinase
K
(
Sigma–Aldrich, USA). Purified esterase was incubated with differ-
3
.3. Kinetic determinations
−
1
◦
ent conc. (5–100 U ␮g proteinase K) at 70 C for 30 min. Esterase
activity was measured with pNP acetate as substrate.
Initial rate of reaction for pNP acetate hydrolysis was calculated
by estimating esterase activity with different substrate concentra-
tions from 25 to 2500 nM Michaelis–Menten constant (Km) and rate
of reaction (Vmax) were determined according to Lineweaver–Burk
plot [22].
4
. Results
4.1. Microbial and phylogenetic characterization and enzyme
production of Rhodococcus sp. LKE-028
3.4. Effect of metal ions, inhibitors and denaturing chemicals
Isolated LKE-028 was found to possess potent esterolytic activ-
Enzyme was incubated for 1 h in 50 mM glycine–NaOH buffer at
ity when qualitatively screened with tributyrin in nutrient agar
◦
2+
2+
2+
2+
3+
2+
medium at 60 ◦C for bright clear zone on the plate. A more visi-
7
0 C with different metal ions (Ba , Ca , Co , Cu , Fe , Hg ,
+
2+
2+
2+
ble clear zone was found with further incubation at 40 ◦C for 4 days.
K , Mg , Mn and Zn ); inhibitors (EDTA, iodoacetic acid, PMSF),
reducing agent (␤-mercaptoethanol) and oxidizing agent (hydro-
gen peroxide) with final concentration of 5 mM and surface active
agents (SDS, Triton X 100, Tween 20 and Tween 80) with final
concentration of 1 and 2% (v/v). Residual activity was measured
following standard assay method with pNP acetate as substrate.
Enzyme activity treated in same way but without any additive was
considered 100%.
Morphological and physiological characteristics of isolate ‘LKE-028’
showed a Coccus morphotype. Based on nucleotide of 16S rRNA
gene sequence of the isolate LKE-028 was found to be a member
of Rhodococcus family (GenBank accession no. GU944774). Taxo-
nomic position is shown in phylogenetic tree (Fig. 1). Strain has
been deposited in ‘MTCC’, Chandigarh (CSIR-IMT, India) with code
name MTCC 5562.
3.5. Effect of sodium chloride on enzyme activity
4.2. Enzyme purification and molecular weight
Effect of salinity on enzyme activity was studied in presence
Enzyme was purified up to 62-fold with 26.7% recovery, spe-
−
1
of different NaCl concentrations ranging from 0 to 10 M. Purified
enzyme was preincubated in 50 mM glycine–NaOH buffer con-
cific activity of finally purified enzyme being 861.2 U mg proteins
(Table 1). Pure enzyme showed a single band on SDS-PAGE to
prove its homogeneity. On the basis of molecular weight marker
ran parallel with proteins during each purification steps in SDS-
PAGE, target protein was found to have molecular weight of 38 kDa.
◦
taining different NaCl concentrations at 70 C for 3 h and then
quantified. Enzyme activity treated in same way without NaCl was
considered 100%.

9
86
L. Kumar et al. / Process Biochemistry 47 (2012) 983–991
Table 1
Summary of the results of the purification of esterase from Rhodococcus sp. LKE-028.
Purification step
Crude extraction
Total activity (U)
Protein (mg)
Specific activity (U mg−1)
Purification fold
Recovery (%)
17,480
14,690
9470
6450
4670
1280
196.7
20.3
10.9
5.4
13.7
74
465.5
590.2
861.2
1.0
5.4
33.9
43
100
84
54.1
36.8
26.7
3
0% (NH4)2SO4 fraction
DEAE ion-exchange
CM-ion-exchange
Sephadex gel-filtration
62.8
Table 2
Effects of different substrates on the activity of the esterase purified from Rhodococcus sp. LKE-028.
