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Process Biochemistry 42 (2007) 1090–1100

Molecular cloning and characterization of thermostable esterase and

lipase from Geobacillus thermoleovorans YN isolated from

desert soil in Egypt

Nadia A. Soliman a,b,*, Michael Knoll b, Yasser R. Abdel-Fattah a,Rolf D. Schmid b, Stefan Lange b,**

a Mubarak City for Scientific Research and Technology Applications, Genetic Engineering and Biotechnology Research Institute, Alexandria, Egyptb University of Stuttgart, Institute of Technical Biochemistry, Stuttgart, Germany

Received 14 January 2007; received in revised form 19 April 2007; accepted 1 May 2007

Abstract

Genes encoding an esterase (EstA) and lipase (LipA) from Geobacillus thermoleovorans YN, a strain isolated from Egyptian desert soil, were

cloned and the respective proteins were expressed in Escherichia coli and characterized. Whereas LipA was cloned directly by PCR amplification

from genomic DNA, a genomic library composed of 3000 clones was screened on tributyrin agar plates to find EstA. An open reading frame of

744 bps encoding a polypeptide of 247 amino acid residues was identified as esterase due to its conserved GXSXG motif and its high similarity

toward other carboxyl esterases. LipA (416 aa residues) is encoded by an ORF of 1251 bps and constitutes a pre-protein with a calculated molecular

mass of 46 kDa including a signal sequence of 28 aa resulting in a mature lipase of 43 kDa. Both, LipA and EstA were sub-cloned and expressed

under control of the temperature-inducible l-promoter and purified by IMAC and gel filtration. The molecular mass of the purified EstA was

29 kDa. Both enzymes were most active at pH �9.5 and remarkably stable at pH 5 and 10.5. Temperature optima and stabilities (up to 70 8C) of

both enzymes as well as their reaction kinetics and substrate spectra were determined.

# 2007 Elsevier Ltd. All rights reserved.

Keywords: Esterase; Lipase; Geobacillus thermoleovorans; Thermophiles; Biocatalysis

1. Introduction

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) cata-

lyse the hydrolysis of ester bonds of triacylglycerols at the

interface between an insoluble substrate and water. This feature

distinguishes lipases from esterases (EC 3.1.1.1), which act

only on water-soluble substrates, such as short chain fatty acid

esters, although also lipases show some activity on water-

* Corresponding author at: Mubarak City for Scientific Research and Tech-

nology Applications, Genetic Engineering and Biotechnology Research Insti-

tute, Bioprocess Development Department, New Burg El-Arab City,

Universities and Research Institutes Zone, Post 21934, Alexandria, Egypt.

Fax: +20 3 4593423.

** Corresponding author at: Institute for Technical Biochemistry, Allmandring

31, 70569 Stuttgart, Germany. Tel.: +49 711 685 63198;

fax: +49 711 685 64569.

E-mail addresses: nadiastuttgart@yahoo.com (N.A. Soliman),

stefan.lange@itb.uni-stuttgart.de (S. Lange).

1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.procbio.2007.05.005

soluble esters. The hydrolytic activity of lipases is significantly

enhanced by the presence of a lipid–water interface, a

phenomena which is called interfacial activation [1,2]. In

non-aqueous media these reactions are reversed due to a

hydrophobic domain (lid), covering the active site of lipases.

The three-dimensional structures of esterases and lipases show

the characteristic a/b fold composed of a-helices and b-sheets

[3], which is also found in haloperoxidases and epoxide

hydrolases. Most of these hydrolases contain the consensus

sequence Gly-X-Ser-X-Gly around the active site serine and

function via a Ser-Asp-His catalytic triad. Additionally the

enzymes can be classified into two groups according to a

sequence motif at the oxyanion hole (either GlyX or

GlyGlyGlyX).

Due to their stability at elevated temperatures, pH values

and organic solvents, lipase/esterase from thermophiles have

become objects of special interest for structural investigation

and for a broad range of biotechnological applications [2,4,5].

They can be used in media containing low water content,

N.A. Soliman et al. / Process Biochemistry 42 (2007) 1090–1100 1091

where they catalyse new reactions such as esterification or

transesterification [6]. These reactions led to many industrial

applications: desymmetrization of prostereogenic compounds

and resolution of racemic mixtures for the preparation of

chiral building blocks for the synthesis of fine chemicals

for the pharmaceutical industry [7,8], synthesis of flavour

esters for the food industry [9], modification of physico-

chemical properties of triglycerides for the fats and oil

industry [10–12] and the synthesis of biopolymers and

biodiesel [13–15].

The thermophile G. thermoleovorans YN was originally

isolated from Egyptian desert by enrichment [16] and identified

by 16S rRNA sequencing as Bacillus thermoleovorans or

recently G. thermoleovorans [17]. Lipase/esterase production

by this strain was optimised in shake flask cultures using a Box-

Behnken experimental design and yielded 495 U/l [18].

Several thermostable lipases have been reported from

thermophile geobacillus such as, G. thermocatenulatus [19–

22], Geobacillus stearothermophilus [23], G. thermoleovorans

ID [24,25] and G. thermoleovorans Tosh. [26], whereas only

few thermostable esterases have been reported from thermo-

phile bacillus. A thermostable esterase from G. stearothermo-

philus was cloned but poorly characterized [27]. Thermostable

esterases from G. stearothermophilus were cloned and

characterized [28]. A thermostable esterase from a moderate

thermophilic bacterial strain was studied [29–31].

In this paper the cloning, purification and characterization of

thermostable esterase and a lipase from G. thermoleovorans

YN was described.

2. Materials and methods

2.1. Bacterial strains, plasmids and growth conditions

G. thermoleovorans YN was isolated from a soil sample collected from

desert and enriched by growing in nutrient broth medium containing olive oil at

50 8C and deposited in the Culture Collection of the Genetic Engineering and

Biotechnology Research Institute, Alexandria, Egypt (GEBRICC). The isolate

was identified by sequencing of 16S rRNA as B. thermoleovorans YN (acces-

sion number AF385083), recently re-classified as G. thermoleovorans YN.

