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Journal of Molecular Catalysis B: Enzymatic 105 (2014) 7481

Contents lists available at ScienceDirect

Journal of Molecular Catalysis B: Enzymatic

journal homepage: www.elsevier.com/locate/molcatb

Purication and biochemical characterization of halophilic,

alkalithermophilic protease AbCP from Alkalibacillus sp. NM-Fa4
Noha M. Mesbah a, , Juergen Wiegel b
a
b

Department of Biochemistry, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt
Department of Microbiology, University of Georgia, Athens, GA 30602, USA

a r t i c l e

i n f o

Article history:
Received 6 December 2013
Received in revised form 6 March 2014
Accepted 28 March 2014
Available online 6 April 2014
Keywords:
Protease
Halophilic
Alkalithermophilic
Wadi An Natrun
Alkalibacillus sp.

a b s t r a c t
An extracellular alkaline, halo- and thermostable protease (AbCP) produced by a novel Alkalibacillus sp.
NM-Fa4, isolated from the alkaline, hypersaline lakes of the Wadi An Natrun, was puried to homogeneity by precipitation with ethanol and anion-exchange chromatography. The molecular weight of the
puried protease was 19.7 kDa. AbCP retains proteolytic activity over broad sodium chloride, pH and

temperature ranges, with maximal activity at 1 M NaCl, pH45 C 9.5 and 5052 C. AbCP was resistant
to phenylmethylsulfonyl uoride (2 mM) and ethylene diamine tetra-acetic acid (2 mM), stimulated by
the reducing agents dithiothreitol (2 mM) and -mercaptoethanol (1% v/v) and inhibited with iodoacetic
acid (5 mM), suggesting that AbCP is a cysteine protease. AbCP showed stability toward anionic surfactants (sodium dodecyl sulfate), oxidizing agents (H2 O2 ), chemical denaturants (urea), and retained most
of its activity in the presence of 1% v/v of the non-ionic surfactant Tween 80. The protease was stable
in 50% mixtures of ethanol and, to a lesser extent, methanol, and was stimulated by Mg2+ , Ca2+ , and
Fe2+ . AbCP shows a broad substrate specicity and hydrolyzes both natural and synthetic substrates.
Based on the LineweaverBurk plot, the Km with casein as substrate was 1.3 0.007 mg/mL and Vmax
was 1111 mg/ml/min. The stability of the enzyme under the combined extreme conditions of high salt
concentration, alkaline pH and high temperature, in addition to being resistant to chemical denaturants, oxidizing agents, surfactants as well as exhibiting broad substrate specicity makes this enzyme a
promising candidate for a variety of biotechnological applications.
2014 Elsevier B.V. All rights reserved.

1. Introduction
There has been a signicant increase in the use of enzymes as
industrial catalysts. The global market for enzymes was estimated
at $3.3 billion in 2010, and is expected to reach $4.4 billion by 2015,
with a compound annual growth rate of 6% over a 5-year period.
Proteases constitute the largest product segment in the industrial
enzymes market. Proteases (EC 3.4.21-24) are a large group of
hydrolytic enzymes that cleave peptide bonds and degrade proteins
into smaller peptides and amino acids. Proteases constitute a big
share of the enzyme market primarily as detergent additives, and
also in the food, organic synthesis and pharmaceutical industries

Abbreviations: -ME, -mercaptoethanol; DTT, dithiothreitol; EDTA, ethylene

diamine tetraacetic acid; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl uoride; SDS, sodium dodecyl sulfate; SDS-PAGE, sodium dodecyl sulfate
polyacrylamide gel electrophoresis.
Corresponding author. Tel.: +20 1227907957; fax: +20 643230741.
E-mail addresses: noha mesbah@pharm.suez.edu.eg, noha.mesbah@gmail.com
(N.M. Mesbah), juergenwiegel@gmail.com (J. Wiegel).
http://dx.doi.org/10.1016/j.molcatb.2014.03.023
1381-1177/ 2014 Elsevier B.V. All rights reserved.

(http://www.reportlinker.com/p0363451-summary/Enzymes-inIndustrial-Applications-Global-Markets.html). Proteases also have

clinical and medical applications such as reduction of tissue
inammation and treatment of burns [1,2]. Proteases from different origins have also been used to produce bioactive peptides
[3,4].
Extremophilic microorganisms from extreme environments
have been an important source of a variety of stable enzymes [59].
Many studies have reported on the activity of halophilic proteases,
alkaliphilic proteases and thermostable proteases [1019]. Most
halophilic proteases belong to the serine protease family, and are
dependent on high salt concentration for structural stability. They
display maximal activity at salt concentrations greater than 1.4 M,
neutral to slightly basic pH and temperatures between 30 and 45 C.
Alkaline proteases are characterized by high activity at alkaline pH,
normally greater than 9.0, and also broad substrate specicity. The
optimal temperature for activity is around 60 C [20]. However,
their activity is greatly reduced in the presence of salt concentrations greater than 0.5 M, thereby limiting their applicability in
processes necessitating low water activity.

