An extracellular solvent stable alkaline lipase from Pseudomonas sp. DMVR46: Partial purification, characterization and application in non-aqueous environment

Article abstract of DOI:10.1016/j.procbio.2014.06.007

The biosynthesis of esters is currently of much commercial interest because of the increasing popularity and demand for natural products among consumers. Biotransformation and enzymatic methods of ester synthesis are more effective when performed in non-aqueous media. In present study, an organic solvent stable Pseudomonas sp. DMVR46 lipase was partially purified by acetone precipitation and ion exchange chromatography with 28.95-fold purification. The molecular mass of the lipase was found to be ～32 kDa. The partially purified lipase was optimally active at 37 °C and pH 8.5. The enzyme showed greater stability toward organic solvents such as isooctane, cyclohexane and n-hexane retaining more than 70% of its initial activity. The metal ions such as Ca²⁺, Ba²⁺ and Mg²⁺ had stimulatory effects on lipase activity, whereas Co²⁺ and Zn²⁺ strongly inhibited the activity. Also lipase exhibited variable specificity/hydrolytic activity toward different 4-nitrophenyl esters. DMVR46 lipase was further immobilized into AOT-based organogels used for the synthesis of flavor ester pentyl valerate in presence of organic solvents. The organogels showed repeated use of enzyme with meager loss of activity even upto 10 cycles. The solvent-stable lipase DMVR46 thus proved to be an efficient catalyst showing an attractive potency for application in biocatalysis under non-aqueous environment.

Full text of DOI:10.1016/j.procbio.2014.06.007

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
An extracellular solvent stable alkaline lipase from Pseudomonas sp.
DMVR46: Partial purification, characterization and application in
non-aqueous environment
∗
Vrutika Patel, Shruti Nambiar, Datta Madamwar
BRD School of Biosciences, Sardar Patel University, Vadtal Road, Satellite Campus, Post Box # 39, Vallabh Vidhyanagar 388 120, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The biosynthesis of esters is currently of much commercial interest because of the increasing popularity
and demand for natural products among consumers. Biotransformation and enzymatic methods of ester
synthesis are more effective when performed in non-aqueous media. In present study, an organic solvent
stable Pseudomonas sp. DMVR46 lipase was partially purified by acetone precipitation and ion exchange
chromatography with 28.95-fold purification. The molecular mass of the lipase was found to be ∼32 kDa.
Received 22 April 2014
Received in revised form 31 May 2014
Accepted 12 June 2014
Available online xxx
◦
The partially purified lipase was optimally active at 37 C and pH 8.5. The enzyme showed greater stability
Keywords:
Organic solvent stable lipase
Pseudomonas sp.
Purification
Microemulsion based organogels (MBGs)
Ester synthesis
toward organic solvents such as isooctane, cyclohexane and n-hexane retaining more than 70% of its
2
+
2+
2+
initial activity. The metal ions such as Ca , Ba and Mg had stimulatory effects on lipase activity,
2
+
2+
whereas Co and Zn strongly inhibited the activity. Also lipase exhibited variable specificity/hydrolytic
activity toward different 4-nitrophenyl esters. DMVR46 lipase was further immobilized into AOT-based
organogels used for the synthesis of flavor ester pentyl valerate in presence of organic solvents. The
organogels showed repeated use of enzyme with meager loss of activity even upto 10 cycles. The solvent-
stable lipase DMVR46 thus proved to be an efficient catalyst showing an attractive potency for application
in biocatalysis under non-aqueous environment.
©
2014 Elsevier Ltd. All rights reserved.
1
. Introduction
(iii) enabling use of hydrophobic substrate, (iv) controlling sub-
strate specificity and (v) thermal stability of enzymes and relative
use of product recovery [4]. Solvent stable lipases are obligatory
in biotechnological applications, specifically in the production of
chiral compounds, high value pharmaceutical substances, modifi-
cations of fats and oils, synthesis of flavor esters and food additives,
production of biodegradable polymers, biodiesel and in synthesis
of fine chemicals [5]. Organic solvents offer advantages on enzyme
activity such as synthesis of esters from their constituent acids
and alcohol, enzyme stability and possess “pH memory” i.e. cat-
alytic activity of enzyme reflects pH of last solution to which it
was exposed [6,7]. So far, the number of solvent tolerant bacte-
rial lipases is inadequate [8], thus it becomes crucial to explore
novel lipases with high activity in organic solvents to expand their
application in practical catalysis.
Substantial efforts are made to exploit new species of microor-
ganisms that secretes solvent-stable lipase. Baharum et al. [9]
reported solvent tolerant lipase produced by Pseudomonas sp. strain
S5 that was stable in organic solvents such as n-hexane, cyclohex-
ane, toluene and 1-octanol. Rahman et al. [10] described organic
solvent tolerant lipase which was not only stable in n-hexane but
its activity was stimulated in presence of n-hexane. Cao et al. [8]
Lipases known as triacylglycerol acylhydrolases (E.C. 3.1.1.3) are
ubiquitous enzymes of considerable physiological significance and
industrial prospective. Lipases catalyze the hydrolysis of triacyl-
glycerols to glycerols and free fatty acids. Compared to esterases,
lipases are activated only when adsorbed to an oil–water inter-
face [1] and do not hydrolyze dissolved substrates in bulk fluid.
Besides, lipases accept a wide range of substrates and are quite
stable in non-aqueous solvents, thus they are frequently used for
the synthesis of enantiopure compounds for industrial applications
[
2,3].