Escherichia coli DH5a [F� endA1 hsdR17 (rk�, mk+) supE44 thi-1 l�

gyrA96 relA1 D (argF-laczya)U169] was used for the cloning steps, the

propagation of all expression vectors and expression of recombinant proteins.

Cells were cultivated at 37 8C and 200 rpm in LBlow salt of the following

composition (g/l): yeast extract, 5; peptone, 10; NaCl, 5. For selection of

plasmids media were supplemented with 100 mg/l ampicillin. Chemically

competent cells of E. coli DH5a were prepared according to [32].

Plasmid pUC19 and pCYTEXP1 were used for cloning and overexpression

[33,34], respectively.

2.2. DNA isolation and manipulation

Genomic DNA from G. thermoleovorans YN was prepared as described

[32]. Plasmid DNA was isolated using the Plasmid Mini or Midi Kit (QIAgen,

Hilden, Germany) according to manufacturer’s recommendations. Extraction of

chromosomal DNA and plasmid DNA from agarose gel was done with the

QIAquick Gel Extraction Kit. Restriction analysis and DNA modifications were

performed using enzymes purchased from MBI Fermentas (MBI Fermentas, St.

Leon-Rot, Germany) according to the recommendations of the manufacturer.

Taq-DNA-polymerase (Eppendorf, Hamburg, Germany), RNase A (Qiagen),

Proteinase K and Lysozyme (Fluka, Seelze, Germany).

2.3. DNA sequencing

Automated DNA sequencing was performed using a 377 DNA sequencer

(Applied Biosystems, Foster City, CA, USA) and a terminator Ready Reaction

Mix with Ampli-Taq-Fs polymerase (Perkin Elmer, Wellesley, MA, USA). M13

forward/reverse sequencing primers were used for the cycle sequencing reac-

tions of the double-stranded templates on both strands. Sequences of the full-

length inserts from genomic libraries were obtained by primer walking strategy

and assembling of the partial sequences using the tool SeqManTM of the

software packet DNA-Star. Sequence homology analysis was done on the

web server of the National Centre for Biotechnology Informations in

the non-redundant nucleotide (nr-nt) or amino acid (nr-aa) databases using

BLASTX, BLASTP and BLASTN programmes [35].

2.4. Construction and screening of a genomic library for lipolytic

activity

Chromosomal DNA of G. thermoleovorans YN was digested with HindIII.

Fragments (5–10 kb) were excised from 1% agarose gels and DNAwas recovered

and ligated into the dephosphorylated HindIII site of pUC19 using T4-ligase.

Chemically competent E. coli DH5a was transformed with the ligation mixture

and plated on LB agar plates containing 100 ml/ml ampicillin and 1% tributyrin

(emulsified with an Ultraturrax) for lipase/esterase activity screening of the

transformants. Plasmids of halo-forming colonies were further analysed by

restriction digestion and sequencing of the insert by primer walking.

2.5. Sub-cloning the genes encoding a lipase and esterase from G.

thermoleovorans YN

The esterase gene was amplified by PCR from genomic DNA of halo-

forming clones of the library. During PCR terminal restriction sites NdeI and

EcoRI were introduced by the primers FEstA (50-ATGGGAATTCCATATGAT-

GATGAAAATTGTTCCGCCG-30) and REstA (50-CCGGAATTCTTAC-

CAATCTAACGATTCAAGAAATG-30). The PCR fragment was ligated into

the respective sites of pCYTEXP1 resulting in pCYTEX-EstA. A modified

version was amplified using the c-terminal primer REstA-6xHis (50-CCGGAATTCCTAATGGTGATGGTGATGGTGCCAATCTAACGATTCAA-

GAAATG-30) in order to introduce a His6-tag for purification. The PCR

products were restricted with NdeI and EcoRI, purified and ligated between

the respective sites of pCYTEXP1 resulting in pCYTEX-EstA-6xhis after

selection of the transformed strains on selective LBamp/tributyrin plates.

A lipase gene was amplified directly from genomic DNA of G. thermoleo-

vorans YN using a pair of primers (FlipA: 50-TATGGGAATTCCATATGAT-

GAAAKGCTGYCGGGT-30; RLipA: 50-CCGGAATTCTTAAGGCCGCAA-

RCTCGCCA-30) designed according to the consensus sequence of lipases from

other thermophilic Geobacillus species (G. stearothermophiles and G. thermo-

leovorans). The restriction sites Nde1 and EcoR1 were incorporated into the

forward and reverse primer sequence, respectively. The PCR fragment was ligated

into the respective sites of pCYTEXP1 resulting in pCYTEX-LipA. Transfor-

mants were validated by restriction analysis and screened for lipase activity on

LBamp plates supplemented with tributyrin. The nucleotide sequence of the G.

thermoleovorans YN lipase gene in pCYTEX-LipA plasmid was determined on

both strands using the primers SDM1 (50-CCAACACTACTACGTTTTAACT-

GAAACAAACTGG-30) and SDM3 (50-GCGAACGCCAGCAAGACGTAGCC-

CAGC-30). In order to allow an IMAC purification of the recombinant lipase, a

His6-tag was introduced into the gene by PCR with a modified reverse primer,

namely RLipA-6xHis: (50-CCGGAATTCCTAATGGTGATGGTGATGGT-

GAGGCCGAAGCTCGCCA-30) containing the codons encoding six histidine

residues. Ligation of the PCR fragment into pCYTEXP1 and transformation of the

resulting construct pCYTEX-LipA-6xhis into E. coli DH5a was done as described

above.