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N.M. Mesbah, J. Wiegel / Journal of Molecular Catalysis B: Enzymatic 105 (2014) 7481

Reports on enzymes that have catalytic activity under more

than one extreme, such as haloalkaliphilic proteases or alkalithermophilic proteases, are scarce [21]. It is assumed that
such polyextremophilic enzymes will have wider versatility and
adaptability to multiple harsh conditions commonly employed in
different industries, such as low water activity, high temperature, alkaline pH, and the presence of detergents, surfactants and
organic solvents. The activity and stability of enzymes are important parameters to determine the economic feasibility in industrial
processes. High stability is critical from the economic point of view
due to reduced enzyme turnover [11]. Therefore, prior to proposing any novel enzyme preparation, it is necessary to determine
the enzyme stability and activity under different and concomitant extreme conditions. Since proteases are the most sought after
enzymes by different industries, obtaining pure protease preparations that are both stable and active under multiple extreme
conditions such as high salt concentrations, alkaline pH, elevated
temperature in addition to the presence of surfactants and oxidizing agents is scientically and industrially signicant.
The goal of the present study was to develop a purication protocol as well as the elucidation of biochemical properties of an
extracellular protease, AbCP, from the novel, halophilic, alkalithermophilic Alkalibacillus sp. isolated from the alkaline, hypersaline
lakes of the Wadi An Natrun, Egypt. AbCP was characterized under
different conditions of salt concentration, pH, temperature, surfactants, detergents and oxidizing agents.
2. Experimental
2.1. Organisms
2.1.1. Isolation and partial characterization
Protease producing strains were isolated from mixed watersediment samples collected from three of the largest lakes of the
Wadi An Natrun in North-western Egypt. Samples were collected
from Lake Fazda (30 19 43.5 N, 30 24 29.68 E), Lake UmRisha
(30 20 48.70 N, 30 23 08.14 E), and Lake Hamra (30 23 48.28 N,
30 19 13.39 ) during November of 2010.
For isolation of protease producing microorganisms, mixed
water-sediment (5 g wet weight) was inoculated into enrichment
medium containing, in g L1 : yeast extract, 5; peptone, 5; KH2 PO4 ,
1; MnCl2 , .018; FeCl2 , 0.025; MgCl2 , 0.5, KCl, 1; NaCl, 100; Na2 CO3 ,
20. The pH of the medium was 9.0 (at 45 C). Enrichment cultures
were incubated at 50 C for 4 days and then transferred into the
same medium three successive times. Pure isolates were obtained
by repetitive dilution to extinction in enrichment medium containing 1% (wt./vol.) agar. Pure isolates were maintained on enrichment
medium containing 1% (wt./vol.) agar. Pure isolates were screened
for extracellular protease production by the protease assay using
the FolinCiocalteau reagent. The isolate showing the maximum
activity was designated strain NM-Fa4, and was used for further
studies. Strain NM-Fa4 was isolated from Lake Fazda.
Preliminary taxonomic characterization was based on cultural
characteristics and 16S rRNA gene sequencing.
2.1.2. Genomic DNA extraction and purication
Isolate NM-Fa4 was grown in 100 mL of enrichment medium
for 4 days at 50 C. Cells were harvested by centrifugation and
washed three times in TrisEDTA buffer (pH 8.0). Genomic DNA was
extracted by the CTAB/NaCl described by Wilson [22].
2.1.3. 16S rRNA gene amplication, sequencing and phylogenetic
analysis
The 16S rRNA gene was amplied by PCR using universal
primers 27F (5 -AGA GTT TGA TCM TGG CTC AG-3 ) and 1492R
(50-GGT TAC CTT GTT ACG ACT T-3 ), and DreamTaqTM Green

