Process Biochemistry 50 (2015) 8696

Contents lists available at ScienceDirect

Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Review

Solvent tolerant lipases: A review

Aliyu Salihu a,b , Md. Zahangir Alam a,
a
Bioenvironmental Engineering Research Centre (BERC), Department of Biotechnology Engineering, Faculty of Engineering,
International Islamic University, Malaysia (IIUM), 50728 Kuala Lumpur, Gombak, Malaysia
b
Department of Biochemistry, Ahmadu Bello University, Zaria, Nigeria

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

Article history: Lipases have proven to be useful in different hydrolytic and synthetic reactions of industrial importance.
Received 28 August 2014 Microbial strains from natural and extreme environments produce lipases with unique characteristics.
Received in revised form 27 October 2014 The ability of lipase to withstand different environmental conditions during reactions, including temper-
Accepted 30 October 2014
ature and pH, is essential. Solvent systems tend to affect lipase-catalyzed reactions, and thus the careful
Available online 6 November 2014
selection of both the medium and the lipase source is necessary. This review considers different solvent
systems used in lipase-catalyzed reactions and some of the enzymatic properties required for function.
Keywords:
Other properties of interest besides enzyme activity include tolerance, stability and compatibility to dif-
Lipase
Organic solvent
ferent reaction media, such as acid, alkaline, salt, organic solvents and other compatible solvents (ionic
Tolerance liquids and detergents). For lipase to be used in a detergent, its thermostability and alkaline tolerance
Alkaline must be well pronounced. In addition, organic solvent stability plays an essential role in employing lipases
Biocatalysis for biodiesel production. Thus, the selection of the lipase for each application is based on specicity and
stability in different solvent systems, which gives lipases many potential applications in aqueous and
non-aqueous biocatalysis.
2014 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
2. Acid tolerant lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
3. Alkaline tolerant lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
4. Salt tolerant lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
5. Organic solvent tolerant lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
6. Lipase compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
6.1. Detergent compatible lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
6.2. Ionic liquid compatible lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
7. Tolerance of lipolytic organisms under different solvent systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
8. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

1. Introduction acylation, and resolution of racemates [1,2]. The boundless appli-

cation potential of lipases is apparent in the production of biofuels,
Lipases are triacylglycerol ester hydrolases (EC 3.1.1.3) that have organic synthetic compounds, detergents, perfumes, cosmetics,
a strong ability to catalyze both hydrolytic and synthetic reac- leather, enantiopure pharmaceuticals, medical diagnostics, foods
tions in nature. These properties allow them to be employed in and feeds [3,4]. The selection of lipase for each of these applications
different biochemical reactions, including esterication, transes- is based on its specicity and stability in different solvent systems.
terication, interesterication, acidolysis, aminolysis, alcoholysis, Microbial sources from fungi, bacteria and archaea produce
lipases with unique features that can be used for biotechnolo-
Corresponding author. Tel.: +60 3 61964571; fax: +60 3 61964442. gical applications. However, microbial lipases are not comparable
E-mail addresses: zahangir@iium.edu.my, zahangir@yahoo.com (Md.Z. Alam). with plant and animal lipases in terms of activity, yield, ease of