2.6. Phylogenetic analysis

Classification of the proteins was done by BLAST searches against the

Lipase Engineering Database (LED, http://www.led.uni-stuttgart.de) [36]. For

phylogenetic tree construction, multiple sequence alignments were performed

N.A. Soliman et al. / Process Biochemistry 42 (2007) 1090–11001092

using Clustal W, Version 1.83 with default parameters, whereas only sequences

annotated as non-putative sequences were used. On the basis of the resulting

multiple sequence alignments a phylogenetic tree was calculated for each

protein by applying the maximum-likelihood method implemented in the

Tree-Puzzle software, Version 5.2.

2.7. Expression and purification of esterase/lipase using IMAC

In order to allow one-step purification by IMAC, the protein variants

containing a c-terminal with His6-tag were expressed. E. coli DH5a carrying

the plasmids (pCYTEX-EstA-6xhis or pCYTEX-lipA-6xhis) containing the

modified genes encoding the esterase and lipase, respectively, were grown in

5 ml LBamp medium overnight at 37 8C. A 0.2 l of LBamp medium, were

inoculated 1:1000 with the overnight culture and cultivated at 37 8C and

170 rpm until an OD578 of 0.7–1.0. Upon induction by a temperature shift

to 42 8C cells were cultivated additional 3 h and finally collected by centrifuga-

tion (10 min, 4000 rpm, 4 8C).

2.7.1. Purification of the esterase

The pellet was dissolved in saline phosphate buffer pH 7.0, sonicated (3�1 min) and after centrifugation the supernatant containing the soluble protein

fraction was separated and applied for purification. The purification procedure

was performed at room temperature and the recombinant protein was purified

under native conditions using TALONTM Metal Affinity Resin (BD Biosciences

Clontech, Heidelberg, Germany) according to manufacturer’s recommenda-

tions at pH 7.0 using imidazole for elution. An 8 ml of the IMAC matrix was

filled in a column and equilibrated with 3 volumes of saline phosphate buffer

(50 mM, pH 7.0). After applying the sample it was washed with saline

phosphate buffer (50 mM, pH 7.0). Fraction-wise elution of bound protein

was obtained by applying 150 mM imidazole in saline phosphate buffer

(50 mM, pH 7.0). Remaining imidazole was removed by gel filtration using

a PD-10 column (Amersham Biosciences, Freiburg, Germany).

2.7.2. Purification of the lipase

Harvested cells were suspended in saline phosphate buffer, sonicated (3�1 min) and centrifuged (14,000 rpm, 4 8C, 20 min). Soluble and insoluble

fractions were applied for purification as described previously in esterase case.

The soluble fraction was used directly, and the insoluble fraction was applied

after solublization in 1% SDS, sonication (3� 1 min) and finally separated by

centrifugation (14,000 rpm, 4 8C and 30 min) after treating with 1% Triton

X-100.

2.8. Protein determination

Protein concentration was determined using the bicinchoninic acid

(BCATM) protein assay kit (Pierce, Rockford, USA) according to the manu-

facturer’s instructions.

2.8.1. SDS-polyacrylamide gelelectrophoresis

SDS-PAGE was performed on 12.5% running gels as described [37], and

resolved proteins visualized by Coomassie staining following standard proce-

dures. A low molecular range protein standard (Bio-Rad Laboratories, Rich-

mond, USA) was used as molecular mass marker. Gels to be stained for lipase

activity were submitted to a renaturation process: after washing the gel for 30–

60 min at room temperature in Tris–HCl (100 mM, pH 7.5) containing Triton

X-100 (0.5%, w/v) it was stained for esterase/lipase activity in developing

solution a-naphthylacetate as substrate.

2.8.2. Determination of hydrolase activity

Esterase activity was assayed and visualized on zymograms using 12.5%

SDS-polyacrylamide gels. The protein samples containing esterase were sepa-

rated by SDS-PAGE and renatured in 100 mM Tris–HCl buffer, pH 7.5

containing 0.5% Triton X-100 for 4 h at 4 8C. The gels were finally incubated

for 5 min at RT in developing solution consisting of 3 mM a-naphthyl acetate,

1 mM Fast Red TR (Sigma), and 100 mM sodium phosphate buffer, pH 7.5.

Esterase activity was detected by the appearance of brown colored bands in

the gels The hydrolysis of p-nitrophenylesters (C2–C18) was measured

spectrophotometrically at 410 nm in a Biochrom 4060 spectrophotometer

(Pharmacia) as follows: 100 ml of the substrate solution (10 mM p-nitrophe-

nylacetate (C2) in DMSO) was added to 900 ml of 50 mM phosphate buffer

containing the tested enzyme Hydrolytic activity toward PNP-palmitate (C16)

was measured in Tris–HCl buffer (100 mM, pH 7.5), gum arabic (0.1%, w/v)

and sodium deoxycholeate (0.8%, w/v) according to the method of [38]. One

unit (U) of hydrolase activity was defined as the amount of enzyme which

released 1 mmol of p-nitrophenol per min under assay conditions.

Hydrolysis of methyl esters (100 mM), triglycerides (20 mM) and menthyl

esters (10 mM) were determined using pH-stat assay (Metrohm, Switzerland).

The substrates were emulsified in distilled water containing gum arabic (20 mg/

ml) as stabilizer using (Ultraturrax T25) homogenizer for 7 min. 20 ml of the

substrate solution was heated to 55 8C and adjusted to pH 7.0. After addition of

20–100 ml of the enzyme solution the activity was measured with pH-stat. The

amount of sodium hydroxide needed to titrate the released fatty acids indicates the

number of units. One unit was defined as the amount of enzyme that released

1 mmol fatty acid per min under the assay conditions. All activity measurements

were performed in triplicates and expressed as the arithmetic mean of estimations.