Specific activity (U mg 1 protein)
−
% Relative activity
Substrate
Specific activity (U mg protein)
−1
% Relative activity
Substrate
pNP acetate
861.2
856.8
491.7
368.5
577.8
109.5
438.3
294.5
72.49
0.0
100
99.5
57.1
42.8
67.1
12.7
50.9
34.2
8.4
Ethyl laurate
Ethyl lactate
Ethyl linolate
Ethyl oleate
Ethyl octanate
Ethyl palmitate
Isopropyl-myristate
Triacetin
0.0
86.1
0.0
55.6
356.5
58.2
0.0
384.9
298.8
0.0
0
10
0
6.5
41.4
6.8
0
44.7
34.7
0
pNP butyrate
pNP carprylate
pNP palmitate
␣
␤
-Napthyl acetate
-Napthyl acetate
Ethyl butyrate
Ethyl caprylate
Ethyl decanoate
Ethyl formate
Ethyl isolaurate
Tributyrin
Trilaurin
Tripalmitin
0
0
0.0
0.0
0
Esterase activity was detected by appearance of a deep purple col-
ored band in gel with activity staining (Fig. 2).
30, 60, 90 and 120 min of pre-incubation. LKE-028 esterase
retained 50% of its residual activity even after 120 min pre-
incubation up to pH 11.0. When pre-incubated at pH 12.0, a
gradual decrease in activity was observed with increased pre-
incubation time and at 120 min it almost lost 75% of its residual
activity.
4.3. Effect of pH and temperature on enzyme activity and stability
Optimum pH for enzyme activity was 11.0 and it remained
◦
Optimum activity of the purified enzyme was found at 70 C
active over a broad range of pH (8.0–12.0) (Fig. 3a) where it
retained almost 80% of its residual activity (activity at pH 11.0
was considered to be 100%). This enzyme was stable in the
pH range between 5.0 and 11.0 for a time span of 120 min.
Fig. 3b illustrates residual activity at different pH values after
(
4
Fig. 4a) and it remained active over a temperature range of
0–90 C, retaining almost 70% of its residual activity (activity at
◦
optimum temperature was considered to be 100%). Thermal stabil-
ity profile of LKE-028 esterase in form of relative activity has been
◦
presented in Fig. 4b. Enzyme was completely stable at 60 C up to
◦
3
h and retained more than 80% activity up to 80 C for 3 h incu-
bation. At 90 C a sharp fall in residual activity was observed with
increase in pre-incubation time.
◦
4.4. Substrate specificity of the LKE-028 esterase
Enzyme activity was studied on different type of substrates
as shown in Table 2. Enzyme was found to be most active
with pNP acetate and pNP butyrate. Increasing length of fatty
acid side chain hindered residual activity of enzyme. ␣-napthyl
acetate was a better substrate than its ␤ isomer. Enzyme showed
relatively lesser activity toward ‘fatty acids from triglycerides’
(Table 2).
4.5. Kinetic determinations
Michaelis–Menten constant (Km) and rate of reaction (Vmax)
were determined according to Lineweaver–Burk plot [22]. Appar-
ent Km and Vmax values, determined for LKE-028 esterase by using
−
1
pNP acetate as substrate (Fig. 5) were 525 nM and 1666.7 U mg
protein respectively.
4.6. Effect of metal ions, inhibitors and denaturing chemicals
Enzyme activity was studied on different metal ions as shown
2
+
2+
in Table 3. Almost 60% of activity was found with Ba and Zn
,
Fig. 2. SDS-PAGE and zymography of the purified esterase from Rhodococcus sp.
LKE-028. Molecular weight marker (lane 1); crude enzyme after precipitation (lane
2+
2+
2+
where as Ca , Mg and Co enhanced enzyme activity (Table 3).