75

DNA polymerase (Fermentas). Sequencing of the PCR products

was accomplished using a 3730xl capillary DNA analyzer (Applied
Biosystems) operated at the Georgia Genomics Facility (University of Georgia, Athens, GA USA). The complete 16S rRNA
gene sequence for strain NM-Fa4 was compared with GenBank
entries by BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
The complete gene sequence was aligned with closely related
sequences using the ClustalX program (http://bips.u-strasbg.fr/
fr/Documentation/ClustalX/). Phylogenetic trees were constructed
using the PHYLIP software package (http://evolution.genetics.
washington.edu/phylip.html). Distances were calculated by using
the JukesCantor algortithm of DNADIST, and branching order
was determined via the neighbor-joining algorithm of NEIGHBOR.
Each tree was a consensus of 100 replicate trees. The 16S rRNA
gene sequence was submitted to GenBank under accession number
KF537622.
2.2. Protease assay
Protease activity was measured by the method described by
Anson [23] using casein as a substrate. Puried enzyme (45 g)
was added to assay mixture consisting of 50 mM TrisCl, pH 9.0
and 1.7 M NaCl. The reaction (200 L total volume) was started
with the addition of casein (0.2% wt./vol., nal concentration).
Reaction mixtures were incubated at 50 C for 20 min. The reaction was stopped by addition of 340 L of 110 mM trichloroacetic
acid solution and centrifuged at 10,000 rpm for 5 min. To 400 L
of supernatant, 800 L of 500 mM Na2 CO3 and 200 L of 0.5 N
FolinCiocalteu reagent were added and mixed thoroughly. Color
developed after incubation at room temperature for 10 min was
measured at 660 nm. All assays were done in triplicate. A negative
control (no protein added) was run for all assays to correct for nonspecic degradation of the substrate. One unit of protease activity
was dened as the amount of enzyme yielding 1 mol of tyrosine
per minute at pH 9.0, 50 C in the presence of 1.7 M NaCl. Specic
activity of protein is expressed as the units of enzyme activity per
milligram protein.
2.3. Protein determination
Protein concentration of samples was determined by the
method of Bradford [24]. Bovine serum albumin was used as a
standard. Protein concentration during different steps of purication was determined by absorbance at 254 nm.
2.4. Enzyme purication
2.4.1. Enzyme production and precipitation
Strain NM-Fa4 was grown in enrichment medium for 4 days
at 50 C with constant shaking (225 rpm) in an InnovaTM 4000
incubator shaker (New Brunswick, NJ). Cells were separated by
centrifugation at 4000 rpm for 20 min. The cell-free supernatant
was mixed with 0.8 volume of ice-cold ethanol and incubated at
20 C overnight. The precipitate was collected by centrifugation.
The enzyme was recovered by re-suspending the precipitate in a
minimal volume of 50 mM TrisCl buffer at pH 9.0. The protein
was dialyzed against the same buffer overnight to remove residual
ethanol and salts.
2.4.2. Anion exchange chromatography
The dialyzed protein was subjected to anion exchange
chromatography on a Q-sepharose column (10 cm 1.6 cm, GE
Healthcare) which had been equilibrated with 50 mM TrisCl,
buffer, pH 9.0. Protein was fractionated by gradient elution using
50 mM TrisCl buffer, pH 9.0, containing an increasing concentration of NaCl (02 M). Fractions were collected at a ow rate

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N.M. Mesbah, J. Wiegel / Journal of Molecular Catalysis B: Enzymatic 105 (2014) 7481

of 2.5 mL/min. Protein content (absorbance at 254 nm) and protease activity were determined. Fractions showing the highest
protease activity were pooled, desalted by dialyzing overnight
against 50 mM TrisCl buffer, pH 9.0, and concentrated by ultraltration (Vivaspin, Sartorius AG). Purity of the protein was checked
by SDS-PAGE.
2.5. Sodium dodecyl sulfate-polyacrlyamide gel electrophoresis
(SDS-PAGE) and zymography
SDS-PAGE was carried out for the control of the purity and
the determination of enzyme molecular weight as described by
Laemmli [25], in a discontinuous system made of a 4% stacking gel
and 12% separating gel. Samples were heated at 100 C for 3 min
in Laemmli sample buffer before electrophoresis. The molecular
weight of the enzyme was estimated using an EZ-Run TM Rec protein
ladder (Fisher Bioreagents).
Zymography was performed on SDS-PAGE by the method of
Schmidt et al. [26], with modications. Polyacrylamide gels (12%)
were copolymerized with 0.1% gelatin. Samples were mixed with
sample buffer without heat denaturation, and electrophoresed at
125 V. After electrophoresis, the gels were washed once in 50 mM
TrisCl buffer, pH 9.0, then immersed in the same buffer containing 2.5% (wt./vol.) Triton-X-100 at room temperature with shaking
for 30 min to remove SDS. Triton-X-100 was removed by washing the gels three times in 50 mM TrisCl, pH 9.0 (10 min each). The
gels were then incubated in 50 mM TrisCl buffer, pH 9.0 containing
1.7 M NaCl at 50 C for 30 min. Finally, the gels were stained with a
0.05% (wt./vol.) solution of Coomassie blue R (10% acetic acid, 50%
methanol) for 30 min, then destained (7% acetic acid, 5% methanol)
for 2 h. Proteolytic activity was observed as a clear band of lysis
against a uniformly blue background.
2.6. Biochemical properties of puried protease
2.6.1. Effect of [Na+ ] on enzyme activity
To determine the effect of [Na+ ] on protease activity, the rate of
casein degradation with puried enzyme was evaluated at different
NaCl concentrations ranging from 0 to 3.2 M.
2.6.2. Effect of pH on enzyme activity and stability
To determine the effect of pH on protease activity, protease
activity was tested over a pH range 7.012.0 (measured at 45 ). For
study of pH stability, the enzyme was incubated for 48 h at room
temperature in buffer adjusted to the test pH, and residual proteolytic activity was assayed using standard assay. The buffer system
used was 50 mM Tris (pH 7.09.0) and 50 mM glycineNaOH (pH
9.012.0).
2.6.3. Effect of temperature on enzyme activity and stability
Protease activity was tested at different temperatures ranging
from 30 to 80 C for 20 min at pH 9.0. Heat stability of the puried protease was determined after incubation at 35, 40, 45, 50,
55 and 65 C for 120 min. Residual proteolytic activity was determined using the standard assay at 50 C. Enzyme that had not been
incubated served as the 100% control.