2.9. Substrate specificity

Substrate specificities of the esterase and lipase from G. thermoleovorans

YN, towards different p-nitrophenyl esters (PNP-acetate (C2), PNP-butyrate

(C4), PNP-caproate (C6), PNP-caprate (C10), PNP-laurate (C12), PNP-myr-

istate (C14), PNP-palmitate (C16) and PNP-stearate (C18)) were analysed

spectrophotometrically.

In addition the activities towards different methyl ester substrates (methyl-

propionate (C3), methyl-butyrate (C4), methyl-caproate (C6), methyl-caprylate

(C8), methyl-caprate (C10), methyl laurate (C12), methyl-myristate (C14),

methyl-palmitate (C16), methyl-stearate (C18), methyl-eicosanoate (C20) and

methyl-decosanoate (C22)) at concentrations of 100 mM, towards triacylgly-

cerols (triacetin (C2), tributyrin (C4), tricaprylin (C8), trilaurin (C12), trimyr-

istin (C14), tripalmitin (C16) and tristearin (C18)) at concentrations of 20 mM

and towards menthyl-esters (menthyl-acetate, menthyl salicylate, menthyl

anthranilate and menthyl isovalerinate) at concentrations of 10 mM were

measured by the pH-stat method at 55 8C and pH 7.0.

2.10. Effect of temperature and pH

Effects of temperature and pH on the enzymes’ activities were measured

using p-nitrophenyl laurate as substrate in the case of esterase and p-nitrophenyl

palmitate in the case of lipase. Temperature optima of both enzymes were

evaluated by testing a wide range of temperature (40–95 8C) and the relative

activities (%) were measured. Thermostability of both enzymes was investi-

gated by measuring the residual activity upon incubation for 30 min at 40–

95 8C. Thermostability at 70 8C was tested by extending the incubation time up

to 60 min. The pH optima were evaluated by testing a pH range of 5.0–10.5.

Enzyme reactions at each pH value were carried out at 60 8C and their relative

activities (%) were measured. In order to determine the stability for both

enzymes at pH values of 5.0 and 10.5, a pre-incubation was performed at each

pH value at room temperature for 120 min. The residual activity (%) was

measured every 20 min at a reaction temperature of 60 8C.

2.11. Effect of metal ions and other reagents

The effects of various metal ions and other reagents on the hydrolytic

activity was assayed at 60 8C, pH 9.5 using p-nitrophenyl laurate and p-

nitrophenyl palmitate as substrate for esterase and lipase, respectively, upon

pre-incubation of the enzyme in each compound for 15 min at room tempera-

ture. The relative activity (%) was calculated in relation to values obtained upon

incubation (15 min, room temperature) of the enzyme without any further

treatment. For the incubation 10 ml of stock solution (100 mM) of each metal

(Mg2+, Ca2+, Cs2+, Zn2+ and Na+) were added to 1000 ml reaction mixture (final

concentration of the metal was 1 mM).

In addition, the effect of some organic solvents and inhibitors (1% of

ethanol, isopropanol, DMSO, b-mercaptoethanol, SDS, DTTand EDTA) on the

enzyme were tested.

N.A. Soliman et al. / Process Biochemistry 42 (2007) 1090–1100 1093

2.12. Kinetic parameters

The Michaelis–Menten kinetic parameters Vmax and Km values of both

enzymes were calculated using p-nitrophenyl acetate as substrate of the esterase

and p-nitrophenyl palmitate as substrate of the lipase. Lineweaver–Burk plots

were used to determine Vmax and Km parameters, assuming that the reactions

followed a simple Michaelis–Menten kinetics.

3. Results

3.1. Cloning of the esterase gene from G. thermoleovorans

YN

Initially a genomic library of G. thermoleovorans YN was

constructed and used to transform E. coli DH5a. In total 3000

transformants were screened on LBamp-tributyrin agar plates for

hydrolytic activity due to expression of a heterologous lipase/

esterase. Two halo-forming clones were found. The one that

formed the strongest halo on the agar plates after 24 h of

incubation at 37 8C was picked and cultivated in liquid LBamp

medium over night. The recombinant plasmid (pUC-P1)

isolated from this transformant contained an insert DNA

fragment of about 3.2 kb as determined by restriction digestion.

The complete nucleotide sequence of the insert was sequenced

by a primer-walking strategy and deposited into GenBank

under accession number (ac:DQ288886, GI:82791235).

Sequence analysis revealed one major ORF of 744 bps, which

encodes a polypeptide of 247 aa residues corresponding to a

molecular weight of 29 kDa. It was identified also due to the

sequence motif GXSXG which is typical to a/b-hydrolases

such as lipases and esterases [3]. The correct start codon ATG

was identified by an upstream located ribosome binding

sequence (50-AGAAAGGAA-30).In addition to the GXSXG motif the esterase contains the

highly conserved GX-motif of the oxyanion hole architecture

Fig. 1. Phylogenetic tree of the homologous fam

and thus could be assigned to the GX class [39]. This esterase

from G. thermoleovorans YN (EstA) was classified according

to a BLAST search against the LED to the homologous family

of Bacillus carboxylesterases (abH11.01). EstA is most closely

related to the carboxylesterase from Geobacillus kaustophilus

HTA426 (YP148898, GI:56421580) with a sequence identity of

99%. A multiple sequence alignment, showing the exact

position of these feature residues and a phylogenetic tree of the

homology family abH11.01 is presented in Fig. 1.

After sub-cloning of the esterase gene its expression was

achieved under control of the temperature-inducible l-

promotor. A zymogram of the cell extract revealed one band

at �30 kDa showing hydrolytic activity (Fig. 2B, lane 3). In

contrast, four bands with molecular weights of about 46, 43, 30

and 27 kDa were detected in the cell extract of G.

thermoleovorans YN (Fig. 2B, lane 1).

3.2. Expression and purification of the recombinant EstA in

E. coli

In order to allow purification of the heterologous esterase by

IMAC, a modified gene which carries the codons encoding a c-

terminal His6-tag was created by PCR. After cloning of the

respective PCR product into pCYTEXP1 the new construct,

namely pCYTEX-EstA was transformed into E. coli DH5a.

