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: GD-95 lipase from Geobacillus sp. strain 95 and its modified variants lacking N-terminal signal peptide and/or 10 or 20 C-terminal amino acids were successfully cloned, expressed and purified. To our knowledge, GD-95 lipase precursor (Pre-GD-95) is the first Geobacillus lipase possessing more than 80 % lipolytic activity at 5 °C. It has maximum activity at 55 °C and displays a broad pH activity range. GD-95 lipase was shown to hydrolyze p-NP dodecanoate, tricaprylin and canola oil better than other analyzed substrates. Structural and sequence alignments of bacterial lipases and GD-95 lipase revealed that the C-terminus forms an α helix, which is a conserved structure in lipases from Pseudomonas, Clostridium or Staphylococcus bacteria. This work demonstrates that 10 and 20 C-terminal amino acids of GD-95 lipase significantly affect stability and other physicochemical properties of this enzyme, which has never been reported before and can help create lipases with more specific properties for industrial application. GD-95 lipase and its modified variants GD-95-10 can be successfully applied to biofuel production, in leather and pulp industries, for the production of cosmetics or perfumes. These lipases are potential biocatalysts in processes, which require extreme conditions: low or high temperature, strongly acidic or alkaline environment and various organic solvents.Extremophiles 11/2013; · 2.20 Impact Factor
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ABSTRACT: Immobilized enzymes are popular as reusable reagents in industrial processes. In this study, α-amylase was used as a model enzyme to evaluate the propensity of synthesized porous chitosan microspheres as immobilization matrix. Chitosan microspheres were synthesized by grafting and covalent gelation technique using acrylamide (AAm) and glutaraldehyde (GA) as chemical agents, respectively. The synthesized chitosan-cl-poly(AAm) demonstrated amylase immobilization capacity of 350mg/g. Furthermore, SEM results supported the porous microsphere structure for chitosan-cl-poly(AAm) with non-aggregated amylase immobilization, which accounts for comparable activity of immobilized amylase (3.28μmol/ml/min) in contrast to free amylase (3.46μmol/ml/min). The immobilized α-amylase was characterized for optimal pH and temperature activity and showed better resistance to temperature and pH inactivation in contrast to free amylase. The immobilized amylase retained more than 60% of its initial activity when stored at 4°C for 30 days and retained 50% of its initial activity after seven successive repeated-use cycles. In conclusion, the study can be used as base for the immobilization of competent industrial biocatalysts in non-aggregated active structure.International journal of biological macromolecules 02/2014; · 2.37 Impact Factor
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ABSTRACT: Bacillus thermocatenulatus lipase 2 (BTL2), a thermoalkalophilic lipase, is the best studied enzyme for it's particular properties that has made it useful in different industries. Displacement of conserved Phenylalanine 17 residue in active site of BTL2 has a critical role in oxyanion hole-forming which is important for the enzyme activity. In this study, to facilitate oxyanion hole formation, Phe17 was substituted with Alanine residue (F17A). The best structures of the opened form of the native and mutated lipases were garnered based on the crystal structures of 2W22. To evaluate catalytic activity, both lipases were docked to a set of ligands using Hex 6.3 software. Following in silico study, both native and mutant btl2 genes were cloned and expressed in Pichia pastoris. Based on the results obtained, the mutation increased lipase lipolytic activity against most of applied substrates especially for Tributyrin and Tricaprylin by 1.9 and 2.15 folds, respectively. However, optimum temperature and pH were the same for both lipases (60 °C and pH 8.0). As previously reported, it is believed that F17A mutation simplifies oxyanion hole formation and declines steric hindrance in the enzyme active site which might ultimately lead to more efficient accessibility of substrates.This article is protected by copyright. All rights reservedBiotechnology and Applied Biochemistry 01/2013; · 1.35 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 184.108.40.206) 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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