http://dx.doi.org/10.1016/j.procbio.2014.10.019
1359-5113/ 2014 Elsevier Ltd. All rights reserved.
 A. Salihu, Md.Z. Alam / Process Biochemistry 50 (2015) 8696
Fig. 1. Application of lipases in different solvent systems. Each line within the main circle represents a particular solvent system, and the number(s) in parenthesis indicate
reference(s) [100110].
purication and molecular modications, and continuous produc-
tion independent of season and stability [5,6]. The environment
where these organisms are isolated occasionally confers some char-
acteristics to the enzymes. Lipases isolated from thermophilic,
halophilic, alkaline and acidic sources tend to be thermostable, salt,
alkaline and acid tolerant, respectively. Bacillus sp. isolated from
soil obtained around edible oil and sh oil processing plants have
acidophilic characteristics, with an optimum pH of 1 [7]. Lipase
from Acinetobacter sp. EH28, which was isolated from oil spill sites,
is thermostable with good tolerance to alkaline and organic sol-
vents [8]. In the case of salt tolerance, Ozcan et al. [9] reported
that archaeal strains have halophilic properties. Thus, lipases are
employed in various processes of industrial relevance, as shown in
Fig. 1.
Lipases with high activity in aqueous media tend to have
lower activities of up to four or ve orders of magnitude in
non-aqueous media. Several factors may contribute to this effect,
including disruption of tertiary structure due to changes in medium
hydrophobicity, limited conformational exibility, desolvation of
the active site resulting in limited enzyme-substrate binding and
interfacial denaturation of the enzyme due to interfacial tension
[10,11].
Lipases with enhanced catalytic potentials are sought for in
different applications. Microbial wild strains often show good char-
acteristics, but using them directly for lipase biocatalytic reactions
is impracticable due to low yield and deactivation. Molecular tech-
niques that involve the cloning and expression of foreign proteins
in appropriate eukaryotic systems have been extensively used
for enhanced lipase catalysis [12]. Other approaches known to
enhance the catalytic efciency of lipases are associated with sol-
vent engineering and molecular imprinting, directed evolution and
mutagenesis [13,14].
This article presents an overview of solvent tolerant lipases by
describing different solvent systems for lipase-catalyzed reactions
87
ranging from acid, alkaline, salt and organic solvents. Each solvent
system has unique features that make lipases versatile in their bio-
catalytic transformations.
2. Acid tolerant lipases
Acid tolerant lipases have the potential to be utilized for
wastewater treatment, leather processing and medical applica-
tions. In the food industry, acidic lipases contribute greatly to the
synthesis of aroma- and avor-containing compounds [15].
Lipase-producing microorganisms undergo several adaptive
processes to circumvent the effect of an acidic environment,
including the selective permeability of the membrane to protons,
fortication of macromolecular structures, production of chaper-
onins and other acid shock proteins such as DnaK, GroEL and HtrA,
changes in membrane fatty acid saturation dominated by monoun-
saturated fatty acids and longer chain fatty acids, and regulation
of genes that contribute to membrane stability, composition and
activity [1618]. Thus, the lipases produced by these organisms
show unique characteristics at low pH.
Enterococcus durans NCIM5427, isolated from sh visceral
waste, produces a lipase that is stable under acidic conditions
from pH 2 to 5, with maximum activity occurring at pH 4.6. This
nding suggests that the source and the nature of the environment
have a great inuence on the characteristics of lipase [19]. Using
babassu oil cake as a renewable substrate, lipase produced by
Penicillium simplicissimum (a wild-type Brazilian strain) has max-
imum activity under acidic pH values. The strain is thermostable,
based on its half-life of more than 5 h at 50 C and pH 5 [2].
Aspergillus oryzae CJLU-31 was isolated from soil containing waste
cooking oil by Zhou et al. [20]; the acid-tolerant lipase produced
by this strain had maximum activity at pH 4 and retained more
than 80% of its activity at lower pH values. Additionally, lipase
from Kluyveromyces marxianus was stable under acidic conditions,
88 A. Salihu, Md.Z. Alam / Process Biochemistry 50 (2015) 8696
retaining more than 85% of its activity after a 24-h incubation.
Maximum activity, as well as 100% retention in activity, appeared
at pH 4. A marked reduction in enzyme activity was observed at
alkaline pH values, with complete deactivation at pH values above
8 after 24 h [21].
Thermostable lipases from Bacillus stearothermophilus SB-1 and
Bacillus licheniformis SB-3 isolated from compost heaps, soils and oil
industry efuents are acid tolerant. The two lipases isolated from
these strains (SB-1 and SB-3) retained 70% and 50% of their activity,
respectively, when incubated in an acidic medium of pH 3 at 50 C
[22].
Ramani et al. [23] showed that Pseudomonas gessardii isolated
from slaughterhouse waste containing beef tallow produced an
acidic lipase. The maximum activity of 139 U/ml was observed at
pH 5 and 37 C. P. gessardii lipase immobilized on functionalized
mesoporous activated carbon showed 100% and 65% hydrolytic ef-
ciencies on waste cooking oil after 21 and 45 cycles, respectively
[24]. Similarly, Bacillus pumilus was found to produce an acid toler-
ant and thermostable lipase with a maximum activity of 1100 U/ml
at 50 C, pH 1, after a 96-h incubation. The lipase showed a 96%
hydrolytic conversion of palm oil containing waste water after 3 h
(pH 1 and 50 C) [7].
Solid-state fermentation of wheat bran containing olive oil
using Aspergillus niger NCIM 1207 for lipase production showed
the highest lipolytic activity at pH 2.5, with a signicant reduc-
tions occurring above pH 3.5. The enzyme retained more than
75% and 90% of its original activity at the extremely acidic pH
values of 1.5 and 2.0, respectively, and more than 70% activity
was retained at pH 2.5 for 24 h [25]. Mhetras et al. [26] explored
the potential of this lipase for isoamyl acetate (avor) synthe-
sis using acetic acid and amyl alcohol at a molar ratio of 1.6:1.
An esterication yield of approximately 99% was achieved after
96 h, with a maximum isoamyl acetate production of 80 g/l. This
efciency makes acidic lipase from A. niger NCIM 1207, the bio-
catalyst of choice for the synthesis of several avor-containing
compounds.
Chiral synthesis of (S)-ketoprofen ester (a non-steroidal anti-
inammatory drug clinically utilized in the racemate form) was
achieved using Candida rugosa lipase (Lipase OF). This enzyme had
its highest hydrolytic activity at pH 4 and enantioselective synthesis
at pH 2.2. However, only 40% and 35% of residual activities were
observed at pH 4 and 2.2, respectively, after the incubation of C.
rugosa lipase at 30 C for 50 h [27]. Wu et al. [28] reported a two-
fold increase in enantioselectivity of Candida antarctica lipase for
the hydrolysis of ketoprofen ethyl ester under acidic medium at
pH 1 compared with neutral pH.
Thus, acid tolerant lipases can be obtained from different
microorganisms, and based on the literature; none of these orga-
nisms were non-GRAS (generally regarded as safe). As such, their
use in different industrial applications poses no threat to human
health.
3. Alkaline tolerant lipases
Microorganisms producing alkaline tolerant lipases have been
isolated from soil, water and several alkaline environments. Lipases
that are alkaline tolerant are highly desirable in detergent formu-
lations because enzyme-based detergents possess higher cleaning
ability than do chemical-based detergents. Other applications
include avor synthesis and bioremediation, as well as food and
feed formulations.
Alkaline lipases with additional properties such as organic sol-
vent tolerance and thermostability are preferable and can be used in
many biocatalytic reactions. Some multifunctional alkaline lipases
are shown in Table 1.
Thus, the physiological and metabolic adaptations of microor-
ganisms aid in their survival and the production of enzymes with
desirable properties. These adaptive characteristics include the
maintenance of homeostasis, which depends on the permeability of
the membrane to ions, the buffering capacity of the cytoplasm, and
the energy-dependent transport of cations (Na+ , K+ and H+ ) across
the membrane [39,40]; the modication of membrane phospho-
lipids and the isomerization of unsaturated fatty acids from cis to
trans [41]; and the induction of specic stress proteins in some alka-
line tolerant lipase-producing microorganisms, including Bacillus
subtilis. Similarly, the expression of stress proteins controlled by
sigma factor ( W ) described by Lonetto et al. [42] as part of the
extracytoplasmic function (ECF) subfamily are associated with the
regulation of gene expression in different alkaline stable microbial
species [43].
In the search for novel sources of lipases with broad spectrum
activity, Romdhane et al. [44] identied Talaromyces thermophilus,
which was isolated from soil samples from the El Hamma ther-
mal station. Lipase production by T. thermophilus was studied using
different carbon sources and wheat bran as a renewable residue,
with the highest lipolytic activity of 60 U/ml. The lipase was sta-
ble in an alkaline pH range of 911 after a 24-h incubation with
an optimum pH of 9.5. In addition, thermostability studies indi-
cated that the enzyme retained its activity over a range of 5060 C.
The T. thermophilus lipase has comparable properties to Lipolase
(a lipase used in detergent formulations) in terms of tolerance to
different surfactants, oxidizing agents and commercial detergent
preparations [44]. Thus, lipase from T. thermophilus has excellent
characteristics for detergent formulations.
Of the 354 alkaline lipase-producing microorganisms isolated
from bay soil samples of Bohai, a lipase from Burkholderia cepacia
LP08 was stable toward various surfactants, oxidizing and bleach-
ing agents, and commercial detergent formulations. Its optimum
activity was observed under alkaline conditions [45].
Yoo et al. [29] reported that a lipase from Ralstonia sp. CS274 has
an optimum pH of 89.5. The puried lipase was tolerant to ionic
and non-ionic surfactants, metal ions and organic solvents, which
conrms its industrial potential, particularly for non-aqueous bio-
catalysis. Staphylococcus aureus strain PP1 was identied among
the dominant strains in soil samples in the groundnut rhizosphere.
The organism produces an alkali-thermostable lipase with an opti-
mum pH of 8. A broad range of stability and activity were observed
between pH 5 and pH 11, and maximum stability was recorded
at pH 9, where the enzyme retained its full activity after 20 h of
incubation at 37 C. However, at pH 8, which appeared to be the
optimum for the enzyme activity, a 90% retention in activity after
20 h of incubation at 37 C was obtained [31].
A new approach using metagenomics was explored by Peng
et al. [46] to analyze the marine environment for alkaline tolerant
lipases. The lipolytic clone Est p6 identied from the metagenomic
library has an optimum pH of 8.6 and >90% of its optimum activity
within a pH range of 810. More than 70% of residual activity was
retained after a 3-day incubation within a pH range of 811 and
maximum stability with 100% retention of enzyme activity was
observed at pH 9.8. Treatment of lipase Est p6 to enhance milk
fat avor resulted in the formation of a special avor associated
with myristic and palmitic acids without affecting the cheesy a-
vor of short chain fatty acids. Based on these results, Peng et al. [46]
concluded that the alkaline nature of lipase Est p6 coupled with
its thermostability imparts this enzyme with application poten-
tial in formulating a number of cleaning products, whereas its
specicity toward myristate and palmitate during milk fat hydrol-
ysis makes it desirable in milk fat avor production. The lipase
produced by Bacillus coagulans BTS-3 was alkaliphilic and ther-
mophilic, exhibiting its highest activity at 55 C and pH 8.5. The
stability of this lipase was apparent within a pH range of 810.5,
 A. Salihu, Md.Z. Alam / Process Biochemistry 50 (2015) 8696 89