After cultivation of the recombinant strains as described in the

methods part a total esterase activity of about 324 � 103 units

was obtained from 200 ml culture as determined with p-

nitrophenyl acetate as substrate.

One-step IMAC-purification of the esterase led to a nearly

homogenous protein of �29 kDa as shown by SDS-PAGE

stained with Coomassie Brilliant Blue (Fig. 2A, lane 4) and

activity staining using FAST Red and a-naphtyl acetate (lane 4

in Fig. 2B, respectively). Remaining imidazole from the elution

ily of Bacillus carboxylesterases (abH11.01).

Fig. 2. SDS-PAGE analysis of the heterologous expression of the G. thermoleovorans YN esterase. (A) The gel was stained with Coomassie Brilliant Blue for protein

detection. (B) The gel was stained with a-naphthyl acetate and Fast Red for detection of hydrolase activity. Lane 1: crude cell extract of WT (wild type) G.

thermoleovorans YN, lane 2: E. coli DH5a/pCYTEX-EstA-his before induction, lane 3: crude cell extract of E. coli DH5a/pCYTEX-EstA-his after induction; lane 4:

EstA-his after IMAC and gel filtration.

Fig. 3. Substrate specificity of the purified esterase (A) and lipase (B) towards

p-nitrophenyl esters.

N.A. Soliman et al. / Process Biochemistry 42 (2007) 1090–11001094

process was removed by gel filtration using a PD-10 column.

The histidine-tagged esterase was finally purified 78-fold with a

yield of 18% resulting in a specific activity of 902 U/mg.

3.3. Biochemical characterization of the EstA

Substrate specificity was initially tested toward several p-

nitrophenyl esters of different chain lengths using a

photometric assay. As shown in Fig. 3A the highest activity

was obtained toward p-nitrophenyl acetate (C2 acyl group).

Also PNP-butyrate, PNP-caproate, and PNP-caprate were

hydrolysed quite well, whereas p-nitrophenol esters with

longer chain lengths were converted only slightly or not at all.

Table 1 represents the activities toward triglycerides, methyl-

and menthyl esters obtained using a pH stat method. Among

triglycerides which were tested using a pH-stat assay, the

activity was observed only toward triacetin and tributyrin (C2

and C4). Also the activities toward methyl esters decreased

with increasing chain lengths (C4–C10) and the least

activities were shown toward menthyl-esters. This confirms

the assumption that the enzyme rather is an esterase than a

lipase.

The effect of temperature and pH on the esterase activity and

stability was tested using p-nitrophenyl laurate. As shown in

Fig. 4A the esterase is most active at temperatures between 60

and 65 8C but strongly decreased at temperatures above

�70 8C. The esterase is fully stable for 30 min at 70 8C; also

extending the incubation time to 60 min does not lead to a

significant inactivation of the esterase (data not shown).

When assayed at various pH values at 60 8C the recombinant

esterase showed high activity mainly in alkaline conditions in a

pH range between 7.5 and 9.5 (Fig. 5A). The esterase showed

high stability in a pH range between 5 and 10.5 (data not

shown). The effects of various reagents, solvents and metals on

the esterase activity are shown in Table 2. Whereas Mg2+, Ca2+,

Cs+ and Na+ ions as well as DTT and b-mercaptoethanol

enhanced the activity, Zn2+ and SDS strongly inhibited the

esterase. The solvents DMSO, ethanol and isopropanol led to a

Table 1

Chain length specificities of the heterologous G. thermoleovorans YN esterase and lipase towards methyl and menthyl esters and triglycerides

Substrate Relative esterase activity (%) Relative lipase activity (%)

Methyl-propionate (C3) 9.5 12.8

Methyl-butyrate (C4) 14.7 12.0

Methyl-caproate (C6) 13.8 22.4

Methyl-caprylate (C8) 5.8 49.6

Methyl-caprate (C10) 1.5 100

Methyl-laurate (C12) 0 23.7

Methyl-myristate (C14) 0 21.2

Methyl-palmitate (C16) 0 20.3

Methyl-stearate (C18) 0 20.0

Methyl-eicosanoate (C20) 0 0.50

Methyl-decosanoate (C22) 0 0.40

Menthyl-acetate 3.3 19.7

Menthyl-salicylate 2.2 14.7

Menthyl-anthranilate 2.8 19.7

Menthyl-isovalerinate 0 16

Triacetin (C2) 100 20.5

Tributyrin (C4) 46.4 21.6

Tricaprylin (C8) 0 44.7

Trilaurin (C12) 0 21.5

Trimyristin (C14) 0 30.1

Tripalmitin (C16) 0 40.3

Tristearin (C18) 0 21.8

The 100% activity corresponds to 5.4 and 109 U/mg protein for esterase and lipase, respectively.

N.A. Soliman et al. / Process Biochemistry 42 (2007) 1090–1100 1095

slight activation of the enzyme, EDTA slightly decreased the

activity.

Kinetic parameters were measured using a spectrophoto-

metric activity assay with p-nitrophenyl acetate as substrate (a

typical substrate for esterase). The esterase exhibited a simple

Michaelis–Menten kinetics for p-nitrophenyl acetate. The Km

value for the conversion of p-nitrophenyl acetate by the esterase

was determined to be 6.74 mM. From a Lineweaver–Burk plot

the respective Vmax value was calculated to be 5350 mmol/

(min mg).

Fig. 4. Effect of temperature on the activity and stab

Fig. 5. Effect of the pH on the activity of t

3.4. Cloning of the lipase gene from G. thermoleovorans

YN

As the esterase of G. thermoleovorans YN showed high

homology with other thermophile esterases, the lipase from G.

thermoleovorans YN amplified directly by PCR with genomic

DNA from G. thermoleovorans YN as template and primers

(Flip and RLip) designed according to the consensus sequence

of lipases from other thermophilic Geobacillus species (G.

stearothermophiles and G. thermoleovorans). The PCR product

ility of the purified esterase (A) and lipase (B).

he purified esterase (A) and lipase (B).