Though solvents are extremely lethal to microorganisms, there
are numerous advantages of conducting enzymatic conversions in
organic solvents such as (i) shifting of thermodynamics equilib-
rium in favor of synthesis, (ii) regiospecificity and stereoselectivity,
∗ Corresponding author. Tel.: +91 2692 229380; fax: +91 2692 236475;
mobile: +91 98256 86025.
E-mail addresses: vrutikaptl 19@yahoo.com (V. Patel),
datta madamwar@yahoo.com (D. Madamwar).
http://dx.doi.org/10.1016/j.procbio.2014.06.007
1
359-5113/© 2014 Elsevier Ltd. All rights reserved.
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sp. DMVR46: Partial purification, characterization and application in non-aqueous environment. Process Biochem (2014),
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studied characterization of an organic solvent stable lipase puri-
fied from Pseudomonas stutzeri LC2-8 and showed its application for
efficient resolution of (R, S)-1-phenylethanol. Dandvate et al. [11]
purified a solvent tolerant lipase from Burkholderia multivorans V2
and showed its application for the synthesis of ethyl butyrate under
non-aqueous environment. Conversely, the stability and reusabil-
ity of the enzyme are of major concern in non-aqueous enzymatic
synthesis. The conceivable answer to this problem is recovery and
reuse by immobilizing in/on a solid support which makes it cost
effective [12,13].
Lipase immobilization is known to allow easier product recov-
ery, flexibility of reactor design and, in some cases, enhanced
storage and operational, thermal and conformational stability [14].
Variety of methods has been used for immobilization of biocat-
alyst such as adsorption, covalent attachment and entrapment
in polymer gels, microencapsulation [15] and sol–gel entrapment
2.3. Identification of solvent stable lipase producing culture using
16S rRNA approach
Bacterial strain designated as DMVR46 was selected on the
basis of its solvent tolerance and was identified using 16S
rRNA gene sequencing. Genomic DNA was extracted using pro-
tocol standardized by Asubel et al. [20] The genomic DNA of
DMVR46 was used as template of PCR reaction (30 ␮L) using uni-
ꢀ
ꢀ
versal primers 8F (5 -AGAGTTTGATCCTGGCTCAG-3 ) and 1492R
ꢀ
ꢀ
(5 -GGTTACCTTGTTACGACTT-3 ). The amplification of 16S rRNA
gene was done in BioRad PCR cycler (Biorad iCycler version 4.006,
Biorad, U.S.A.). Each PCR cycle (total 35 cycles) consisted of 1 min
denaturation step at 94 C, followed by 1 min annealing step at 55 C
and 1 min elongation step at 72 C, with an initial denaturation step
at 94 C for 5 min and a final extension step at 72 C for 15 min. PCR
products were resolved on 1.2% low melting agarose gel in 1× TAE
buffer and was visualized with ethidium bromide staining in Gel
Documentation (Alpha-Inotech, U.S.A.). The amplified PCR prod-
◦
◦
◦
◦
◦
[
16]. One of the well explored methods for immobilization in non-
aqueous biocatalysis is encapsulation of enzyme in microemulsion
based organogels (MBGs). The arrangement of MBGs consists
of solid network of gelatin/water rods stabilized by monolayer
of surfactant, in co-existence with a population of conventional
water-in-oil microemulsion droplets. Thus, enzyme immobilized
in MBGs has considerable advantages in organic media as direct
exposure of enzyme to organic solvent is nullified [17,18].
uct was subjected to sequencing by automated DNA Analyzer 3730
using ABI PRISM® BigDye
TM
cycle sequencing kit (Applied Biosys-
tems, USA). The nearly complete sequence (>95%) was submitted
to Genbank at NCBI. BLAST (n) program at NCBI server was used
to identify and download nearest neighbor sequence from BLAST
database [20].
In this paper, we report production, partial purification and
characterization of solvent tolerant lipase produced from solvent
stable Pseudomonas sp. DMVR46 isolated from oil contaminated
soil. There are scanty reports for the production of pentyl valerate
ester immobilized in MBGs from purified lipase. Thus, we intend
to use this lipase for esterification of pentanol and valeric acid
to produce pentyl valerate, a compound with fruity aroma used
in industries. The main purpose for the study was exploitation of
DMVR46 lipase in non-aqueous environment and its reusability.
2.4. Optimization of process parameters for maximum lipase
production
Optimization of process parameters for lipase production from
isolate DMVR46 was aimed to evaluate the effect of a single param-
eter at a time, and later manifesting it as standardized condition
before optimizing the next parameter. The isolate DMVR46 was cul-
tured in 100 mL medium containing 0.5% (m/v) peptone, 0.3% (m/v)
yeast extract and 1% (v/v) tributyrin oil with pH 7.0 and incubated at
◦
◦
◦
◦
◦
different temperature (30 C, 37 C, 40 C, 45 C and 50 C) to deter-
mine the effect of temperature. Similarly, to test the effect of initial
pH the culture was investigated at varying pH ranging from 6.0 to
2
. Materials and methods
◦
◦
2.1. Chemicals
11.0 incubated at 37 C. The standardized temperature 37 C and pH
.0 were used in each step for optimizing the effect of additional
8
DEAE-cellulose and gelatin were procured from Sigma–Aldrich
Germany). Tributyrin oil and bovine serum albumin (BSA) were
obtained from HiMedia (India). All p-nitrophenyl esters were pur-
chased from Sigma–Aldrich (Germany). Sodium bis-2-(ethylhexyl)
sulfosuccinate (AOT), valeric acid, pentanol and pentyl valer-
ate were obtained from Fluka (Switzerland). All other solvents
methanol, butanol, iso-propanol, ethanol, acetone, cyclohexane,
iso-octane, chloroform) used during the experiment were of
HPLC/GC grade.