Table 1
Some of the multifunctional alkaline lipases reported in the literature.

Organism Source pH optimum and Temperature optimum Organic solvent tolerance Reference
stability and thermostability

Acinetobacter Oil rich soil pH 10 50 C After 1 h of incubation: [8]

sp. EH28 stable at pH 9 (100%
activity for 24 h) and
pH 10 (97% activity for
24 h)
stable at 50 C (100%
activity) for 1 h 30 min
100% and 130% activities in 15 and
30 mM DMSO, respectively
85% activity in 15 mM methanol
96% activity each in 15 mM ethanol
and isopropyl alcohol
102% and 104% activities in 15 mM
n-Hexane and acetone, respectively

Ralstonia sp. Soil isolates pH 8.09.5 5055 C After 24 h of incubation: [29]

CS274 Highly stable within
the optimum range and
80% activity
at pH 12 after 24 h
50, 15 and 10 min, as
half-lives for 60 C,
70 C and 80 C,
respectively
60.8% activity each in methanol and
n-Hexane (25%, v/v)
82.5% activity in DMSO (25%, v/v)
47% activity in ethanol (25%, v/v)
59.8% activity in acetone (25%, v/v)

Bacillus Mangrove pH 8.0 45 C After 30 min of incubation: [30]

licheniformis ecosystem 67.9% and 22.6% 82 min and 48 min as 62% activity in isopropanol (1:1, v/v)
MTCC 6824 activities at pH 9 and half-lives for 45 C and 71% activity in methanol (1:1, v/v)
10, respectively, after 55 C, respectively <50% activity in ethanol (1:1, v/v)
20 min
Staphylococcus Arachis pH 8 Broad range (3060 C) After 1 h 30 min of incubation: [31]
aureus hypogaea stable at pH 9 (100% 65.3% and 62.6% 80% activity with ethanol (30%, v/v),
rhizosphere activity from 520 h) activities for 60 C and DMSO (30%, v/v), methanol (30%, v/v),
soil samples and pH 8 (90% activity 70 C, respectively, isopropanol (50%, v/v), toluene (30%,
from 520 h) after 1 h v/v) and ethylene glycol (50%, v/v)

Bacillus subtilis Hot mineral pH 8 70 C After 30 min of incubation: [32]

DR8806 spring >90% activity at pH half-life of 2 hr 25 min 90% activity with methanol (20%,
range of 8 to 10 after at 70 C; 90% and 87.5% v/v), ethanol (20%, v/v), hexane (20%,
1h activities for 75 C and v/v), acetone (20%, v/v), butanol (20%,
80 C, respectively, v/v), diethyl alcohol (20%, v/v), isoamyl
after a 1-h incubation alcohol (20%, v/v), isopropanol (20%,
v/v) and heptane (20%, v/v)

Bacillus ther- El Carrizal hot pH 910 60 C After 1 h of incubation: [33]

moleovorans springs 80% activity at pH 100% activity at 90% activity with methanol (70%,
CCR11 range (511) after a 3040 C, 75% at v/v), ethanol (70%, v/v), 2-propanol
26-h incubation 5060 C after a 1-h (70%, v/v) and acetone (70%, v/v)
incubation

Bacillus Bulgarian hot pH 7.59.0 7580 C After 30 min of incubation: [34]

stearother- spring 100% stable at pH Half life of 3 h at 70 C 80% activity with methanol (5 mM) and
mophilus MC range of 711 after 40% activity with isopropanol (5 mM),
7 30 min of incubation glycerol (5 mM) and acetone (5 mM)

Burkholderia Dining hall soil pH 8.59.0 6570 C After 6 h of incubation: [35]

cepacia S31 samples 100% stable at pH 80% activity at 70 C
range of 89 for 12 h after incubation for 1 h,
96% and 85% activity
after incubation for
12 h at 50 C and 55 C,
respectively
100% activity with methanol (25%,
v/v), glycerol (25%, v/v), n-Hexane
(25%, v/v), butanol (25%, v/v), toluene
(25%, v/v) and ethyl acetate (25%, v/v)