Table 2

Effect of metal ions and various enzyme inhibitors on esterase and lipase enzyme activities

Treatment Residual esterase activity (%) Residual lipase activity (%)

None 100 100

MgCl2 (1 mM) 170 96.05

CaCl2 (1 mM) 152 102.12

CsCl2 (1 mM) 148 97.93

ZnCl2 (1 mM) 17 14.4

NaCl2 (1 mM) 122 94.8

DMSO (1%) 117 94.12

DTT (1%) 179 96.48

SDS (1%) 39 99.51

EDTA (1%) 94 98.12

b-Mercaptoethanol (1%) 146 99.08

Isopropanol (1%) 102 93.74

Ethanol (1%) 113 98.8

Enzyme activities were assayed spectrophotometrically using PNP-laurate and/or PNP-palmitate as substrate upon incubation of the enzyme (15 min) in each

compound; the applied reaction conditions were pH 9.5 and 60 8C.

Fig. 6. Phylogenetic tree of the homologous family of Staphylococcus aureus like lipases (abH15.01).

N.A. Soliman et al. / Process Biochemistry 42 (2007) 1090–11001096

N.A. Soliman et al. / Process Biochemistry 42 (2007) 1090–1100 1097

was sub-cloned into pCYTEXP1 under control of the

temperature-inducible l promoter. Sequence analysis of the

cloned fragment revealed one major open reading frame of

1251 bps, which encodes a polypeptide of 416 aa residues. The

sequence has been submitted to GenBank and is available under

accession number GI:83939851.

Based on In silico data, a 28 aa constitute a putative signal

peptide and a cleavage site between Ala28 and Ala29 was

expected at the N-terminus. Thus, the deduced sequence of the

mature lipase contains 388 amino acids and corresponds to a

calculated molecular mass of 43.14 kDa whereas the calculated

molecular weight of the pre-protein, which is composed of 416

aa residues, was calculated to be 46 kDa. The thermostable

lipase from G. thermoleovorans YN (LipA) was classified to the

homologous family of Staphylococcus aureus like lipases

(abH15.01) by applying a BLAST search against the LED.

Within the conserved pentapeptide GXSXG the first glycine is

replaced by alanine, which can also be found in other sequences

within this homologous family. The lipase could be assigned to

the GX class of the oxyanion hole architecture. LipA shows

highest sequence identity to the lipase of Bacillus sp. Tosh.

(GI:20501953) with a sequence identity of 99%. A multiple

sequence alignment, showing the exact position of the above

mentioned residues and a phylogenetic tree of the homologous

family abH15.01 is provided in Fig. 6.

3.5. Expression and purification of the recombinant LipA

in E. coli

For IMAC purification of the recombinant lipase a modified

construct containing six c-terminal histidine residues was

expressed in a 400 ml scale. After 3 h of induction, cells were

collected; the soluble and insoluble fractions were separately

purified by IMAC and gel filtration as described in the methods

part. In the crude extract from a 400 ml culture about

2335 � 103 units of lipase were obtained in total as determined

with p-nitrophenyl palmitate as substrate. The lipase was

purified 130-fold by IMAC followed by gel filtration with a

yield of 2.1% and a final specific activity of 3586 U/mg. SDS-

PAGE analysis was done before (Fig. 7A) and after purification

Fig. 7. SDS-PAGE analysis of different samples taken during the purification proces

before purification. Lanes 1 and 3: soluble fraction after resuspension, sonication an

extract upon addition of 1% SDS, sonication, renaturation and centrifugation. (B) Pu

fractions after gel filtration. In both gels lanes 1 and 2 were stained with Coomassie B

Red for detection of hydrolase activity.

(Fig. 7B) of both, soluble (lanes 1 and 3) and insoluble (lanes 2

and 4) fractions. Activity staining revealed that the soluble

fraction contained only one active protein of�43 kDa (Fig. 7A,

lane 3), whereas the insoluble fraction contained two active

proteins, one of �43 kDa and one with a slightly higher

molecular weight (Fig. 7A, lane 4); both bands still were

present after purification (Fig. 7B, lane 4). Staining with

Coomassie Brilliant Blue confirmed the high purity of the

lipase after purification (Fig. 7B, lanes 1 and 2).

3.6. Characterization of the LipA

The lipase showed a very high specific activity toward all

tested p-nitrophenyl esters (C2–C18); the specific activity

increased with the chain length of the substrates (from C2 to

C10) with a remarkable preference of C10 as shown in Fig. 3B.

Among other substrates (methyl esters, menthyl esters and

triglycerides) which were tested by a pH-stat assay, the lipase

showed a preference to long chain fatty acids rather than short

chain fatty acids. The highest activities were observed towards

methyl caprate (C10), methyl caprylate (C8), tricaprylin (C8)

and tripalmitin (C16). The activities toward methyl esters

decreased from C12 to C22; the least activities were found

toward menthyl esters (Table 1).

The effect of temperature and pH on the lipase activity and

stability were tested spectrophotometrically using p-nitrophe-

nyl palmitate as substrate. As shown in Fig. 4B the lipase was

most active in a temperature range between 60 and 65 8C. At

70 8C it was stable for half an hour; whereas extension of the

incubation at 70 8C up to an hour did not lead to a significant

loss of activity. A drop of activity was induced by incubation at

temperatures above 75 8C (data not shown).

When assayed at various pH values and at 60 8C, the

recombinant lipase showed high activity in alkaline conditions

up to pH 10.5 (Fig. 5B). The range of its pH stability was shown

to be from pH 5 to 10.5 (data not shown).