to identify potential activators or inhibitors. The effect of chloride

salts of K+ , Mg2+ , Ca2+ , Fe2+ , and Hg2+ on protease activity was
determined at 1, 5 and 10 mM cation concentration. Activity in the
absence of any cations was taken as 100%.
2.6.5. Effect of surfactants and detergents
Effect of the surfactants Triton-X-100 (Fluka), Tween 20
(SigmaAldrich), Tween 80 (Oxford chemicals), SDS (Fluka), the
oxidizing agent H2 O2 , and the chemical denaturant urea on enzyme
stability and activity was studied by pre-incubating the enzyme
for 20 min at 50 C in the presence of 1% and 5% (vol/vol) nal
concentration of each agent. Residual activity was measured at
pH 9.0, 50 C. The activity of the enzyme without any surfactant/denaturant was taken as 100%.
2.6.6. Effect of organic solvents
Proteolytic activity was measured by the standard assay in the
presence of 50% organic solvents vol/vol at 50 C in 50 mM TrisCl
buffer, pH 9.0. Ethanol, methanol, isopropanol, butanol, benzene,
chloroform and hexane were used as organic solvents in the reaction mixture. Stability is expressed as the remaining proteolytic
activity relative to a non-solvent control.
2.6.7. Protease activity against different substrates
Substrate specicity was studied using different protein substrates including casein, gelatin, bovine serum albumin, wheat
gluten, skimmed milk, yeast extract, Casamino acids, tryptone,
peptone and hemoglobin. Each substrate was added to a nal concentration of 0.5% (wt./vol.).
2.6.8. Amidolytic activity toward synthetic substrates
Protease activity was determined by measuring p-nitroaniline
liberation from the synthetic peptide substrates l-lysine pnitroanilide, N-succinyl-l-phenylalanine-p-nitroanilide, l-alanine
p-nitroanilide, l-leucine p-nitroanilide, and l-glutamic acid -(4nitroanilide). All synthetic peptides were purchased from Sigma
Aldrich. A stock of 20 mM solution of each substrate was prepared
in 50 mM TrisCl, pH 9.0. Where necessary, the stock solution was
prepared by dissolving the required amount in a minimal amount of
ethanol and made up to nal volume with 50 mM TrisCl, pH 9.0. The
reaction mixture consisted of 50 mM TrisCl, pH 9.0, 1.7 M NaCl and
10 mM synthetic peptide substrate. Reactions were started with
the addition of enzyme (45 g), and incubated at 50 C for 20 min.
Each reaction was terminated by addition of acetic acid solution
(30% v/v) to a nal concentration of 6%, and liberated p-nitroaniline
was monitored by absorbance at 410 nm. No-enzyme controls as
well as reactions containing an equivalent amount of ethanol were
run to account for non-specic degradation of the substrate and
potential stimulation of the enzyme by ethanol. One unit of enzyme
activity is dened as the amount of enzyme yielding 1 mol of pnitroaniline per minute at pH 9.0, 50 C in the presence of 1.7 M
NaCl. Specic activity of protein is expressed as the units of enzyme
activity per milligram protein
2.7. Determination of kinetic parameters

2.6.4. Effect of metal ions and enzyme inhibitors

All inhibitors were purchased from SigmaAldrich. Effects of different inhibitors such as PMSF, EDTA, DTT, iodoacetic acid and -ME
on activity of the protease were determined. The puried protease
was incubated at 50 C for 20 min with each inhibitor at nal concentrations of 2 and 5 mM (1 and 5% vol/vol for -ME). The protease
assays were started by addition of casein substrate.
The effect of metal ions on puried protease activity was tested
by the standard protease assay in the presence of different ions

The effect of increasing substrate concentration on velocity of

the enzyme-catalyzed reaction was assayed at pH45 C 9.0, 50 C

in the presence of 1.7 M NaCl and with casein as a substrate. The
concentration of the casein substrate was increased from 0 to
30 g/100 mL. The value of the Michealis Menten constant (Km ) and
maximal velocity were determined from the LineweaverBurke
plot. The catalytic constant kcat was calculated by dividing the Vmax
by molar concentration of enzyme.

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100
100

77

Alkalibacillus salilacus DSM 16460T(HM179185)

Alkalibacillus halophilus DSM 17369T (DQ359731)

Alkalibacillus flavidus DSM 23520T (EU874387)

73

Alkalibacillus silvisoli DSM 16460T (AB264528)

Alkalibacillus filiformis DSM 15448T (AJ493661)

68
66

Alkalibacillus haloalkaliphilus DSM 5271T (NR041985)

Alkalibacillus sp. NM-Fa4 (KF537622)
Bacillus subtilis DSM 10T (AJ276341)
0.01

Fig. 1. Neighbor-joining tree based on 16S rRNA gene sequences showing the position of strain NM-Fa4 in relation to the type species of the genus Alkalibacillus.
GenBank accession numbers for the sequences are shown in parentheses. The tree
was rooted with the 16S rRNA gene sequence of Bacillus subtilis DSM 10T as outgroup. Numbers at nodes denote bootstrap values based on 100 replicates. Bar, 1
nucleotide substitution per 100 bases.