Streptomyces Soil samples pH 8 30 C After 48 h of incubation: [36]

sp. CS268 100% stable at pH 100% stable up to 55 C 100% activity with acetone (25%, v/v),
range of 49 for 1 h for 2 h benzene (25%, v/v), toluene (25%, v/v),
octane (25%, v/v) and hexane (25%, v/v)

Pseudomonas Putrid cutting pH 11 70 C After 24 h of incubation: [37]

aeruginosa oil 80% activity at pH After 15 min of 90% activity with ethanol (25%, v/v),
san-ai range (610) incubation, at 70 C and acetone (25%, v/v), chloroform (25%
after a 3-h incubation 60 C only 30% and 75% v/v) and n-Hexane (25%, v/v)
of the activity
remained

Pseudomonas Soil samples pH 9 45 C After 24 h of incubation: [38]

sp. strain S5 86 and 64% activities at 86% and 52% activity at 100% activity with pentanol (25%,
pH 8.0 and 7.0, 37 and 50 C, v/v), chloroform (25% v/v),
respectively, after 30 of respectively, after cyclohexane (25%, v/v), octanol (25%,
min incubation 30 min of incubation v/v) and n-dodecane (25%, v/v)

and half-lives of 2 h and 30 min were observed at 55 C and 60 C, but was enhanced in the presence of different concentrations of
respectively [47]. methanol (from 5 to 25%, v/v). Additional experiments revealed that
The optimum activity of alkaline lipase from Streptomyces sp. the enzyme catalyzes a transesterication reaction of soybean oil
CS268 was recorded at pH 8. The lipase activity was not only stable with methanol, and complete bioconversion was achieved in 12 h
90 A. Salihu, Md.Z. Alam / Process Biochemistry 50 (2015) 8696
[36]. Thus, alkaline stability and organic solvent tolerance of the
lipase from Streptomyces sp. CS268 show its potential to be utilized
in various applications.
4. Salt tolerant lipases
Lipases with good stability at extreme conditions offer signi-
cant contributions and are suitable for harsh industrial processes.
Archaea, bacteria and fungi exist in diverse environments that were
thought to be too harsh for survival [48]. The enzymes produced by
these organisms can be utilized in a number of industrial appli-
cations, including the food industry for avor formation and the
production PUFA-enriched oils, and where some unique character-
istics are required, such as biodiesel production and the synthesis
of ne chemicals.
The isolation of a lipase-producing bacterium from Maharla salt
lake was carried out using culture-based and titrimetric methods.
Based on molecular and morphological characterizations, Bacillus
vallismortis BCCS 007 was identied as the highest lipase-producing
strain among the isolates. The organism was moderately halophilic,
requiring NaCl concentrations of 110% for growth [49]. Generally,
halophilic microorganisms can be grouped based on their salt tol-
erance into four groups as described by Larsen [50], where those
requiring 1% NaCl for their growth and metabolism are consid-
ered non-halophilic; slightly halophilic and moderately halophilic
are stimulated in the presence of 23% and 510% NaCl, respec-
tively; and a higher than 10% NaCl requirement indicates extreme
halophilia.
Halophilic isolates from soil of Yuncheng salt lake were identi-
ed as Idiomarina sp. W33, with an optimal lipase activity at 60 C,
pH 79 and 10% NaCl. The unique characteristics of lipase pro-
duced by this strain in terms of thermostability, alkaline, organic
solvent and salt tolerance in combination with its specicity toward
long-chain substrates make it appropriate for enzyme-catalyzed
transesterication reactions for biodiesel production. The lipase
was used in free and Celite immobilized forms for the transesteri-
cation of Jatropha oil, and the maximum yield of fatty acid methyl
ester was 84% and 91%, respectively [51].
The lipase produced by Bacillus sp. VITL8 isolated from a
hydrocarbon-contaminated site retained >55% of its activity at
60 C, pH 10 and 3 M NaCl concentration. The lipase was stable
in different organic solvents, with 25% and 20% improvements in
enzyme activity in 25% (v/v) of acetonitrile and butanol, respec-
tively. The hydrolytic specicity of the enzyme was toward long
chain fatty acids (olive oil and sunower oil). Thus, the halotoler-
ant lipase produced by Bacillus sp. VITL8 could be utilized in various
green technological processes of industrial importance [52].
A salt tolerant strain of the genus Haloarcula, G41, with lipoly-
tic potential was isolated from soil samples of Yuncheng Salt Lake.
Media containing 20% NaCl or 15% Na2 SO4 enhanced lipase pro-
duction with an optimum activity at 70 C, pH 8, and 15% NaCl [53].
In addition to these properties, lipase from Haloarcula sp. catalyzed
the transesterication of soybean oil with methanol in the presence
of tert-butanol; the biodiesel yield was >80% [53].
The lipase expressed from a metagenomic library of Haliclona
simulans had high specicity (KM of 1.093 mM1 and Vmax of
50 mol min1 ) toward long chain triglycerides. Maximum lipase
activity was observed at 40 C, pH 7, in a medium containing 5 M
NaCl. Thus, the halophilic nature of H. simulans coupled with its
specicity and solvent tolerance give it additional advantages for
utilization in various applications [54].
Ozcan et al. [9] carried out extensive screening studies of 118
archaeal strains isolated from different halophilic environments in
Turkey for lipolytic enzyme production. Five of the 18 isolates that
tested positive were coded as A43, B53, E7, A138 and B49. The ve
strains showed increasing lipase activities in the presence of NaCl
up to 4 M, with an optimum temperature of 6065 C and pH 88.5.
A lipase from Marinobacter lipolyticus SM19 showed potential
application in the enrichment and synthesis of unsaturated fatty
acids. An improvement of approximately 45% in the eicosapen-
taenoic acid content of sh oil was obtained when incubated with
the enzyme. Other properties of interest include its activity on acyl
substrates of short-to-medium chain length, thermostability with
a temperature optimum of 80 C and stability in various organic
solvents [55].
Thus, lipases produced from salt tolerant organisms have advan-
tages, particularly with regard to their thermal stability and organic
solvent tolerance, in addition to their halophilic characteristics
and are equally applicable in industrial processes requiring these
unique characteristics.
5. Organic solvent tolerant lipases
Recent advances in biosynthetic applications require non-
aqueous environments. Several advantages that outweighed the
disadvantages have been reported for biocatalysis in organic sol-
vents, including the high dissolution of hydrophobic compounds,
shift of thermodynamic equilibrium toward synthesis rather than
hydrolysis, minimum water-dependent unwanted reactions, less
or an absence of contamination by microorganisms, modication
of enzyme specicity to particular substrates, ease of recovery and
reusability of enzymes, possible enhancement of thermostability
and feasibility of catalyzing reactions that are impracticable in
aqueous media [10,11].
Several lipases with high stability in organic solvents have been
reported. Some researchers have explored this unique property by
determining their practical applications, as shown in Table 2.
Among the lipase producing organisms isolated from Jatropha
seed cakes by Bose and Keharia [56], Pseudomonas aeruginosa AAU2
produces a lipase that is stable in organic solvents with a log P of
3.1 and it retains more than 70% of its activity after a 24-h incu-
bation. Generally, organic solvents with a high log P are preferably
used in lipase-catalyzed reactions in non-aqueous medium because
of their little or no effect on stripping the essential water from lipase
molecules. Enhanced P. aeruginosa AAU2 lipase activity (102%) was
observed in the presence of a higher concentration of iso-octane
(75%, v/v), which was suggested to be a result of conformational
changes associated with the interaction of amino acid residues,
specically hydrophobic ones, present in the lid that covers the
enzymes active site, causing the enzyme to exist in an open con-
formation [59]. Thermostable lipase from Pseudomonas uorescens
P21 was also found to be moderately stable after a 2-h incubation
in non-polar solvents, including heptane, hexane and styrene, with
a residual activity of 91.4%, 94.1% and 83.5%, respectively [60].
Mander et al. [61] studied the organic solvent tolerance of lipase
from Streptomyces sp. CS133. An approximately two-fold enhance-
ment in lipase activity was observed in organic solvents with
log P 0.87. Thus, an increase in residual activities was apparent in
n-Hexane (log P 3.5) and octane (log P 4.9) after a 48-h incubation
at 30 C and pH 8. Non-polar solvents tend to have a stabiliz-
ing effect compared to their polar counterparts. The enhancement
of Streptomyces sp. CS133 lipase activity in organic solvents can
be ranked as n-Hexane (log P 3.5) > octane (log P 4.9) > benzene
(log P 2.0) > heptane (log P 4.0) > toluene (log P 2.5) > xylene (log P
3.1) > dichloromethane (log P 1.25) > diethylether (log P 0.87). Inter-
estingly, a methanol concentration range of 530% (v/v) resulted in
enhanced lipase activity (up to 15%) after a 24-h incubation, and a
more than two-thirds retention of enzyme activity was observed
after 48 h. The lipase-catalyzed transesterication of soybean oil or
olive oil with methanol for biodiesel production was achieved, and
 A. Salihu, Md.Z. Alam / Process Biochemistry 50 (2015) 8696 91