inducers, nitrogen sources and carbon sources. The effect of differ-
ent inducers for lipase production was studied by addition of cotton
seed oil, soybean oil, sunflower oil, tributyrin oil, maize oil, olive oil
and groundnut oil (each at initial concentration of 1% (v/v). Nitro-
gen sources such as peptone, yeast extract, tryptone, casein, urea,
ammonium sulphate and ammonium nitrate were used to observe
the influence of nitrogen sources (1%, m/v) on lipase production.
To test the influence of additional carbon sources, media were sup-
plemented with dextrose, lactose and sucrose at 1% (m/v). The best
carbon, nitrogen and inducer sources obtained were further tested
for varied concentration of 0.2%, 0.5%, 1%. 1.5% and 2%. All experi-
ments were performed in triplicates and the results shown are the
average of three independent experiments. Data are represented
as mean with standard deviation.
(
(
2
.2. Screening of organic solvent tolerant lipolytic microorganism
Soil samples were collected from various oil spilling sites near
industrial area of Kadi, Ahmedabad, Gujarat, India. Solvent toler-
ance was determined by plate overlay method as described by
Ogino et al. [19]. Five microliters of overnight grown cultures was
transferred to tributyrin agar plates (1% (m/v) tributyrin oil, 0.3%
2
.5. Growth profile for lipase production
A Erlenmeyer flask of 500 mL capacity containing 200 mL of pro-
(
m/v) yeast extract and 0.5% (m/v) peptone extract, 1.5% (m/v)
duction medium (0.3% (m/v) yeast extract, 0.5% (m/v) peptone as
a basal medium and 1.5% (m/v) tryptone, 0.5% (m/v) dextrose and
1
culture of DMVR46 to obtain an initial culture density (A600) 0.05
and incubated at 37 C in an orbital shaker (150 rpm). The samples
agar–agar). The plates were kept for 20 min until the drops get
dry followed by flooding with 18 mL of different solvents like
iso-octane, cyclohexane, toluene, isopropanol, methanol, DMSO.
% (m/v) cotton seed oil) was inoculated with an overnight grown
Colonies were examined for solvent tolerance after incubation at
◦
◦
3
7 C for 24 h. The ability of the cultures to grow and produce lipase
were withdrawn at regular intervals of 24 h and analyzed for cell
growth and enzyme activity. The enzyme activity was estimated
in presence of solvents was observed.
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sp. DMVR46: Partial purification, characterization and application in non-aqueous environment. Process Biochem (2014),
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from supernatant (crude lipase) obtained upon centrifugation and
cell growth was observed by suspending pellet in distilled water.
concentration of 0.3 mg/mL and the lipase activity was measured
according to the pNPP method [22].
2.6. Partial purification of lipase
2.8. Synthesis of pentyl valerate under water restricted
environment using free and immobilized DMVR46 lipase
Solvent tolerant lipase from DMVR46 was purified in two
sequential steps. Step 1: The culture supernatant was obtained by
centrifugation at 10,000 × g (Kubota, Model 6500 Japan) for 20 min
followed by precipitation using acetone as solvent. Chilled acetone
was added slowly to the culture supernatant of strain DMVR46
upto 60% (v/v) concentration with continuous stirring and kept
Pentyl valerate was synthesized by the condensation of pen-
tanol and valeric acid using free as well as immobilized lipase as
described by Dandavate et al. [23]. For immobilization, the par-
tially purified lipase was first entrapped in a surfactant solution
comprising of 0.1 M sodium bis (2-ethylhexyl) sulfosuccinate (AOT)
with Wo = 60 in isooctane by vigorously mixing the two solutions
to create a clear reverse micellar system of AOT/50 mM phosphate
buffer (pH 8.5) – lipase/isooctane (1 mL). The amount of purified
lipase used for immobilization was 108 ␮L having specific activity
of 64.57 U/mg. This system was then polymerized by the addition
◦
at −20 C for O/N to allow protein precipitation. The precipitates
◦
were harvested by centrifugation at 10,000 × g for 20 min at 4 C
and resuspended in 50 mM sodium phosphate buffer (pH 8.0). Step
2
: The enzyme containing solution was applied to a DEAE-cellulose
column previously equilibrated with 50 mM sodium phosphate
buffer (pH 8.0). The flow rate was adjusted to 15 mL/h (approx.)
having the fraction volume of 1 mL and each fraction was fur-
ther assayed for enzyme activity and protein content. The lipase
preparations (crude and purified fractions) were electrophoresed
on SDS-PAGE as described by Laemmli [21]. The protein bands on
the gel were visualized upon silver staining.