The effects of various reagents, solvents and metals on lipase

are shown in Table 2. Like the esterase Zn2+ strongly inhibited

the lipase, whereas Ca2+ led to some activation. The other tested

substances (b-mercaptoethanol, SDS, ethanol, EDTA, Cs+,

s of the G. thermoleovorans YN lipase. (A) Crude extracts upon cell harvesting

d centrifugation of the cell pellet; lane 2 and 4: insoluble fraction of the crude

rified cell extract fractions. Soluble (lanes 1 and 3) and insoluble (lanes 2 and 4)

rilliant Blue for protein detection, lanes 3 and 4 with a-naphtyl acetate and Fast

N.A. Soliman et al. / Process Biochemistry 42 (2007) 1090–11001098

DTT, Mg2+, Na+, DMSO and isopropanol) showed slight

inhibition varying between 1% and 7%, respectively.

Kinetic parameters were measured using a spectrophoto-

metric activity assay with p-nitrophenyl palmitate as substrate

(a typical substrate for lipase). The lipase exhibited a simple

Michaelis–Menten kinetics with a Km value of �1.1 mM and a

Vmax value 25,200 mmol/(min mg).

4. Discussion

A Geobacillus isolate from Egyptian desert soil, identified on

the basis of its 16S rRNA as G. thermoleovorans YN showed

extracellular lipase/esterase activities. The production of

esterase/lipase was optimised in shake flask cultures using a

Box-Behnken experimental design and led to a yield of 495 U/l

[18]. It leads to a magnification of the total lipolytic activity and

in due course released many active proteins. Separation of the

proteins in the cell extract by SDS-PAGE followed by activity

staining (zymogram) revealed several hydrolases with molecular

weights of 46, 43, 30 and 27 kDa. The appearance of several

active protein bands produced by YN host reflects the presence of

many active enzymes, but does not reflect the correlation

relation. However, purification of any of the respective enzymes

turned out to be difficult due to their low expression levels and

strong interactions with other proteins and lipid materials. By this

way the discrimination between these enzymes and the

production of each protein individually by native host cell is

considered a difficult task. Only cloning allowed and facilitated

the individual discrimination and production of proteins

respective to EstA and LipA. Thus, genes encoding one esterase

of 29 kDa and one lipase of 43 kDa were cloned into E. coli

revealed two halo forming clones. One of them contained an

insert of 3174 bps, which encoded an esterase gene. The ORF

including the conserved motive GXSXG was sub-cloned into

pCYTEXP1 and expressed under control of the temperature-

inducible l promoter. Comparison of the deduced esterase amino

acid sequence with other esterases in Genbank showed its

homology of 97% to the esterase of G. stearothermophilus IFO

12550 [27]. Both enzymes differ only in three out of 247 amino

acids.

Using the genomic library we succeeded in cloning of an

esterase gene but not the one encoding a lipase. This might be

related to cell lysis due to possible toxicity of the lipase in E.

coli cells [40]. To overcome this problem the gene encoding a

lipase of G. thermoleovorans YN has been cloned directly from

DNA by PCR using primer sequences based on homology

searches among several lipases. According to its sequence and

biochemical properties G. thermoleovorans YN lipase belongs

to subfamily I.5. This subfamily includes lipases identified in

G. thermocatenulatus, G. stearothermophilus and G. thermo-

leovorans. Lipases of those host organisms are originally

synthesized as pre-proteins with a signal peptide sequence that

is cleaved to yield the mature lipase of 388 amino acid residues.

This signal peptide is needed for proper targeting of the protein,

and is removed before the mature protein is released into the

external fluid [41]. The amino acid sequence homology among

the three Bacillus lipases is more than 91%. Their deduced

molecular weight is approximately 43 kDa and their pI value is

around 6.2. A comparison of the deduced amino acid sequence

of G. thermoleovorans YN lipase with other lipases in GenBank

database showed homology toward lipases of G. thermo-

leovorans Tosh. [26] of 99%, G. stearothermophilus [42,43]

and G. thermoleovorans [24] of 95%. In total these enzymes

differ only in 17 out of 388 amino acids in mature enzyme.

Although many lipases from other genera contain the conserved

pentapeptide Gly-X-Ser-X-Gly around the active serine, this is

not the case with the Geobacillus lipases. The conserved

pentapeptide Ala-X1-Ser-X2-Gly is found. However, in sub-

family I-5, X1 is a His residue and X2 is a Gln residue.

Despite the high sequence homology, the lipases show

different pH optima. Whereas the lipases from G. stearother-

mophilus and G. thermocatenulatus exhibited maximum

activities at pH 9.5 and 8.5, respectively [20,23], the maximum

activity of G. thermoleovorans lipase is at neutral pH of �7.5

[44]. This group of Geobacillus lipases is thermostable as it

retains activity at temperatures above 50 8C.

Both, esterase and lipase were expressed as a fusion with a c-

terminal His6-tag for one step purification by IMAC. Upon

over-expression of the enzymes, the soluble cytoplasmic

protein fractions were applied for purification and analysed

by SDS-PAGE. Activity staining of the purified fractions

revealed one active band containing either esterase or lipase

with the expected molecular weights 29 and 43 kDa,

respectively. Upon expression of the lipase also the insoluble

fraction contained another active band with a molecular weight

slightly higher than 43 kDa. This band was also found when all

fractions were analysed separately (supernatant, periplasm,

soluble cytoplasm and insoluble cytoplasm), and even in the

cellular crude extracts that were analysed periodically up to 3 h

after induction (data not shown). Whereas the periplasmic

expression is due to the signal peptide, lipase activity in the

supernatant is probably due to lysis of some cells. In absence of

the signal peptide, expression was found neither in the

periplasm nor in the supernatant (data not shown).

An explanation for the second active band might be that this

band constitutes the pre-protein which is formed mainly in

insoluble form, and was not processed completely to the mature

lipase. However, the apparent molecular weight of this pre-

protein is less than the calculated molecular weight of 46 kDa.