Table 2
Some organic solvent tolerant lipases and their applications.

Organism Stability in organic solvents Incubation Application Reference

period (h)
Stimulatory effect Mild inhibitory Inhibitory effect (<90%
(100% relative effect (90% relative activity)
activity) relative activity)

Acinetobacter Dimethylsulfoxide Ethanol (15 mM, Methanol (15 mM, 1 Synthesis of ethyl caprylate (fruity-owery [8]
sp. EH28 (15 mM, 30 mM), 30 mM), isopropyl 30 mM), DMF (30 mM), avor) based on esterication of 1.3:1 of caprylic
n-Hexane (15 mM, alcohol (15 mM), isopropyl alcohol acid and ethanol in cyclohexane medium
30 mM), acetone DMF (15 mM) (30 mM) 63% and 90% conversion to ester of ethyl
(15 mM, 30 mM) caprylate was obtained by free lipase and
immobilized lipase, respectively

Ralstonia sp. Methanol (515%, 25% (v/v) of glycerol, 24 Lipase produced by Ralstonia sp. CS274 catalyzed [29]
CS274 v/v) DMSO, methanol, biodiesel production of soybean oil or palm oil
ethanol, acetone, with methanol
n-Hexane Maximum conversion was achieved after 12 h
and 36 h for palm oil and soybean oil,
respectively; using pH 8.0, 5% methanol and 20%
water content

Streptomyces 25% (v/v) of 25% (v/v) of methanol, 48 Lipase produced by Streptomyces sp. CS268 [36]
sp. CS268 acetone, benzene, ethanol, DMSO, catalyzed transesterication of soybean oil or
n-Hexane, toluene, acetonitrile olive oil with methanol for biodiesel production
octane 24 h is the critical bioconversion limit for
transesterication of soybean oil and olive oil

Haloarcula sp. 35% (v/v)* of 35% (v/v)** of DMSO, <120* Transesterication was carried out in a mixture [53]
G41 glycerol, n-Hexane methanol, ethanol, <48** of soybean oil (6 mmol, 5.4 g), methanol
acetone, tert-butanol, (24 mmol, 960 l), and water (500 l) in
chloroform, benzene tert-butanol (6 ml)
Maximum yield of Biodiesel was 80.5% and 89.2%
for free and immobilized Haloarcula sp. G41
lipase, respectively

Idiomarina sp. 50% (v/v) of 50% (v/v) of 50% (v/v) of DMSO, >240 Biodiesel production was carried out using 6.7 g [51]
W33 n-Hexane glycerol, methanol, ethanol, 120 Jatropha oil and 0.54 g methanol (molar ratio of
cyclohexane tert-butanol, 48 methanol to oil, 4:1; and water content, 0.7%
acetone (w/w)) at 60 C with 180 rpm orbital shaking
84% and 91% biodiesel yields were achieved by
free and immobilized Idiomarina sp. W33 lipase,
respectively.