Enzyme was completely inhibited by well-known serine inhibitor
2
); active fractions after anionic exchange chromatography (lane 3); active fractions
(
PMSF) indicating that this enzyme belonged to serine hydrolyses
after cationic exchange chromatography (lane 4); purified esterase after molecular
sieving chromatography (lane 5); zymogram of the purified esterase (lane Z).
family (Table 4). LKE-028 esterase has also been found stable in

L. Kumar et al. / Process Biochemistry 47 (2012) 983–991
987
(
b) 110
(
a)110
100
100
9
0
0
0
0
0
0
0
0
90
80
70
60
50
40
30
20
8
7
6
5
4
3
2
pH 5.0
pH 6.0
pH 7.0
pH 8.0
pH 9.0
pH 10.0
pH 11.0
pH 12.0
4
5
6
7
8
9
10
11
12
13
0
20
40
60
80
100
120
pH
Time (Min.)
Fig. 3. Effect of pH (a) and stability (b) on the activity of esterase from Rhodococcus sp. LKE-028.
(
b)
(
a)
100
100
8
6
4
2
0
0
0
0
8
6
4
2
0
0
0
0
0
6
7
0 C
0
0 C
0
80 C
0
9
0 C
0
2
0
40
60
80
100
120
-20
0
20
40
60
80
100 120 140 160
0
Temperature ( C)
Time (min)
Fig. 4. Effect of temperature on activity (a) and stability (b) of esterase from Rhodococcus sp. LKE-028.
presence of both oxidizing and reducing agents and retained more
than 60% activity in presence of ␤-mercaptoethanol and hydrogen
peroxide. In presence of surfactants like SDS, Tween 20, Tween 80
and Triton X 100 (1% and 2%) enhancement of enzyme activity has
been observed.
4.7. Effect of sodium chloride on enzyme activity
Purified esterase remarkably retained 100% activity when incu-
bated with 10 M NaCl. Activity enhanced about 50% from that
Table 4
Effects of inhibitors and surfactant on the activity of the esterase purified from
Rhodococcus sp. LKE-028.
Table 3
Inhibitors/reducing/oxidizing
agent/surfactant
Specific activity
(U mg protein)
% Relative
activity
Effects of metal ions on the activity of the esterase purified from Rhodococcus sp.
LKE-028.
−
1
Control
EDTA
Iodoacetic acid
PMSF
␤-Mercaptoethanol
H2O2
861.2
769.0
726.8
15
571.1
677.5
951
1162.6
1095.4
1179.8
1162.6
1205.6
1194.4
100
−
Metal ions
Control
Specific activity (U mg 1 protein)
% Relative activity
89.1
84.4
1.7
66.3
78.6
110.43
135
127.2
137
135
861.2
1444.2
769
726.8
769
810.3
1073.9
1492.4
688.9
375.4
291.9
100
2+
Ca
167.7
89.3
84.4
89.1
94.1
124.7
173.3
80
2
+
Hg
3
+
Fe
K
+
SDS
2+
Cu
Co
Triton X 100 (1%)
Triton X 100 (2%)
Tween 20 (1%)
Tween 20 (2%)
Tween 80 (1%)
Tween 80 (2%)
2+
2
+
Mg
2
+
Fe
2+
Zn
43.6
33.9
140
138.7
2+
Ba

9
88
L. Kumar et al. / Process Biochemistry 47 (2012) 983–991
Table 6
0
0
0
0
0
0
0
.018
.015
.012
.009
.006
.003
.000
Effects of proteinase K on the stability of the esterase purified from Rhodococcus sp.
LKE-028.
Y = 0.0568X+0.0006
R = 0.9815
2
−1
)
Proteinase K (U ␮g
Specific activity
% Relative activity
−
1
(
U mg protein)
K = 525 nM
Vmax = 1666.7 U mg
m
-1
Control
5
861.2
861
774.2
715.6
659.6
218.7
100
99.9
89.9
83.1
76.6
25.4
1
2
0
0
50
00
1
1
.1–1.7-fold increase in specific activity (with respect to control)
was observed over a period of 10 days.
4.9. Stability of enzyme against protease
0.00
0.05
0.10
0.15
1/S]
0.20
0.25
0.30
Enzyme was found to be active after treatment with proteinase
[
K. LKE-028 esterase retained over 75% of its residual activity up
−
1
to 50 U ␮g proteinase K (Table 6). A sharp loss in activity was
Fig. 5. Lineweaver–Burk plot of esterase from Rhodococcus sp. LKE-028 for the
kinetic analysis with pNP acetate.
observed with further increase in proteinase-K concentration up
−1
to 100 U ␮g
.