◦
of 14% gelatin (1.5 mL) maintained at 55 C followed by vigorous
mixing to obtain homogenous AOT based organogel (MBGs). The
gel was poured into plastic Petri plates, dried overnight, and cut
into small pieces (1 cm × 1 cm with 110 ± 10 ␮m thickness). The
reaction mixture consisted of 20 mL cyclohexane and equimolar
concentrations (100 mM) of pentanol and valeric acid. The ester-
ification reaction was initiated by addition of free lipase and the
pieces of MBGs to the reaction mixture in glass-stoppered flasks
2.7. Characterization of purified lipase
◦
kept on orbital shaker at 37 C and 150 rpm. At every 24 h interval
1
00 ␮L of the reaction mixture was withdrawn and analyzed by gas
2
.7.1. Effect of pH and temperature on lipase activity and
chromatograph.
thermal-stability of lipase
Optimum pH of the solvent tolerant lipase was determined by
measuring the enzyme activity over a pH value ranging from 6.0 to
2.9. Reusability studies
◦
1
0.0 at 37 C for 30 min. Different buffers used for determination
of lipase activity were: pH 6.0–8.5 sodium phosphate buffer, pH
For reusability studies, upon completion of each cycle for ester
9
.0–10.0 glycine–NaOH buffer.
production the immobilized enzymes was recovered by washing
with neat cyclohexane twice for removal of unwanted substrate or
product formed, air dried and reused for next cycle of esterification.
This procedure was repeated for several cycles.
The effect of temperature on the lipase was studied by carrying
out the enzymatic reactions at different temperature in the range of
◦
3
0–50 C at pH 8.5 (50 mM sodium phosphate buffer). The reactions
were carried out for 0–30 min and proceed for enzyme activity. The
thermal-stability of the lipase was assayed at various temperatures
◦
2.10. Analytical procedures
ranging from 30 to 50 C for different time interval from 0 to 4 h. At
each time interval, 1 mL sample was pipette out and then assayed
for residual activity, which was expressed as percentage of initial
activity.
2
.10.1. Lipase assay
The pH stat method: Lipase activity was measured under con-
trolled temperature using a pH 718 STAT Titrino Titrator (Metrohm,
Switzerland). The buffer used for the activity determination com-
prised of 10% (m/v) Gum Arabic, 0.2% (m/v) bile salt, and 20% (m/v)
CaCl , 5% (v/v) cotton oil as substrate and 0.1 M NaOH as the titrant.
The insoluble triglycerides were dispersed by vigorous stirring. One
unit of enzyme activity was defined as the amount of enzyme that
2.7.2. Effects of metal ions, inhibitors and surfactants on the
lipase activity
2
The effects of different metal ions (Ca2+, Mg2+, Fe2+, Co2+, Zn2+
2
+
and Ba ) and inhibitors (EDTA and ␤-mercaptoethanol) were
investigated by pre incubating the purified lipase with 10 mM solu-
◦
liberates 1 ␮mol of fatty acid/min at 37 C [24].
◦
tions of these ions or inhibitors at pH 8.5 for 30 min at 37 C.
In order to check hydrolytic activity of lipase exclusively for
different p-nitrophenyl esters lipase activity was measured with
spectrophotometric method using p-nitrophenyl esters as sub-
strate [22]. The substrate with a final concentration of 0.3 mg/mL
was dissolved in 1 mL of isopropanol and mixed with 9 mL of
50 mM sodium phosphate buffer (pH 8.5) containing gum Arabic
(0.1%) and Triton X-100 (0.6%). The reaction mixture was com-
posed of 240 ␮L of substrate solution and 10 ␮L of appropriately
Similarly, the effects of surfactants (SDS, CTAB, Triton X-100, Tween
2
0 and Tween 80) at the concentration of 0.5% were investigated.
2.7.3. Stability of lipase DMVR46 in different organic solvents
The effects of different organic solvents on the activity and the
stability of the purified lipase were investigated following method
defined by Ogino et al. [19]. The stability of lipase in organic sol-
vents was investigated by appropriately mixing 3 mL of purified
enzyme and 1 mL of solvent in crew cap vials to obtain a final solvent
concentration of 25% (v/v). The solution was incubated in shaker
◦
diluted enzyme solution, and incubated at 37 C for 10 min. The
p-nitrophenol (p-NP) produced in the reaction mixture was quanti-
fied spectrophotometrically at 410 nm. One unit of enzyme activity
was defined as the amount of enzyme that liberated 1 ␮mol p-
nitrophenol/min under standard assay conditions.
◦
(
150 rpm) at 37 C for 0–4 h and lipase activity was assayed in the
aqueous phase.
2.7.4. Substrate specificity to various p-nitrophenyl esters
2.10.2. Protein assay
The substrates, p-nitrophenyl fatty acid esters, of varying chain
length (C2, C4, C8, C12, C16 and C18) were used at the final
Soluble protein was estimated by Lowry’s method [25] using
bovine serum albumin as standard.
Please cite this article in press as: Patel V, et al. An extracellular solvent stable alkaline lipase from Pseudomonas
sp. DMVR46: Partial purification, characterization and application in non-aqueous environment. Process Biochem (2014),
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2
.10.3. Quantification of ester production by gas chromatography
Quantification of accumulated ester was done by using gas chro-
matograph using FID as detector described by Raghavendra et al.
26]. After initiation of esterification, 100 ␮L sample was periodi-
[
cally collected and analyzed by gas chromatograph (Perkin Elmer,
Model Clarus 500, USA) equipped with the flame ionization detec-
tor and a 30 mRtx-R-20 (cross bond 80% dimethyl-20% diphenyl
polysiloxane) capillary column. Nitrogen served as a carrier gas at
a split flow rate of 90 mL/min. The injector and detector tempera-
◦
tures were 250 and 280 C respectively and oven temperature was
◦
◦
programmed to increase from 100 to 160 C at the rate of 20 C/min,
◦
◦
◦
from 160 to 280 C at the rate of 2 C/min and from 165 to 175 C
at the rate of 1 C/min. Ester identification and quantification was
◦
Fig. 1. Phylogenetic tree based on 16S rRNA gene sequence of Pseudomonas sp.