2.8. Whole genome sequencing, identication and analysis of

putative protease gene
Genomic DNA was extracted from one liter of isolate NM-Fa4 by
the CTAB/NaCl method of Wilson [22]. Whole genome sequencing
was performed with an Illumina MiSeq instrument operated by
the Georgia Genomics Facility at the University of Georiga (see
http://dna.uga.edu/illumina-library-preparation/overview,
for
details). The Velvet and A5 pipelines were used for assembly
[27,28] and the FastX-Toolkit was used for assessment of data
quality (http://hannonlab.cshl.edu/fastx toolkit/). Genes were
identied using the RAST 9 pipeline [29] and manual curation.
Sequencing data were manually examined for putative proteases. The putative amino acid sequence of the protease was
compared against the National Center for Biotechnology Information (NCBI) protein database using the Basic Local Alignment Search
tool for proteins (BLASTp). Multiple sequence alignment was performed with ClustalW.
3. Results
3.1. Partial characterization of strain NM-Fa4
Halophilic, alkalithermophilic bacterium NM-Fa4, stains Grampositive and is an aerobic, rod-shaped rmicute. According to the
16S rRNA gene sequence, the strain was identied as Alkalibacillus
sp., and was related to Alkalibacillus haloalkaliphilus (97% sequence
identity) (Fig. 1) [30].
Strain NM-Fa4 grows well in enrichment medium between 35
and 53 C with optimal temperature between 47 and 50 C. There
was no growth at 55 C. Strain NM-Fa4 grew at NaCl concentrations
between 0.9 and 3.4 M, with an optimum at 1.7 M, and between the
pH range 8.5 and 10, with an optimum at pH 9.

Fig. 2. SDS PAGE of puried protease, AbCP. (A) Assessment of homogeneity and
molecular weight of AbCP by 12% SDS PAGE. Lanes 13 represent: molecular
weight marker, ethanol precipitated protein, AbCP (30 g). (B) Zymogram of AbCP.
(C) Zymogram of supernatant from Alkalibacillus sp. NM-Fa4 culture. The white,
unstained region in the gel shows hydrolysis of gelatin by the enzyme.

hydrolysis on the gelatin zymogram, indicating at least 6 proteases

produced by the strain (Fig. 2C). Two of these bands, sized approximately 40 and 50 kDa, did not appear on the SDS-PAGE with ethanol
precipitated protein, indicating that they were not precipitated by
treatment of the culture supernatant with ethanol.
3.3. Effect of sodium chloride concentration on enzyme activity
AbCP showed maximum activity at 1 M NaCl, and retained 61%
of activity at 3.1 M NaCl. The enzyme showed 25% relative activity
in the absence of NaCl and lost proteolytic activity when the NaCl
concentration exceeded 3.4 M (Fig. 3).
3.4. Effect of pH and temperature on enzyme activity and stability
AbCP showed maximum activity at pH value of 9.5 and retained
greater than 50% of activity over the pH range 711.5 (measured
at 45 C) (Fig. 4). AbCP showed great stability when incubated for
48 h at room temperature over the pH range 8.512, retaining over
95% of initial activity at pH values 8.59.5. AbCP retained 50% of
its proteolytic activity after incubation at pH 12 for 48 h at room
temperature (Fig. 4).
AbCP showed maximal activity at 5052 C, and was active
between 30 and 70 C (Fig. 5). Relative activities at 37 and 62 C
were 69 and 60% respectively. Activity decreased beyond 65 C but
AbCP retained 32% activity at 70 C (Fig. 5). Heat stability assays
of the puried enzyme showed that it retained more than 99% of
activity after incubation at 45 C for 2 h. At 50 C, AbCP retained
more than 84% of activity after incubation for 2 h. More than 50%

3.2. Enzyme purication and molecular weight

Extracellular protease from strain NM-Fa4 was precipitated
from 700 mL of culture supernatant by a treatment with 0.8
volumes of ice-cold ethanol and incubation at -20 C overnight,
yielding a 4.5 fold purication and 48.4% enzyme yield (Table 1).
The precipitated fraction was dialyzed against 50 mM TrisCl buffer
(pH 9.0) and subjected to anion exchange chromatography on a
Q-sepharose column leading to 9.8 fold purication (Table 1).
The enzyme migrated as a single band approximately 20 kDa
in SDS-PAGE, and was designated AbCP (Fig. 2A). Gelatin zymography conrmed the proteolytic nature of the protein (Fig. 2B).
The enzyme also migrated as a single band, approximately 20 kDa
in size on a native polyacrylamide gel, indicating homogeneity of
the protein. The culture supernatant showed at least 6 zones of

Fig. 3. Effect of NaCl on protease activity. Assays were performed at pH45 C 9.0 and
50 C. Activity at 1 M NaCl was taken as 100%, and was equivalent to 84 U/mg. Each
value represents the mean of three assays, bars denote standard error.