Table 5
5. Discussion
Effects of NaCl on the activity of the esterase purified from Rhodococcus sp. LKE-028.
−
NaCl (M)
Control
Specific activity (U mg 1 protein)
% Relative activity
Not many extremophilic esterases from thermophiles have
861.2
1731
100
201
been reported, except there are few reports of esterase activity
in some Rhodococcus sp. [23–25]. This paper describes purifica-
tion and characterization of a salt, organic solvent and protease
stable thermoalkaliphilic esterase from a thermophilic Rhodococ-
cus sp. LKE-028, a soil bacterium, isolated from Gangotri region
of Uttarakhand Himalaya. LKE-028 esterase was purified from cul-
ture supernatant in four steps and achieved approximately 4 times
1
2
3
4
5
1
M
M
M
M
M
1301.2
1216.8
1165.2
1080.8
869.8
151.1
141.3
135.3
125.5
101
0 M
recovery in comparison with reported esterase BSE01 [10] and
without NaCl when incubated with 2.0 M sodium chloride (Table 5).
−1
higher specific activity (861.2 U mg
protein) (Table 1) with 2
times recovery in comparison with Thermobifida fusca esterase
26].
LKE-028 esterase displayed high activity under neutral to alka-
4
.8. Effect of organic solvents on enzyme activity and stability
[
Purified LKE-028 esterase showed stability in presence of
line condition and no activity below pH 5.0. Highest level of activity
was measured at pH 11.0. Interestingly, enzyme was extremely sta-
ble, with little loss in activity after incubation up to 2 h in alkaline
conditions up to pH 11.0. pH stability profile of Rhodococcus sp.
LKE-028 esterase showed that enzyme retained over 50% activity
at broad range between pH 6.0 and 11.0 (Fig. 3) up to 2 h at room
temperature compared to thermophile Anoxybacillus gonensis A4
esterase [27]. LKE-028 displayed alkaliphilic properties that have
been previously detected only in enzymes isolated from extreme
environments [14]. Industrial processes often require different pH
as well as high temperatures and majority of known enzymes need
to be stabilized under these conditions; therefore, there is a great
interest in enzymes that are derived from extremophiles and stable
without pretreatment [6,28,29,30]. LKE-028 esterase was optimally
DMSO, benzene, toluene, methanol, ethyl alcohol, acetone, isoamyl
alcohol. Enzyme retained considerable activity after 10 days of
incubation in presence of isoamyl alcohol, DMSO and benzene com-
pared to other solvents (Fig. 6). In case of DMSO and benzene a
1
60
40
20
00
1
1
1
◦
◦
active at 70 C (Fig. 4). At 90 C LKE-028 displayed a half-life (t1/2)
8
0
0
0
0
0
of 50 min and no significant reduction in activity was observed at
◦
8
4
0 C. Enzyme was active in broad range of temperature between
6
4
2
◦
0 and 120 C. Manco et al. investigated thermostability of esterase
Est1 and Est2 from Bacillus acidocaldarius [31,32]. Est2 had a t1 of
/2
Control
Ethyl alcohol
◦
Isoamyl alcohol
Acetone
10 min at 90 C and 30% of the initial activity was recovered after
Est1 had been incubated for 90 min at 75 C. Sobek and Gorisch [33]
Toluene
Methanol
DMSO
Benzene
◦
Ethyl acetate
Glacial acetic acid
reported that residual activity of Sulfolobus acidocaldarius esterase
Pyridine
declined to 92% after purified enzyme had been incubated for 1 h
◦
at 90 C. These results provide strong evidence that esterase from
-1
0
1
2
3
4
5
6
7
8
9
10 11
Rhodococcus sp. LKE-028 possesses unique properties for large-
scale applications at extreme pH and temperature.
Time (days)
Substrate specificity of purified esterase from Rhodococcus sp.