DMVR46. (GenBank accession No. KF636492) and sequence of closest phylogenetic
neighbors obtained by NCBI BLAST (n) analysis, numbers in the parenthesis indi-
cate accession numbers of corresponding sequence. The tree was constructed using
MEGA 5.0 software. Bacillus subtilis strain NR 102783 has been taken as an out-group.
done by comparing the retention time and peak area of the sample
with a standard. Pure pentyl valerate (>98%) was used as external
standard.
3
. Results and discussion
of 79.54 U/mL was observed on 2nd day with cell mass of 16.571
(
including dilution factor) absorbance at 600 nm (assay done using
3
.1. Isolation and identification of solvent tolerant lipase
pH STAT method [24]). On 3rd day decline in lipase activity as well
as growth was observed (Fig. 3). The decrease of lipase production
and growth of the bacterial cells at the later stage could be possibly
due to pH inactivation, proteolysis, or both [28].
producing bacteria
Lipases are inducible enzymes that are produced in the pres-
ence of lipids such as oils, triaclyglycerols, fatty acids and glycerols
in addition to carbon sources [27]. Organic solvents are often lethal
to microorganisms and thus can denature or inactivate enzymes.
Therefore, we assumed that organic solvent tolerant bacteria are
ought in producing enzyme that can tolerate the toxic effect of
organic solvent to a certain degree and will be very useful in indus-
try. In the present study, we have screened solvent tolerant lipase
explored by its ability to grow on tributyrin agar plates flooded with
different solvents. Out of all isolates showing clearance zone on
tributyrin agar plate, the isolate DMVR46 showed the highest lipase
activity (9 U/mL), and hence was used for further studies. Organic
solvent tolerant lipase produced by strain DMVR46 was identified
as Pseudomonas sp. on the basis of 16 S rRNA gene sequencing fol-
lowed by BLAST analysis which showed sequence homology (100%)
with the complete 16S rRNA gene sequence of Pseudomonas sp. The
sequence of strain DMVR46 was deposited in NCBI with an Acces-
sion number KF636492. Phylogenetic affiliation of isolate DMVR46
with related Pseudomonas sp. is shown in Fig. 1.
3
.3. Purification of solvent tolerant lipase DMVR46
The extracellular lipase secreted by DMVR46 was purified by
acetone precipitation and DEAE-cellulose anion-exchange chro-
matography. About 28.95 fold purification with 29.74% recovery
was achieved (Table 1). The purified lipase showed a single band
on SDS-PAGE (Fig. 4). The purified lipase was homogenous and its
molecular mass was estimated to be ∼32.0 kDa.
Cao et al. [8] reported purification of lipase from P. stutzeri
by acetone precipitation with 32.8% recovery. Ogino et al. [19]
reported purify cation of Pseudomonas aeruginosa LST-03 lipase
by ion-exchange and hydrophobic interaction chromatography
achieving 34.7-fold purification and 12.6% yield. Rahman et al. [10]
succeeded in getting higher recovery but they employed affinity
chromatography in combination with ion-exchange chromatogra-
phy. P. aeruginosa PseA lipase was purified by ultrafiltration and
gel exclusion chromatography with 8.6 fold purification and 51.6%
recovery [29]. Meanwhile, Pseudomonas mendocina PK-12CS lipase
was purified to 240 fold with 14.8% recovery using acetone precip-
itation and anion exchange chromatography [30].
3.2. Growth profile for lipase production
Various physico-chemical parameters were scrutinized for max-
imum lipase production. The optimum temperature and pH for
initial lipase activity was found to be 37 C and pH 8.0 (Fig. 2(a) and
3.4. Characterization of purified lipase
◦
(
b)). Cao et al. [8] showed maximum lipase production at pH 8.0
3.4.1. Effect of pH and temperature on activity and stability of the
lipase
◦
and at 30 C from P. stutzeri LC2-8. Among various inducers tested
for lipase production, cotton seed oil (1% (v/v)) with an activity
of 48 U/mL (Fig. 2(c)) was the best inducer followed by soya bean
and maize oil. The best lipase production (75.54 U/mL lipase activ-
ity) was obtained with 1.5% tryptone (Fig. 2(d)) as organic nitrogen
supplement. However, all the inorganic nitrogen sources resulted in
the reduction of bacterial growth as well as lipase production. Also,
as shown in Fig. 2(e) cotton oil used at the concentration of 1% (v/v)
and tryptone with concentration of 1.5% (m/v) stimulated lipase
activity. Using optimized conditions the maximum lipase activity
The optimum pH for purified lipase was found to be 8.5 (Fig. 5)
and it retained 100% of its maximum activity, while remarkable
drop was obtained below pH 8.0 and above pH 9.0 indicating alka-
line nature of enzyme. Gilbert et al. [31] reported P. aeruginosa EF2
lipase with a maximum activity in the range of pH 8.5–9.0.