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Table 1
Purication of protease from Alkalibacillus sp. NM-Fa4.
Purication step

Total enzyme (U)

Culture supernatant
Ethanol precipitate
Q-sepharose

1323
640
205

Total protein (mg)

140
15
2.2

Specic activity (U/mg)

Fold purication

Yield (%)

9.5
42.7
93.2

1
4.5
9.8

100
48.4
15.5

Table 2
Stability and activity of protease in the presence of enzyme inhibitors.
Relative activity (%) standard errora

Inhibitor

Control
PMSF
EDTA
DTT
Iodoacetic acid
-ME

2 mM

5 mM

100 0.9
99 0.7
117 2.6
172 3.3
32 0.9
146 3.5(1% vol/vol)

100 1.4
94 2.2
94 2.3
130 3.8
30 0.5
167 4.6(5% vol/vol)

a
Assays were performed at 50 C, pH 9.0 in the presence of 1.7 M NaCl. One
hundred percent activity was equivalent to 81 U/mg.

Fig. 4. Effect of pH45 C on protease activity () and stability (), at 50 C in the pres
ence of 1.7 M NaCl. Activity at pH45 C 9.5 was taken as 100%, and was equivalent to
76 U/mg. For assays of pH stability, the enzyme was incubated at room temperature in buffer adjusted to the test pH for 48 h, then activity was assayed at 50 C
after addition of substrate. Each value represents mean of three assays, bars denote
standard error.

of the original activity was retained after 2 h of incubation at 55, 60

and 65 C (Fig. 5).

Table 3
Effect of metal ions on protease activity.
Metal Ion

Relative activity (%) standard errora

Control
KCl
MgCl2
CaCl2
FeSO4
HgCl2

100
138
114
157
123
80

1 mM

1.5
4.4
4.5
5.0
3.8
2.9

5 mM

10 mM

100 2.2
69 1.4
119 2.3
119 4.2
155 2.6
No activity

100 2.5
64 1.3
165 1.8
74 1.6
82 3.2
No activity

a
Assays performed at 50 C, pH 9.0, in the presence of indicated concentration of
cation. Control was performed in the absence of cations. Protein preparations were
dialyzed before the assay. One hundred percent activity was equivalent to 86 U/mg.

3.5. Effect of metal ions and enzyme inhibitors

At pH45 C 9.0 and 50 C in the presence of 1.7 M NaCl, AbCP

was inhibited by the cysteine protease inhibitor iodoacetic acid,
retaining only 30% of initial activity in the presence of 5 mM of the
inhibitor, whereas EDTA, DTT, and -ME had stimulatory effects on
activity (Table 2). The serine protease inhibitor PMSF had only a
slight effect on activity, the enzyme retained 94% of initial activity
in the presence of 5 mM of the inhibitor.
No activity was observed in the presence of 5 mM of HgCl2 , but
the enzyme retained 80% of its activity at 1 mM of HgCl2 (Table 3).
KCl was inhibitory to the enzyme at concentrations greater than
1 mM, where as CaCl2 and FeSO4 were inhibitory at concentrations
greater than 5 mM. AbCP was also inhibited in the presence of 5 mM

of Zn2+ , Cs2+ , Ba2+ , Cu2+ , Sr2+ , Mn2+ , Ag+ , and Al3+ , retaining less
than 15% of its initial activity. However, MgCl2 had a stimulatory
effect, addition of 10 mM to the reaction mixture increased the protease activity by 65% compared with a control with no Mg2+ added
(Table 3).
3.6. Effect of surfactants and detergents

At pH45 C 9.0, 50 C and 1.7 M NaCl, activity of AbCP was stable

and enhanced in the presence of the anionic surfactant SDS and
the oxidizing agent hydrogen peroxide, showing 197 and 134% of
its initial activity after pre-incubation with 5% (vol/vol) of either
agent, respectively (Table 4).
The enzyme was stimulated slightly in the presence of 1%
(vol/vol) Triton-X-100, but was completely inhibited in the presence of 5% of the detergent. AbCP was sensitive to the polysorbate
surfactants Tween 20 and Tween 80, retaining only 44 and 55% of its
Table 4
Activity of protease in the presence of different surfactants and detergents.
Agent

Fig. 5. Effect of temperature on protease activity () and stability () at pH45 C 9.0
and 1.7 M NaCl. Activity at 50 C was taken as 100%, and was equivalent to 90 U/mg.
For assays of temperature stability, the protein was incubated at the specic temper
ature at pH45 C in the presence of 1.7 M NaCl for 2 h, and then activity was assayed
at the respective temperature after addition of substrate. Each value represents the
mean of three assays, bars denote standard error.