Pseudomonas 50% (v/v) of 50% (v/v) 50% (v/v) of n-Heptane, 24 P. aeruginosa AAU2 lipase catalyzed biodiesel [56]
aeruginosa iso-octance, xylene cyclohexane n-Hexane, toluene, production using Jatropha oil and methanol
AAU2 benzene, chloroform, (1:4 M ratio) led to maximum yield of 4050%.
dichloromethane, Stepwise addition of methanol at 0, 12, 24 and
methanol 48 h under the same conditions enhanced the
biodiesel yield to 90% after 72 h

Phorbol ester, the toxic component present in

Jatropha seed cake, which makes it unsuitable as
animal feed, was hydrolyzed using P. aeruginosa
AAU2 lipase. The lipase showed 86.49%
degradation of Phorbol 12-myristate 13-acetate
after 12 h incubation as analyzed by HPLC

Pseudomonas 25% (v/v) of 25% (v/v) of 25% (v/v) of methanol, 48 The reaction medium for lipase catalyzed [57]
aeruginosa LX1 n-Hexadecane, n-Hexane, DMF, ethanol, tert-butanol, biodiesel production contains soybean oil
iso-octane, glycerol DMSO acetone, acetonitrile (5 mmol, 4.5 g), methanol (15 mmol, 600 L) and
water (0.27 g) dissolved in tert-butanol (5 ml);
the reaction was allowed to proceed for 72 h
Maximum biodiesel production of 71.5% and 80%
was achieved by free and immobilized lipase,
respectively

Pseudomonas 25% (v/v) of 25% (v/v) of 25% (v/v) of nonane, 24 Kinetic resolution of (R, S)-1-phenylethanol was [58]
stutzeri LC2-8 isopropanol, n-Heptane iso-octane, n-Octane achieved based on the esterication of
acetone, methanol, 1-phenylethanol in the presence of P. stutzeri
ethanol, DMF, LC2-8 lipase in n-Hexane using vinyl-acetate as
DMSO an acyl donor. After 24 h of reaction time (30 C,
200 rpm), the racemic mixtures were resolved
with 99.9% enantiomeric excess of
(S)-1-phenylethanol with an E-value of 200.
(R, S)-1-phenylethanol is used in the cosmetics
and pharmaceutical industries
*, **, , ,
indicate different incubation periods for organic solvent tolerance.
92 A. Salihu, Md.Z. Alam / Process Biochemistry 50 (2015) 8696

Table 3
Mechanism of solvent tolerance in various lipolytic organisms.

Microorganism Possible mechanism [16,97,98,41,99]

Lipase producing gram a. Changes in cell morphology and membrane surface hydrophobicity
negative bacteria (e.g.,
Acinetobacter sp., Burkholderia b. Homeoviscous adaptation
sp., Pseudomonas sp.) Changes associated with isomerization (cis unsaturated fatty acids to trans unsaturated fatty acids, catalyzed
by cistrans isomerase), saturated-to-unsaturated fatty acid ratio, length of the acyl-chains, increased levels of
PlsX (fatty acid/phospholipid biosynthesis) and FabF [3-oxoacyl-(acylcarrier-protein) synthase II]

c. Expression of stress response genes (e.g., groES, groL, grpE, dnaK, rpoH)

d. Expression of the cfa (cyclopropane fatty acids) synthase gene

e. Lon ATP-dependent protease synthesis enhances extracellular encapsulation by polysaccharide and

decreased permeability

f. Solvent emulsifying/deactivating/solubilizing enzymes/substances

g. Putative efux pump (HAE1)

Lipase producing gram positive a. Filamentation minimizes the exposure of cell surface to environmental stressing agents
bacteria (Bacillus sp.,
Staphylococcus sp., b. Homeoviscous adaptation
Streptomyces sp.)
c. Alteration of membrane composition

d. General stress regulon activation (sigma regulon and Hsp33 stress protein)

e. Sporulation by formation of endospores

f. Solvent emulsifying/deactivating/solubilizing enzymes

g. Putative efux pump (HAE2)