The optimum temperature for lipase activity was observed to
◦
be 37 C as indicated in Fig. 6(a). Whereas, Cao et al. [8] showed
◦
lipase from P. stutzeri LC2-8 having optimum activity at 30 C and
organic solvent stable P. aeruginosa LST-03 lipase screened by Ogino
Table 1
Purification of lipase produced by Pseudomonas sp. DMVR46.
Purification steps
Total activity (Units)
Total protein (mg)
Specific activity (U/mg)
Fold purification
Yield (%)
Crude enzyme
Acetone precipitation
DEAE-cellulose
3951
2780.4
1175.3
1764.5
112.5
18.2
2.23
24.71
64.57
1
100
70.37
29.74
11.08
28.95
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Fig. 2. Effect of (a) pH, (b) temperature, (c) carbon inducer, (d) nitrogen supplements, (e) different substrate i.e. tryptone (%) and cotton seed oil (%) concentration on lipase
production by DMVR46.
◦
Fig. 3. Growth profile of lipase produced by Pseudomonas sp. DMVR46. The production was carried out at 37 C under shaking condition (150 rpm) in presence of cotton seed
oil with pH 8.
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Fig. 4. SDS-PAGE of lipase DMVR46 at different stages of purification. Lane M:
protein marker; Lane 1: supernatant; Lane 2: lipase concentrated by acetone pre-
cipitation; Lane 3: lipase purified by DEAE-cellulose.
Fig. 6. (a) Temperature profile of purified DMVR46 lipase. The effect of temperature
on lipase activity was studied by carrying out the enzyme reaction at different tem-
◦
perature in the range of 30–50 C at pH 8.5 using sodium phosphate buffer (50 mM).
The reaction was carried out for 4 h. (b) Thermal stability of purified lipase was
measured by incubating the lipase in 50 mM sodium phosphate buffer (pH 8.5) at
◦ ◦ ◦ ◦ ◦
0 C, 37 C, 40 C, 45 C and 50 C for 4 h. Residual lipase activity (%) was calculated
3
relative to the initial activity.
Fig. 5. Effect of pH on activity of lipase obtained from purified Pseudomonas sp.
DMVR46. The enzyme was added to various buffer systems (pH 6.0–10.0) of 50 mM
◦
at 37 C for 30 min. The buffer systems used were pH 6.0–8.5 sodium phosphate
buffer and pH 9.0–10.0 glycine–NaOH buffer.
◦
et al. [19] showed maximum activity at 37 C. The trend for ther-
mal stability of purified lipase on different temperature is shown in
Fig. 6(b). Lipase DMVR46 retained approximately 68% of its initial
◦
activity at 37 C when incubated for 4 h. With increase in temper-
◦
ature, at 40 C enzyme retained only 28% of initial activity, while
◦
at 50 C enzyme was drastically inactivated as shown in Fig. 6(b).
Similar trends of reports were seen in organic solvent-stable LST-03
◦
lipase having maximal activity at 37 C as reported by Ogino et al.
[
19]. Thermal stability profile of PseA purified by Gaur et al. [29] was
◦
found to be stable at 40 C upto 4 h. Sharma et al. [32] reported that
Pseudomonas sp. AG-8 lipase has optimal activity at 45 C. In con-
◦
Fig. 7. Substrate specificity of purified DMVR46 lipase against various fatty acid
esters. The activity of lipase toward different p-nitrophenyl esters were determined
and calculated relative to the maximum activity measured toward p-nitrophenyl
caprylate (taken as 100%). The composition of the reaction mixture was 0.1 mg/mL
of lipase and 0.3 mg/mL of synthetic pNP esters (where C2 = p-nitrophenyl acetate,
C4 = p-nitrophenyl butyrate, C8 = p-nitrophenyl caprylate, C12 = p-nitrophenyl lau-
rate, C16 = p-nitrophenyl palmitate, C18 = p-nitrophenyl sterate).
trast, organic solvent stable LST-03 lipase showed a lower stability
◦
(
below 40 C for 10 min) then DMVR46 lipase.
3.4.2. Substrate specificity
Substrate specificity of lipases may be attributed to alterations
in the geometry and dimensions of their active sites [33]. The sub-
strate specificity of purified lipase was determined by testing the
lipolytic activities against p-nitrophenyl fatty acid esters according
to the spectrophotometric method [22]. The trend for hydrolytic
activity of enzyme toward substrate is evident from Fig. 7. The
result indicates higher hydrolytic activity for long chain substrate
different substrates were found to be (p-nitrophenyl butyrate)
p-NPB 200 ␮M, (p-nitrophenyl caprylate) p-NPC 5 ␮M, (p-
nitrophenyl laurate) p-NPL 100 ␮M, p-NPP 50 ␮M and (p-
nitrophenyl stearate) p-NPS 20 ␮M. Thus, from the results it could
be concluded that i) no interfacial activation was noted with short
and moderate chain substrates, while activation was observed at
point using substrate of long chain (p-NPP) and ii) the lack of inter-
facial activation of lipases could be caused not only by the structural
features of the enzyme but also by very low CMC values of the
substrate [34–36]. Generally, lipases from Pseudomonas sp. prefer
(
p-nitrophenyl palmitate) p-NPP; this can be explained in the terms
of lipase structure. Lipases exist in two forms, closed (inactive) and
open (active) forms. Pseudomonas sp. lipase has been characterized
of lacking interfacial activation and ‘lid’ in contrast to most of other
lipases [34]. The CMC (critical micelle concentration) values for
Please cite this article in press as: Patel V, et al. An extracellular solvent stable alkaline lipase from Pseudomonas
sp. DMVR46: Partial purification, characterization and application in non-aqueous environment. Process Biochem (2014),
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Table 2
Table 3
a
◦
Organic solvent stability of Pseudomonas sp. DMVR46 lipase at pH 8.5 and 37 C.