LKE-028 was studied by using various pNP and ethyl esters of
Fig. 6. Effect of different organic solvents on the stability of esterase from Rhodococ-
cus sp. LKE-028.

L. Kumar et al. / Process Biochemistry 47 (2012) 983–991
989
Table 7
Comparative feature regarding various characteristics of esterase from Rhodococcus sp. LKE-028.
Strain/source
Optimum
NaCl conc. limit (%, M)
Reference
◦
Temperature ( C)
pH
11
Rhodococcus sp. LKE-028
70
40
30
80
45
45
60-80
50
47
–
10.0 M
0.5 M
Present work
Xiao-Yan, et al. [53]
Atta et al. [54]
Du Plessis et al. [56]
Rao et al. [55]
Sana et al. [15]
Thalassobacillus sp. strain DF-E4
Penicillum notatum NRRL-1249
Thermus scotoductus SA-01
Haloarcula marismortui (ATCC 43049).
Bacillus species BSE-01
8.5
5.5
7.0
9.5
8.0
5.5
9.0
10.0
–
–
3.4 M
1.0 M
Anoxybacillus gonensis A4
Ozlem et al. [27]
Elend et al. [52]
Elend et al. [52]
Bresler et al. [24]
Esterase EstA3 from metagenome (drinking water)
Esterase EstCE1 From metagenome (soil)
Rhodococcus sp. strain MB1
–
–
–
straight chain fatty acids ranging in chain length from C2 (acetate)
to C14 (myristate). Our enzyme showed substrate specificity on a
set of substrates typical for lipases and esterases, including pNP and
ethyl esters. pNP and ethyl esters with acyl chain lengths bigger
than C8 were not best substrates for LKE-028. Maximum enzyme
activity was observed toward pNP esters and ethyl esters of chain
length ranging from C2 and C6 (Table 2), found to be typical sub-
strates for esterases. pNP esters were hydrolyzed by LKE-028 at
higher rates compared to ethyl esters. Further titration tests using
triglycerides as substrates confirmed that only short chain sub-
strates were converted by LKE-028 esterase. Specific activities of
LKE-028 esterase with triacetin, tributyrin and tripalmitin were
similar to most of the tested substrates, indicating LKE-028 esterase
was also able to hydrolyze long chain fatty acids. These results indi-
cated that enzyme belonged to esterase class rather than lipases.
Broad substrate specificity of this robust LKE-028 enzyme was
remarkable and they could be of use to produce a diverse range
of high-value products.
from thermoacidophilic euryarchaeon Picrophilus torridus [45], Est1
and Est2 from B. acidocaldarius [31,32], thermostable esterase from
S. acidocaldarius [33] and thermostable SsoP1 [8]. In contrast, lipase
from Acinetobacter calcoaceticus was not inhibited by PMSF, possi-
bly caused by an active serine buried deeply within the molecule
[46]. There are increasing evidences that a disulfide bridge is essen-
tial for functionality of some esterases. Most of the esterases and
lipases have a serine residue at active site of the enzyme [47]. But
lipases can be distinguished from esterases by having a hydropho-
bic domain (lead) covering active site [48–50]. Therefore, lipases are
either slightly or not inhibited by PMSF because the “lead” struc-
ture makes active site of enzyme inaccessible to the reagent. So
as to determine the presence of a thiol group in esterase molecule,
enzyme was incubated with ␤-mercaptoethanol at a concentration
of 5 mM (Table 4) and was found to be non-influencive. From this
result, it could be inferred that thiol group was present or critical
for catalytic function.