maximum production was reached after 24 h and 36 h for soybean
oil and olive oil, respectively [61].
Bacillus sphaericus MTCC 7542 produces an organic solvent tol-
erant lipase that is stable in both polar and non-polar organic
solvents, with a residual activity ranging from 80 to 95% after a
12-h incubation. This result suggests that the lipase could be used
efciently for biodiesel production via enzyme-catalyzed transes-
terication [62]. Additionally, B. subtilis DS9 isolated from marine
mud samples was found to grow in the presence of 10% (v/v) of
hydrophobic organic solvent with log P 2.5. The lipase synthe-
sized by this strain was not only stable but was enhanced in the
presence of 25% (v/v) xylene, heptane, n-Hexane, isooctane and
n-Decane [63].
A recombinant lipase from B. cepacia strain G63 had a maxi-
mum lipase activity of 863 U/ml and was stable in the presence
of 50% (v/v) methanol, ethanol, tert-butyl alcohol and glycerol for
48 h. When the enzyme was tested for biodiesel production using
soybean oil and methanol, an 88% conversion into biodiesel was
achieved after 72 h [64].
Similarly, Ebrahimpour et al. [65] showed that a recombinant
lipase from Geobacillus sp. strain ARM had a 1623-fold higher
enzyme activity than did the wild-type. The characterization of this
lipase revealed that it is thermostable, organic solvent tolerant and
1,3-regiospecic with respect to triolein.
The lipase-producing yeast Rhodotorula mucilagenosa P11I89 is
tolerant to methanol. Methanol is commonly used as the acyl accep-
tor in esterication and transesterication reactions but is often
inhibitory to lipases [66]; thus, R. mucilagenosa P11I89 lipase was
used for production of fatty acid methyl ester (FAME) using differ-
ent molar ratios of palm oil and methanol (1:3, 1:4, 1:5, 1:6). It was
determined that a higher molar ratio of methanol results in higher
FAME yields [67].
A cold-active lipase produced by Stenotrophomonas maltophilia
CGMCC 4254, as reported by Li et al. [68], retained its activity
when incubated in 20% and 50% (v/v) non-polar organic solvents
(log P 2) for 7 days. However, only 94% of the original activity
was retained in less polar organic solvents (log P < 0) at a 20% (v/v)
concentration after 24 h. Incubating the lipase with organic sol-
vents at absolute concentration (100%) revealed a >50% retention
in the original activity for all of the solvents tested. Cavicchi-
oli et al. [69] reported that reactions catalyzed by cold stable
enzymes tend to be stereospecic because most undesirable reac-
tions occur at elevated temperatures. The potential of cold stable
lipases in various synthetic reactions requiring mixed aqueous-
organic or non-aqueous solvents could be linked to their exibility
in counteracting the effect of low water activity in various solvent
systems.
Rational design approaches are effective in enhancing the lipase
stability in organic solvents, as reported by Park et al. [70]. Using
data obtained from directed evolution, the stability of lipase in polar
organic solvents can be increased by identifying the target amino
acid residues that interact with the surrounding medium and those
residues that do not participate in the formation of secondary struc-
tures and by introducing some hydrophilic amino acids (such as
Asp, Gln, Glu, Ser, Asn) that increase the chance of hydrogen bond
formation [71]. A computer-based in silico method of mutating Asn
to Gln and Asp to Glu resulted in an increased formation of hydro-
gen bonds of varying length. The mutation sites (D223E, D265E,
N97Q, N264Q, and N292Q) of mutant C. antarctica B lipase led to an
improved stability in 80% methanol compared with the wild-type
strain [70].
Kawata and Ogino [72] used site-directed mutagenesis to iden-
tify the specic mutation sites contributing to the organic solvent
tolerance of P. aeruginosa LST-03 lipase. Single mutations of S155L,
G157R, S164K, S194R and D209N of the LST-03 lipase gene resulted
in an increase in the number of hydrogen bonds and the for-
mation of salt bridges, as well as increased packing of the inner
hydrophobic nucleus, which collectively contributed in a several-
fold increment in organic solvent stability observed in the mutated
lipase.
 A. Salihu, Md.Z. Alam / Process Biochemistry 50 (2015) 8696
Lipase from recombinant P. uorescens JCM5963 was stable in
various organic solvents at 30% (v/v) concentration after a 24-
h incubation. Interestingly, a stimulatory effect was observed in
all hydrophilic organic solvents, including ethanol, methanol, iso-
propanol, glycerol and acetone [73]. In the case of recombinant
Bacillus sphaericus 205y lipase, the activity was not just stable but
enhanced in the presence of methanol, DMSO, p-xylene and n-
Decane, with a slight decrease in residual activity in ethanol (96%)
[74]. Yang et al. [75] examined the structureorganic solvent rela-
tionship of recombinant C. antarctica lipase A. The residual activity
associated with hydrolytic and esterication potential of this lipase
was more pronounced in the presence of acetonitrile followed by
acetone. This was attributed to alteration of the secondary con-
formation of the enzyme, where treatment with acetonitrile or
acetone resulted in increased -helixes with decreased random coil
conformation.
6. Lipase compatibility
The interest in different applications of lipases has motivated
researchers to investigate the stability and activity of lipase-
catalyzed reactions under unconventional conditions. Those most
notable were the compatibility of lipases in detergency and in ionic
liquids, which are of considerable importance.
6.1. Detergent compatible lipases
Enzyme-based detergents have been developed with supe-
rior cleaning properties compared with synthetic detergents [76].
The main constituents of detergents include surfactants, builders,
bleaching agents, optical brighteners, corrosion inhibitors, foam
regulators and enzymes [77]. Lipase and protease are the two
enzymes used in the formulations. The lipases aid in removing
oily stains and sebum from clothes that are not easily removed by
conventional washing processes.
Some of the commercially available lipases used in detergent
formulation include Lipolase , produced by Novo Nordisk, which
was obtained from Thermomyces lanuginosus expressed in A. oryzae;
Lipomax from Pseudomonas alcaligenes; and Lumafast from Pseu-
domonas mendocina [78,79]. Several factors are considered for the
selection of detergent-compatible lipases, including activity and
stability under washing conditions of pH 811 and temperatures
of 3060 C, broad substrate specicity, high stain removal poten-
tial, safety, cost-effectiveness and washing time, and compatibility
with various components of the detergent [76,80,81].
Lipase from Ralstonia pickettii was studied for its effectiveness
as an additive in detergent formulations. The effect of 8 different
commercial detergents (Ariel, Rin, Henko, Surf Ultra, STGC, Triton-
X, SDS, Labolene) on lipase stability was studied. The detergents
did not have inhibitory effects; instead, an increase in enzyme
activity was observed after a 1-h incubation. When cotton fab-
rics stained with olive oil were examined, a mixture of R. pickettii
lipase (150 U) with Ariel or Surf Ultra improved the removal
efciency by 26% or 30%, respectively, and further increases in
enzyme concentration had no effect. The optimum temperature
for olive oil removal in all cases was 40 C [82]. A lipase pro-
duced by Bacillus smithii BTMS 11 could be used as an additive
in detergent formulation based on its compatibility and stability
in different commercial detergents, including Surf excel (pH 9.7),
Henko stain champion (pH 10.1), Tide (pH 9.9), Ujala washing pow-
der (pH 9.8), Ariel compact (pH 9.9) and Sunlight extra bright (pH
10) after 3 h of incubation. Stain removal evaluation using white