Control
SDS
Triton-X-100
H2 O2
Tween 20
Tween 80
Urea

Relative activity (%)a

1%

5%

100 2.2
166 3.3
112 0.6
112 3.3
53 2.1
94 3.4
100 2.3 (2 M)

100 1.7
197 4.5
No activity
134 2.4
44 2.0
55 2.2
144 3.2 (4 M)

a
Assays performed at 50 C, pH 9.0 in the presence of 1.7 M NaCl. One hundred
percent activity was equivalent to 82 U/mg.

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79

Table 5
Effect of organic solvents on protease activity.
Solventa

Relative activity (%) standard errorb

Control
Ethanol
Methanol
Isopropanol
Butanol

100
123
74
47
37

3.2
2.6
2.1
0.6
1.3

Solvents were added to 50% (vol/vol) nal concentration.

Assays performed at 50 C, pH 9.0 in the presence of 1.7 M NaCl. One hundred
percent activity was equivalent to 82 U/mg.
b

initial activity in the presence of 5% of either surfactant. The enzyme

was stable and activity was stimulated by the chemical denaturant
urea, showing 144% of its initial activity in the presence of 4 M urea
(Table 4).

Fig. 6. Effect of casein substrate concentration on the reaction velocity of AbCP.

Values for Km and Vmax were calculated by linear regression analysis of the data
using Microsoft Excel.

3.7. Effect of organic solvents

AbCP activity was enhanced in the presence of ethanol showing
123% of its initial activity, and retained 74% of activity in the presence of methanol (50% vol/vol nal concentration of each solvent,
Table 5). The enzyme lost over 50% of activity in the presence of
isopropanol and butanol (Table 5) and showed no activity in the
presence of benzene, chloroform or hexane.
3.8. Determination of protease specicity
AbCP showed strong hydrolytic activity against casein, peptone,
skim milk, bovine serum albumin (BSA) and wheat gluten (Table 6),
and was particularly active on gelatin, exhibiting a specic activity
of 182 U/mg. AbCP did not show any activity with yeast extract
or hemoglobin, and weak activity against tryptone and Casamino
acids (Table 6).
Protease specicity in the P1 position was analyzed using different synthetic p-nitroanilide substrates. AbCP showed broad
specicity against synthetic substrates with the aromatic amino
acid N-succinyl-l-phenylalanine (specic activity 77 2 U/mg), the
hydrophobic amino acid l-alanine (specic activity 67 1.7 U/mg),
and the basic amino acid l-lysine (specic activity 66 0.5 U/mg).
A slightly weaker amidolytic activity was seen with the pnitroanilides containing hydrophobic l-leucine (specic activity
46 3 U/mg) and the acidic l-glutamic acid (specic activity 42 3 U/mg). These data indicated that AbCP prefers both
hydrophilic and hydrophobic residues. Moreover, the enzyme
showed good activity with a p-nitroanilide containing a bulky aromatic group.
3.9. Kinetic parameters
Using casein as a substrate, AbCP obeyed MichealisMenten
kinetics (Fig. 6). The value of the Km obtained from linear

regression
from
the
LineWeaver
Burke
plot
was
1.3 0.007 mg/mL. The protease had a Vmax of 1111 mg/ml/min.
3.10. Whole genome sequence analysis of Alkalibacillus sp.
NM-Fa4 and identication and analysis of putative protease gene
The genome of Alkalibacillus sp. NM-Fa4 has an estimated size
of 6 Mbp and contains 9202 putative coding sequences. Fortyone putative proteases were annotated in the Alkalibacillus sp.
NM-Fa4 genome, including 8 putative serine alkaline proteases,
5 putative metalloproteases, 2 putative cysteine proteases and
5 putative carboxyl-terminal proteases. The remaining putative
enzymes belonged either to the CAAX amino terminal protease
family, membrane bound zinc metalloprotease family (htpX), and
Lon-like proteases. Of the two putative cysteine proteases identied, one had a predicted molecular weight of 86.2 kDa, and the
other 19.7 kDa. Analysis of the puried protein by both native and
SDS-PAGE and experimental data, suggest that the latter protein
was AbCP. This conclusion was further supported by that the other
proteases annotated had estimated molecular weights greater than
30 kDa. The putative amino acid sequence of AbCP was 177 amino
acids long and has an estimated isoelectric point of 4.84 (estimated
by the Compute pI/Mw tool at www.expasy.org). BLAST analysis of
the amino acid sequence showed that it had 84% sequence identity with a putative cysteine protease from Alkalilimnicola ehrlichii
MLHE-1 (WP 0011628525) and 73% identity to putative cysteine
protease from Methylomonas methanica (WP 013819676). In addition, AbCP exhibited 70 and 60% sequence identity with cysteine
protease from Methylophilus methylotrophus (WP 01897339) and
Teredinibacter turnerae (WP 018274867), respectively. Catalytic
residues Gln42 , Cys79 , His112 , and Asp145 [31] are present in AbCP,
and are also found in other cysteine proteases analyzed (Fig. 7).
4. Discussion

Table 6
Substrate specicity of protease.
Substratea

Specic activity (U/mg) standard errorb

Casein
Peptone
Skim milk
Casamino acids
Tryptone
BSA
Wheat gluten
Gelatin

88
95
81
60
2.2
95
95
182

a
b

1.4
1.1
0.5
0.4
0.9
0.6
1.4
3.2

Substrates added to 0.5% (wt./vol.) nal concentration.