Effect of various metal ions, inhibitors and surfactants on DMVR46 lipase activity
at pH 8.5 and 37 C.
◦
Organic solvents (25%)
Log P
Relative lipase activity (%)
Metal ions
Relative lipase activity (%)
Control
Methanol
Ethanol
Butanol
Iso-propanol
Toluene
–
100
8.90
30.5
0
40.8
0
45.23
120.41
80.52
78.98
0
b
Control
100
−0.76
−0.24
0.89
0.28
2.64
−0.23
4.7
3.2
3.5
2.0
2+
Ca
135.1
119.4
98.3
7.7
3.6
110.3
40.44
98.89
95.7
2
+
Mg
2
+
Fe
2+
Co
Zn
2+
Acetone
2+
Ba
Iso-octane
Cyclohexane
n-Hexane
Chloroform
EDTA
-Mercaptoethanol
2
PMSF
Iodoacetate
SDS
36.12
80.65
137.5
92.49
96.39
12.5
Note: Data are means of triplicate determinations. The purified enzyme and organic
solvents were mixed in a 3:1 ratio, and the mixture was incubated at 37 C with
shaking at 150 rpm for 4 h and assayed for lipase activity. Activities of DMVR46 in
presence of various organic solvents are shown as a value relative to those in the
absence of organic solvent (control).
*
◦
*
Triton X-100
*
Tween 20
*
Tween 80
*
CTAB
a
Lipase DMVR46 was incubated with various metals, inhibitors (10 mM) and
*
detergents (0.5%) with the superscript in 50 mM sodium phosphate buffer (pH
◦
8
.5) at 37 C for 30 min.
b
achieved in isooctane, cyclohexane and n-hexane with the rela-
tive lipase activity of 120%, 80% and 78% respectively after 4 h.
The activation of lipase could be explained by the interaction of
organic solvents with hydrophobic amino residues present in the
lid that covers the catalytic site of the enzyme, thereby main-
taining the lipase in its open conformation [43]. When organic
solvents such as toluene, butanol and chloroform was added
to purified lipase solution and incubated for 1 h, the enzyme
got drastically inactivated. The possible reason for this may be
the incubation of enzyme in polar solvents (Log P values <3.0),
remove water molecules necessary for its catalytic function which
leads to decrease in activity of enzyme. The results suggested
DMVR46 lipase exhibited fairly good stability, retaining more than
70% of its activity in presence of organic solvents with Log P
value of 3.00 or higher. The reasonably high stability of DMVR46
lipase in organic solvents makes it potentially useful for practi-
cal application in many synthetic reactions in non-conventional
media.
Lipase activity is shown as value relative to the initial activity without addition
of effectors (control).
small or medium chain fatty acids triglycerides or their methyl
esters [19,37,38]. However, lipase from P. aeruginosa AAU2 [39],
Pseudomonas alcaligenes EF2 [31], Pseudomonas sp. S5 [10] and Pseu-
domonas cepacia [33] shows specificity for longer chain fatty acids
substrates.
3.4.3. Effects of metal ions, inhibitors and surfactants on lipase
activity
Among the tested metal ions Ca2+, Mg2+ and Ba2+ significantly
stimulate activity while Zn2+ and Co strongly inhibit lipase activ-
2+
ity (Table 2). Many lipases have been found to display enhanced
2+
2+
activity in the presence of Ca [10,11,29,31]. The presence of Ca
,
2+
2+
Ba and Mg increase lipase activity this may be due to bind-
ing of metal complex to the active site of the enzyme which leads
2+
to the conformational changes in protein [10]. Alternatively, Ca
might complex with fatty acids produced during catalysis elim-
inating the possibility of product inhibition [40]. The chelating
agent EDTA significantly reduce the lipase activity suggesting that
purified lipase may be a metalloenzyme, whereas in presence of
disulphide reducing agent ␤-mercaptoethanol slight reduction in
activity was observed. PMSF, a serine inhibitor has a marginal effect
on DMVR46 lipase at 10 mM concentration exhibiting 95% of rel-
ative lipase activity. This may be attributed to the fact that the
catalytic serine residue is inaccessible owing to the presence of lid
covering the active site which is a characteristic feature for most of
the lipases [8,29,39].
3.5. Kinetic study
The maximum specific activities (Vmax) and Michaelis constants
(Km) for free and immobilized lipase were estimated from dou-
ble reciprocal plots of thee initial rates of pNPP hydrolysis. The
Km of immobilized lipase (0.45 mM) was ∼3-folds lower than that
of free lipase (1.437 mM), while the V
max
of immobilized lipase
(257 ␮mol/mg/min) was ∼9-folds higher than that of free lipase
(25.18 ␮mol/mg/min). The lower K value indicated the higher
m
affinity of enzyme for substrate and higher Vmax value indicates
Surfactants facilitate access of substrate to the enzyme by reduc-
ing the interfacial tension between oil and water increasing the
lipid–water interfacial area where catalytic reactions takes place
higher activity of enzyme. The immobilized enzyme was found to
be 35 times more efficient catalyst for pNPP hydrolysis in compar-
ison to that of free lipase. Immobilization of lipase in organogels
induces some structural changes, which probably is responsible
for enhancing its esterification activity. The changes in parame-
ters suggest that immobilization of lipase resulted in an increased
affinity for the substrate by improving accessibility of the active
site [23].