This might be due to proteolytic degradation of the insoluble

form of the lipase, as reported elsewhere [19,45].

Both recombinant enzymes showed the same temperature

and pH optimum of 60–65 8C and pH 9–9.5, respectively. This

is in accordance to BTID-A and BTID-B lipases from G.

thermoleovorans ID-I [25] and Est30 and Est55 esterases from

G. stearothermophilus [28]. In contrast to other homologous

enzymes they show a high thermostability: even after exposure

to 70 8C for 60 min no loss of activity was observed, whereas

BTID-A and BTID-B lost 25% of activity after exposure to only

60 8C for 30 min. Also the reported half lives of the lipases

from G. stearothermophilus L1 and G. thermocatenulatus were

clearly lower: only 30 min at 60 8C and 30 min at 62 8C,

respectively [20,23]. The esterase from G. stearothermophilus

[30] and Geobacillus sp. [46] lost 35% and 50% activity upon

N.A. Soliman et al. / Process Biochemistry 42 (2007) 1090–1100 1099

exposure to 70 8C for 60 min. Whereas at 60 8C both, Est30 and

Est55 were stable for more than 2 h, at 70 8C their half lives

concerning thermal inactivation at 70 8C was 40 and 180 min

for Est55 and Est30, respectively [28].

The activities of the esterase and lipase from G. thermo-

leovorans YN were enhanced by Ca2+ but strongly inhibited by

Zn2+, which is in accordance to the results of Lee et al. [25] and

Nthangeni et al. [47]. SDS showed very strong inhibitory effect

on the esterase (60%), whereas the lipase showed certain

stability against it. Similarly BTID-B lipase was inhibited by

SDS, whereas the effect on BTID-A lipase was negligible.

Immediate inactivation of BTL2 from G. thermocatenulatus by

SDS was shown by the work of Rua et al. [20]. The stability or

enhancement of enzyme activity in presence of organic solvents

and reducing agents is generally considered as a valid feature,

as it is a prerequisite for the synthesis of chiral compounds in

non-aqueous solvents [48]. However, reducing agents such as

DTT and b-mercaptoethanol often cause reduction of lipase

activity, especially of those lipases belonging to subfamily I.5:

treatment of lipases isolated from G. thermoleovorans and B

stearothermophilus with b-mercaptoethanol or DTT reduced

their activities by approximately 15% [23,44]. Whereas the

recombinant lipase of G. thermoleovorans YN was only

slightly inhibited, the esterase was even strongly activated by

these reducing agents. In addition both recombinant enzymes of

G. thermoleovorans YN showed stability towards solvents such

as DMSO, isopropanol and ethanol. Also BTL2 showed

stability toward these solvents, but it decreased after 1 h

incubation [20]. The same agents (DMSO, isopropanol,

ethanol) enhanced BTID-B lipase whereas BTID-A lipase

inhibited [25]. Whereas b-mercaptoethanol did not show any

effect on the activity of the B. licheniformis lipase, DTT caused

activation at all concentrations [47]. Only a slight inhibiting

effect was shown by EDTA on the activities of both, esterase

and lipase from G. thermoleovorans YN, contradictory BTID-

A and -B lipases strongly inhibited by EDTA [25], whereas a

recombinant lipase from B. licheniformis was activated by

EDTA [47].

Substrate specificity: as expected the esterase from G.

thermoleovorans YN showed preference for substrates with

short chain fatty acids rather than the long chain fatty acids.

Using PNP-esters as substrates its activity decreased from C2 to

C16; uncommonly some activity toward PNP-palmitate (C16

acyl group) was detected. The same was found [29] in case of an

esterase from a newly isolated moderate thermophilic

bacterium: it showed the highest activity on PNP-acetate

(C2) and no activity against PNP-palmitate (C16) [29]. On the

other hand Est55 exhibits its maximum activity towards C4 and

C6, a sharply decreasing activity from C8 to C12 and nearly no

activity toward C14 and C16. In case of Est30 maximal activity

was observed toward C6, followed by a gradual decrease of

activity (C8–C12) until complete loss of activity toward C18

[28].

In addition the G. thermoleovorans YN esterase showed

some activity toward methyl ester substrates with a preference

of C4 and C6 acyl groups. The highest activity was measured

against the triacylglycerol substrates triacetin (C2) and

tributyrin (C4). Similarly, also the esterase of G. stearother-

mophilus showed a preference for triglycerides with short chain

rather than long chain fatty acids [27].

In contrast, the lipase from G. thermoleovorans YN

showed activity toward all PNP-esters, with an increase by

increasing chain length and a marked preference of C10.

Similarly, BTL2 showed high activity toward PNP-caprate

(C10) [22], whereas L1 lipase showed high activity toward

PNP-caprylate (C8) [23]. In addition the G. thermoleovorans

YN lipase showed a broad range of activities toward other

substrates; highest activities were obtained against methyl

caprate (C10), methyl caprylate (C8), tricaprylin (C8) and

tripalmitin (C16). The same was found previously for BTID-A

and -B, which both showed a broad preference of

triglycerides, with the highest activity toward tricaprylin

(C8) and tricaprin (C10) [25].

In conclusion, the two enzymes described in this study

represent an example for the high potential of microorganisms

surviving in the heat and aridity of the desert, which is

generally rather inhospitable, as source of novel thermostable

enzymes. Although both enzymes share some properties with

other enzymes, both of them are very stable in high

temperatures up to 70 8C and surprisingly also at high pH

values. Due to this and other unusual properties of the enzymes

such as their high resistance against a variety of substances the

enzymes are valuable candidates for industrial applications in

biocatalysis.

Acknowledgements

Authors would like to thank the Ministry of Scientific

Research and Mubarak City for Scientific Research and

Technology Applications for financial support of this work

including a grant for a research stay of Nadia A. Soliman in

Germany.

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