High level expression and characterization of a novel thermostable, organic solvent tolerant, 1,3-regioselective lipase from Geobacillus sp. strain ARM.
ABSTRACT The mature ARM lipase gene was cloned into the pTrcHis expression vector and over-expressed in Escherichia coli TOP10 host. The optimum lipase expression was obtained after 18 h post induction incubation with 1.0mM IPTG, where the lipase activity was approximately 1623-fold higher than wild type. A rapid, high efficient, one-step purification of the His-tagged recombinant lipase was achieved using immobilized metal affinity chromatography with 63.2% recovery and purification factor of 14.6. The purified lipase was characterized as a high active (7092 U mg(-1)), serine-hydrolase, thermostable, organic solvent tolerant, 1,3-specific lipase with a molecular weight of about 44 kDa. The enzyme was a monomer with disulfide bond(s) in its structure, but was not a metalloenzyme. ARM lipase was active in a broad range of temperature and pH with optimum lipolytic activity at pH 8.0 and 65°C. The enzyme retained 50% residual activity at pH 6.0-7.0, 50°C for more than 150 min.
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ABSTRACT: Thermophilic bacteria are being extensively used in biotechnology. Species of the genus Geobacillus rank among the most prospective thermophiles. The current methods of genetic and metabolic engineering of these microorganisms are considered in the article. Examples of their use in various branches of biotechnology are presented.Russian Journal of Genetics: Applied Research. 05/2014; 4(3):218-226.
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ABSTRACT: An unconventional aqueous two‐phase system (ATPS) composed of polyethylene glycol (PEG) and sodium carbonate was developed and optimized by employing response surface methodology for separation of Rhizopus niveus lipase. A five‐level central composite design was applied to evaluate the optimal level of three process variables in order to obtain maximum lipase separation. Experimental data were analyzed by regression and a polynomial model was created which was found significant. The maximum partition coefficient was achieved with the system PEG 4000/sodium carbonate. Validation experiments confirmed the high accordance of predicted and experimental results. The optimized ATPS can be applied as a suitable cost‐effective system for lipase extraction. Aqueous two‐phase systems are promising alternatives to conventional enzyme purification methods which often involve several cost‐ and time‐consuming steps. Optimization of lipase partitioning using a polyethylene glycol (PEG)/carbonate system is described. Low‐molar‐mass PEG and sodium carbonate enabled better partition and may be utilized as a low‐cost system for lipase purification.Chemical Engineering & Technology 07/2014; 37(7). · 2.18 Impact Factor
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ABSTRACT: The objective of the present study was the isolation, molecular cloning and biochemical characterization of a thermophilic organic solvent-resistant lipase from Bacillus sp. DR90. The lipase gene was expressed in Escherichia coli BL21(DE3) using pET-28a(+) vector. The purification of recombinant lipase was conducted by nickel affinity chromatography and its biochemical properties were determined. The lipase sequence with an ORF of 639 bp contains the conserved pentapeptide Ala-His-Ser-Met-Gly. His-tagged recombinant lipase had a specific activity of 1,126 U/mg with a molecular mass of 26.8 kDa. The cloned lipase was optimally active at pH 8.0 and 75 °C representing high stability in broad ranges of temperature and pH. High performance liquid chromatography was used to determine the major compounds released during the lipase-catalyzed reaction of p-nitrophenyl derivatives as well as the substrate specificity. The purified lipase showed high compatibility towards various organic solvents, surfactants and commercial solid/liquid detergents; therefore the recombinant DR90 lipase could be considered as a probable candidate for future applications, predominantly in detergent processing industries.The Protein Journal 07/2014; · 1.04 Impact Factor
High level expression and characterization of a novel thermostable, organic
solvent tolerant, 1,3-regioselective lipase from Geobacillus sp. strain ARM
Afshin Ebrahimpoura, Raja Noor Zaliha Raja Abd. Rahmana, Mahiran Basrib, Abu Bakar Salleha,⇑
aFaculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
bFaculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
a r t i c l e i n f o
Received 5 January 2011
Received in revised form 23 March 2011
Accepted 25 March 2011
Available online 29 March 2011
Organic solvent tolerant
a b s t r a c t
The mature ARM lipase gene was cloned into the pTrcHis expression vector and over-expressed in
Escherichia coli TOP10 host. The optimum lipase expression was obtained after 18 h post induction incu-
bation with 1.0 mM IPTG, where the lipase activity was approximately 1623-fold higher than wild type. A
rapid, high efficient, one-step purification of the His-tagged recombinant lipase was achieved using
immobilized metal affinity chromatography with 63.2% recovery and purification factor of 14.6. The puri-
fied lipase was characterized as a high active (7092 U mg?1), serine-hydrolase, thermostable, organic sol-
vent tolerant, 1,3-specific lipase with a molecular weight of about 44 kDa. The enzyme was a monomer
with disulfide bond(s) in its structure, but was not a metalloenzyme. ARM lipase was active in a broad
range of temperature and pH with optimum lipolytic activity at pH 8.0 and 65 ?C. The enzyme retained
50% residual activity at pH 6.0–7.0, 50 ?C for more than 150 min.