[
(
41]. Non-ionic detergent Triton X-100 stimulated lipase activity
Table 2). Whereas, Tween 80 has modest influence on lipase activ-
ity by reducing it, while anionic surfactant sodium dodecyl sulphate
SDS) showed 20% decrease in activity. Cationic surfactant (CTAB)
(
completely inactivated the enzyme, as CTAB has been thought to
destroy the conformation of lipase [42].
The effect of temperature on affinity of free and immobilized
enzyme can be seen in Arrhenius plot (Fig. 8) The free and immobi-
lized lipase exhibited a linear relationship in the temperature range
3.4.4. Stability of lipase in organic solvents
◦
of 30–40 C and the corresponding activation energies were calcu-
High activity and stability of lipases in organic solvents is con-
lated to be 9.2 kJ/mol for free lipase and 2.48 kJ/mol for immobilized
lipase. The activation energy of the immobilized lipase is lower
in comparison to that of free lipase which suggests the change in
conformation of enzyme during immobilization.
sidered as novel attributes. Effects of different organic solvents
25%) on the stability of Pseudomonas sp. DMVR46 lipase are
shown in Table 3. The enzyme was found to be quite stable and
active in most of the organic solvents. The highest stability was
(
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sp. DMVR46: Partial purification, characterization and application in non-aqueous environment. Process Biochem (2014),
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Fig. 8. Arrhenius plot for initial hydrolysis rate of pNPP catalyzed by free and
immobilized lipase. Activation energy for free (ꢀ) and immobilized lipase (ꢁ) was
calculated to be 9.2 and 2.48 kJ/mol, respectively.
Fig. 10. Reusability of DMVR46 lipase immobilized into AOT-based organogels. The
reaction was carried out at 37 C and 150 rpm.
◦
high reusability. The MBGs were subjected to reusability examina-
tion for determining the efficiency of immobilization. The enzyme
preparation was given solvent washes after every cycle where the
reaction time for each cycle was 4 days and reused in fresh media
supplemented with substrates. The reusability studies showed that
immobilized enzyme was stable up to 6 cycles of esterification, dur-
ing which no significant decrease in esterification efficiency was
noticed. However, activity started declining slowly after 6th cycle
(Fig. 10). This may be due to accumulation of water formed as a by-
product of reaction. Lipase DMVR46 exhibited 70% of its residual
activity even after 10th run indicating meager loss of activity.
4. Conclusion
In this study, an extracellular lipase from solvent tolerant
Fig. 9. Time course of pentyl valerate production catalyzed by free and immobilized
Pseudomonas sp. DMVR46 was purified following simple purifica-
tion procedure with 29.74% recovery. The molecular mass of the
lipase was found to be ∼32.0 kDa by SDS-PAGE. It exhibited opti-
DMVR46 lipase using pentanol (0.1 M) and valeric acid (0.1 M). The reaction was
◦
carried out in presence of cyclohexane as solvent at 37 C with shaking (150 rpm).
◦
mum activity at pH 8.5 and 37 C. Among various p-nitrophenyl
3.6. Application of purified lipase for ester synthesis under water
esters with different chain lengths, the lipase showed maximum
activity on p-nitrophenyl palmitate (C16). The enzyme exhibited
significant stability in presence of iso-octane and cyclohexane
restricted environment using free and immobilized DMVR46 lipase
The biosynthesis of esters is currently of much commercial
interest as the fatty acid esters synthesized using enzymes offer
better odor and flavor characteristics when compared to those
produced chemically [44]. Lipase from DMVR46 was used for the
synthesis of industrially important ester “pentyl valerate”. Fig. 9
shows the trend of synthesizing ester using free and immobilized
lipase. The partially purified lipase from DMVR46 exhibited signifi-
cant esterification efficiency with 88% ester production after 4 days
of reaction when immobilized into AOT-organogels. While the free
lipase showed only 47% of ester synthesis after 4 days of reaction.
The immobilized enzyme has been more active than crude extract.
This phenomenon can be attributed to the fact that the catalytic
activity of lipase extract is lower than immobilized form due to
the better dispersion of enzyme molecules on the support surface
which improves its activity in organic medium [45]. Moreover, the
immobilization of lipase in MBGs has been reported to improve
the catalytic efficiency of enzyme by providing protection against
inhibitory effect of organic solvents as well as aid in reusability
of lipase facilitating easy recovery by simple filtration [23]. Hence,
DMVR46 lipase may prove to be promising in esterification and
standardization of reaction parameters may increase its efficiency
as a biocatalyst.
and was activated by Ca2+, Ba and Mg but SDS and EDTA
has negative influence on its activity. The partially purified lipase
showed significant esterification activity for synthesis of pentyl
valerate and revealed improved catalytic efficiency upon immo-
bilization in microemulsion based organogels. These results make
solvent-tolerant lipase DMVR46 more potentially valuable for
biotechnological applications in non-aqueous catalysis.
2+
2+
Acknowledgements
The authors are thankful to University Grants Commission
UGC) (grant no. F. 42-167/2013 (SR)) New Delhi, India for financial
assistance. The authors would also extend their acknowledgment
to Dr. Yogesh Souche of NCCS, Pune, India for 16S rDNA sequencing.
(
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