Organic solvents can be advantageous in various industrial
enzymatic processes. Reaction media used in biocatalytic esteri-
fication and trans-esterification contains less than 1% water. Use of
organic solvents could increase solubility of non-polar substrates
and thermal stability of enzymes, decrease water-dependent side
reactions, or eliminate microbial contamination [51]. Organic sol-
vents affected performance of enzymes in different ways. In this
study, LKE-028 esterase depicted high stability in presence of many
water miscible organic solvents. In presence of DMSO and ben-
zene, LKE-028 esterase retained over 100% relative activities even
after 10 days (Fig. 6). Remarkable stability of EstB1 and EstB2
esterase activity toward organic solvents was reported by Kar-
pushova et al. [18]. LKE-028 esterase performed even better as
activity significantly increased with 33.3% (v/v) DMSO. Methanol
and ethyl alcohol slightly enhanced activity of our enzyme while
alcohols inhibited EstB1 and EstB2. Metagenome derived esterase
reported by Elend et al. [52] expressed maximum activity after 1 h
incubation with 15% and 30% (v/v) DMSO, while LKE-028 esterase
retained over 100% relative activity after incubation for 10 days as
similar to BSE01 esterase from Bacillus sp. reported by Sana et al.
[15].
It is known that metal ions play important role in maintaining
structural stability and protein integrity through binding to amino
acid residues with negative charges in specific sites [34]. In pres-
2
+
2+
ence of Ca and Mg our esterase activity was enhanced up to
1
2+
67.7 and 173.3% (Table 3). Stimulatory effect of Ca might be
attributed to better alignment of enzyme on substrate molecule and
neutralization of fatty acids liberated from substrate [35]. The same
stimulatory effect of Ca2 was recorded for Avena fatua esterase
+
2+
[
36]. Other metals such as Co2+ and Mg had significant effect on
enzyme activity in our study (Table 3). These observations reflect
similarities those described by Mohamed et al. [36], Baigori et al.
[
37] and Nourse et al. [38]. Interestingly, LKE-028 was inhibited by
2
+
2+
2+
addition of Ba or Zn ions and slightly inhibited with Cu in
hydrolysis medium (Table 3). These results are comparable with
esterases from turkey pharyngeal tissue and Cucurbita pepo which
were fully inhibited by the presence of Cu2 or Zn using pNP
esters as substrate [39,40]. From literature data it is evident that
many lipases and esterases are activated or inhibited by metal ions.
Intracellular esterase from Serratia marcescens 345 was activated
+
2+
2
+
by Mg ions [41], acetylcholinesterase is activated by monovalent
Na , K and divalent Ca and Mg cations [42] and in case of pan-
creatic lipase both alkali and alkali earth metal ions, especially Na ,
Ca and Mg stabilized the enzyme [43]. Several esterases have
been reported to be Ca dependent [44]. Zn ions strongly inhib-
ited EstB1 and EstB2 esterase activity, whereas addition of Ca and
Mg ions slowly deactivated both esterases [18]. Catalytic triad
conserved in esterases contains an active serine residue and there-
fore serine inhibitor such as PMSF inhibits activity of these serine
hydrolases. This was substantiated by the inhibition of LKE-028 in
the presence of 5 mM PMSF (Table 4). Inhibition in the presence of
PMSF was also observed for the thermostable esterase EstA and EstB
+
+
2+
2+
Remarkable stability of EstB1 and EstB2 esterase and BSE01
esterase activity toward NaCl was reported by Karpushova et al.
[18] and Sana et al. [15] due to their marine origin. LKE-028 esterase
activity was increased significantly with 1 M NaCl and retained
100% activity up to 10 M NaCl (Table 5), and displayed 10 times
more halotolerancy than previously reported esterases [15,18].
Comparative features regarding various characteristics of esterase
LKE-028 has been given in Table 7.
+
2+
2+
2+
2+
2+
2+
High salt tolerance of LKE-028 indicates extremophilic nature
of this halophilic enzyme. Thermostable, salt and organic solvent
tolerant enzymes might be applied to treat industrial effluents

9
90
L. Kumar et al. / Process Biochemistry 47 (2012) 983–991
treatments, to produce inter-esterification substances in food
industry, or to synthesize useful chemical compounds and non-
aqueous biocatalysis for non-natural hydrolysis of substrate as well
as modification of molecules. Stability of LKE-028 activity in pres-
ence of protease, organic solvents and salt, as well as in alkaline pH
at high temperatures makes it a potential candidate for its applica-
tion as a non-aqueous biocatalyst.
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