? 2011 Elsevier Ltd. All rights reserved.
Nowadays, lipases (EC 18.104.22.168) have developed into the most
widely used class of enzymes in biotechnology and synthetic or-
ganic chemistry because of their ability to catalyze a broad range
of novel and important reactions in aqueous and nonaqueous med-
ia. They are used to hydrolyze ester bonds of a variety of nonpolar
substrates at high activity, regioselectivity, and stereoselectivity.
Moreover, they are able to catalyze wide range of ester and amide
bonds formation in nonpolar solvents. The reaction can be de-
signed and optimized to produce a variety of novel products by
changing substrate structure, solvents, additives, water activity,
pressure, temperature, and the biocatalyst itself. The lipase used
in each application is selected based on its activity, stability and
selectivity (Hasan et al., 2006; Dizge et al., 2009).
Ironically, many of the industrial processes in which lipases
would offer clear sustainable advantages do not operate under
mild conditions. Finding enzymes that work optimally in harsh
conditions and not losing their activities is a tall order. Thermosta-
ble enzymes in comparison to mesophilic enzymes display higher
resistance to chemical denaturants and withstand higher substrate
concentrations. They catalyze the reactions at higher process rates
due to a decrease in viscosity and an increase in diffusion
coefficient of substrates at high temperature. The reactions result
in higher process yield due to increased solubility of substrates
and products and favorable equilibrium displacement in endother-
mic reactions (Vieille and Zeikus, 2001).
Furthermore, lipases that can function as biocatalysts in nearly
anhydrous organic solvents offering new possibilities such as shift-
ing of the thermodynamic equilibria in favor of synthesis, enabling
the use of hydrophobic substrates, controlling or modifying en-
zyme selectivity by solvent engineering, suppressing undesirable
water dependent side reactions, improving thermal stability of
the enzymes and decreasing microbial contamination. Exploiting
such advantages is often limited by the low stability and/or activity
of biocatalysts in these systems. Since most lipases easily denature
in organic solvents and therefore lose their catalytic activities, it is
necessary to find lipases that are stable in nonaqueous systems
(Ogino, 2008; Xu et al., 2010).
Substrate specificity of lipases is often very important to their
applications for analytical and industrial purposes. Position-spe-
cific lipases (particularly 1,3-specifics) are the key point for
specialty structured lipids production. Structured lipids are re-
ferred particular molecular species of triacylglycerols (TAGs) with
defined molecular structure. Molecular structure of TAGs (i.e. com-
position and positions of the fatty acids in the molecule) influences
their functionalities including metabolic fate in organisms (i.e.
digestion and absorption) as well as their physical characteristics
(e.g. melting points and crystallinity). Consequently, it is possible
to control the behavior of TAGs by designing structured lipids with
particular chemical structure, thereby improving the nutritional
0960-8524/$ - see front matter ? 2011 Elsevier Ltd. All rights reserved.
⇑Corresponding author. Tel.: +60 389466091; fax: +60 386566038.
E-mail addresses: email@example.com (A. Ebrahimpour), rnzaliha@bio-
tech.upm.edu.my (R.N.Z.R.A. Rahman), firstname.lastname@example.org (M. Basri),
email@example.com (A.B. Salleh).
Bioresource Technology 102 (2011) 6972–6981
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