pieces of cotton fabrics revealed the effectiveness of B. smithii lipase.
The enzyme was found to be stable over a range of pH of 710
and a temperature range of 3070 C, making it suitable for both
warm and hot washing conditions [83]. Liu et al. [84] reported that
93
Fusarium solani N4-2 lipase isolated from Soda Lake has properties
that make it suitable for use in detergent formulations. Comparative
experiments with a known commercial lipase (Lipolase ) indicated
that commercial detergent, ionic and non-ionic surfactants have
no inhibitory effects on the lipase. In addition, a higher residual
activity of approximately 99% was observed in the presence of
sodium hypochlorite as an oxidizing agent, compared to 46% in
the Lipolase . Because the enzyme mixture in detergents contains
both lipases and proteases, incubating F. solani N4-2 lipase for 1 h
at 30 C, pH 9, with commercial proteases (Protamex (1.5 AU/mg)
and Alcalase (2.4 AU/mg)) retained 100% and 78% activities,
respectively, compared with 100% and 74% in Lipolase . Similarly,
incubating T. thermophilus lipase at 40 C for 1 h in various commer-
cial detergents showed residual activities of 85%, 80%, 75%, 65% and
53% for Axion, New Det, Dixan, Ariel and OMO, respectively [38].
Statistical optimization for determining the efciency of utiliz-
ing lipase from A. niger MTCC 2594 in detergent formulations was
carried out by Saisubramanian et al. [85]. The percentage of oil
removal under the optimized conditions of 1% detergent, 75 U of
lipase, pH of 9.5 and a temperature of 25 C was 31% higher than
when detergent was used alone under the same conditions. Staphy-
lococcus sp. SL1 lipase retained 100% activity in the presence of
Axion and Ariel compared to 65% retained by Lipolase when incu-
bated for 1 h at 50 C [86]. In the detergents Dixan and Nadhif, Cherif
et al. [86] showed that SL1 was 20% more stable than Lipolase .
Thus, the choice of lipases for use in detergent formulations is
dependent on stability and activity as well as compatibility under
the harsh conditions associated with the washing process, the com-
position of detergents, and the temperature and pH.
6.2. Ionic liquid compatible lipases
Ionic liquids (ILs) are organic salts of cations and anions that
exist in liquid form at room temperature with unique properties,
including high thermal stability, excellent ionic conductivity, low
vapor pressure and a high dissolution potential, with the abil-
ity to reduce the purication and separation steps [87]. Based on
these properties, ILs have received considerable attention for use
as media and stabilizing media for chemical and biochemical trans-
formations with remarkable results [88].
ILs compatible with lipases include 1-butyl-3-methyl-
imidazolium hexauorophosphate [BMIM][PF6 ], 1-ethyl-3-
methylimidazolium triuoromethyl sulfonate [EMIM][TfO], 1-
hexadecyl-3-methylimidazolium triimide ([C16 MIM][NTf2 ]), and
1-octadecyl-3-methylimidazolium triimide ([C18 MIM][NTf2 ])
[89]. Diego et al. [90] reported maximum biodiesel production and
operational stability of immobilized C. antarctica lipase B during
transesterication of triolein with methanol in the presence of
[C16 MIM][NTf2 ] and ([C18 MIM][NTf2 ]). Following an intensive
screening study of 23 ILs by Ha et al. [91], C. antarctica lipase
showed the highest biodiesel yield in the presence of [EMIM][TfO].
Immobilized C. antarctica lipase was used for the transesterication
of sunower and waste cooking oil with methanol in 10 different
ILs. The maximum conversions of fatty acid methyl esters obtained
by C. antarctica lipase in 3-octyl-1-methylimidazolium hexauo-
rophosphate ([OMIM+ ][Pf6 ]) and 3-octyl-1-methylimidazolium
bis{(triuoromethyl)sulfonyl}imide ([OMIM+ ][NTf2 ]) were better
than for the reference solvents (isopropanol and methyl tert-butyl
ether). The biphasic nature of the system makes product recovery
and purication easy [92].
Hydrophobic ([BMIM][NTf2 ] and [BMIM][PF6 ]) and hydrophilic
([BMIM][BF4 ]) ILs were compatible with Pseudomonas cepacia
lipase during transesterication of soybean oil with methanol [93].
ILs that are active at room temperature were examined as solvents
for biodiesel production by B. cepacia lipase. ILs containing Tf2 N
and PF6 had a better stabilization effect on lipase activity due their
94 A. Salihu, Md.Z. Alam / Process Biochemistry 50 (2015) 8696
anionic nature, and a maximum biodiesel yield of 82% was obtained
in the presence of 1-octyl-3-methylpyridinium tetrauoroborate
[OMPy][BF4 ] [94]. Methanolysis of corn oil was catalyzed by Penicil-
lium expansum lipase in the presence of n-Hexane, tert-butanol and
IL [BMIM][PF6 ]. The percentage of FAME produced after 25 h of reac-
tion was 70% in [BMIM][PF6 ], compared to 19% and 1% in n-Hexane
and tert-butanol, respectively. Similarly, hydrolytic reactions of p-
nitrophenyl palmitate by P. expansum lipase were 12 times higher
in the IL than in n-Hexane [1]. Thus, the high yields during both
hydrolytic and synthetic reactions demonstrated the importance
and stabilization effects of ILs on lipases. Several reports demon-
strated the use of ILs as reaction media; their use as coating material
has also been demonstrated by Lee and Kim [95], where P. cepacia
lipase was mixed with 1-(3 -phenylpropyl)-3-methylimidazolium
hexauorophosphorate ([PPMIM][PF6 ]) at 53 C and the mix-
ture solidied on cooling. The coated P. cepacia lipase showed an
improved enantioselectivity without loss of enzyme activity.
7. Tolerance of lipolytic organisms under different solvent
systems
Both recombinant and wild solvent-tolerant lipases presented
in this review were obtained from different microorganisms that
tolerate various conditions of acidity, alkalinity, and organic sol-
vents. The rst defense mechanisms that most organisms adopt
under different environmental conditions are to prevent solvent
efux from the cells, followed by changes associated with mem-
brane composition to maintain the conguration and integrity of
the membrane bilayer [96]. Only organisms that develop these
physiological and biochemical adaptations thrive under that par-
ticular environment. The adaptation of microorganisms under
extreme conditions for lipase production could be linked to both
constitutive and inducible strategies for growth and metabolism
where the F1 F0 -ATPase proton pump contributes in the exchange of
excess protons to maintain the intracellular and extracellular envi-
ronments. Others include alterations of membrane composition,
production of chaperones and shock proteins, and expression of
some regulatory proteins [16]. Depending on the condition, Table 3
shows the different metabolic changes that occur when organisms
are grown under extreme conditions and in which the lipases and
other metabolites tend to have unique properties in terms of sta-
bility and tolerance.
8. Conclusion
Lipases have been used in different hydrolytic and synthetic
reactions, and the solvent systems determine the favored type of
reaction. Tolerance, stability and compatibility to different reac-
tion media are the key requirements for lipases. For lipases to
be utilized in waste treatment, leather processing, avor synthe-
sis, biodiesel production, and detergent formulation, their unique
properties and tolerance will aid in selecting and determining their
effectiveness. The isolation of microbial strains from extreme envi-
ronments assists in producing lipases with unique characteristics.
Advances in biotechnology associated with cloning, expression, and
mutagenesis as well as directed evolution contribute to conferring
different properties to microorganisms. Thus, the ability of lipases
to be stable in acid, alkali, salt, and organic solvents without deteri-
orating in ILs and detergents contributes immensely to their wide
industrial application.
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