Assays performed at 50 C, pH 9.0 in the presence of 1.7 M NaCl.

Halophilic alkalithermophiles are an unusual group of

extremophiles capable of robust growth under the combined
extremes of high salinity, alkaline pH and elevated temperature
[32]. Halophilic alkalithermophiles are potential sources for novel
enzymes that are stable and function under multiple extreme
conditions where other enzymes would be inactivated and/or
denatured.
Alkalibacillus sp. NM-Fa4, isolated from the alkaline, hypersaline
Wadi An Natrun lakes, produced an alkaline, thermostable, saltstable, surfactant, denaturant and oxidizing agent stable protease,
AbCP.

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80

N.M. Mesbah, J. Wiegel / Journal of Molecular Catalysis B: Enzymatic 105 (2014) 7481

AbCP
A. ehrlichii
Me. methanica
M. methylotrophus
T. turnerae

AbCP
A. ehrlichii
Me. methanica
M. methylotrophus
T. turnerae

*
20
*
40
*
60
*
MEVQDLPDDVLTYLLPSRYCESDRFMQMAWSIVGDARPGHDQVARITDWIRENVRFNPDSPHFQLSAVELNQARE
VEVQDLPDDALTYLLPSRYCESDRFLDMAWSIVGDAHPGYDQVARIEAWIREHVHFNPDSPHFQLSAVELNQARE
EEVQNLPNAVLTYLLPTRYCESDRFIDLARELVEHALPGYDQVAAINEWIRQNVRFNPDSPYFQMSAVEVNQARE
EEIQHLPPHVLTYLLPSRYCESDKFIDLSKEIVAHVQPGYDQVSAIEQWIRQNIRFNPDSPHFQLSAVEVNQQRE
ISVQNLPDSVLKYLLPSRYCESDCFNEMATEITQNQSPGYDQVAAVEHWLRQTIRFEPGSSSFPSSAIEVNKVRV
e6Q LP vLtYLLP3RYCESD F 6a 6v
PGyDQVa 6 W6R2 6rFnPdSp Fq SA6E6Nq Re
80
*
100
*
120
*
140
*
GVCREFAHLGIALCRGLCIPARMVVGFLHGLEPMDFHAWFEAYIGGRWVAFDATQPEDCGKRVTVACGRDAADVA
GVCREFAHLGIALCRALCVPARMAVGYLHGLQPMDFHAWFEAYVGGRWCVFDATQPPGRRGRVTVAYGRDAADVA
GVCRDLAHLGIALCRALCIPARMVVGYLHGLQPMDFHAWFEAYVGGRWYSFDPTQPEPRGGRVVVAYGHDAADVA
GVCRELAHLGIALCRGLCIPARMVVGYLHGLQPMDFHAWFEAYVNGRWYTFDPSQSESVGGRITVAYGRDAADVA

160

IFNQFGPPMYPTRIGVTVEEVEGPPAV
AbCP
IYNQFGPPMYPTRISVTVDRLEGPPAV
A. ehrlichii
IFTQFGPALSPIHMAVTVEALSGPPEMe. methanica
M. methylotrophus IFNQFGPALYPEVMQVRVTQLAAAPVIYNQFGPPVFPYEEAVQVTLSDSS--T. turnerae
Fig. 7. Multiple sequence alignment of the putative amino acid sequence of AbCP with those of putative cysteine proteases from Alkalilimnicola ehrlichii (WP 0011628525),
Methylomonas methanica (WP 013819676), Methylophilus methylotrophus (WP 01897339) and Teredinibacter turnerae (WP 018274867). Catalytic residues are highlighted in
black. Gray blocks denote highly conserved regions.

AbCP showed activity over a broad range of sodium chloride

concentrations, pH values and temperatures. Few studies report
on proteases that are concomitantly salt, alkaline and temperature stable. Bacillus cereus S1U1 produces an extracellular protease
active and stable at pH 9.0 and a temperature range of 4555 C,
and retained 67% of activity at 1.2 M NaCl [33]. Bacillus sp. SM2014
produced a protease showing optimal activity at pH 1011 and 60
to 70 C. The enzyme showed optimal activity at 0.2 M NaCl, and
retained 93% of activity at 1.5 M NaCl [21]. The serine protease from
Bacillus subtilis AP-MSU 6 showed optimal activity at pH 9.0, 0.5 M
NaCl and 40 C, and rapidly lost activity at temperatures greater
than 45 C and NaCl concentrations greater than 0.5 M [15]. Bacillus sp. EMB9 produced a serine protease with a pH optimum of
9.0, temperature optimum of 55 C, but a NaCl optimum of 0.2 M
[34].
AbCP was stable in the presence of the metal chelator EDTA and
the inhibitor PMSF indicating that it is neither a metalloprotease
nor a serine protease. The reducing agents DTT and -ME had a
stimulatory effect on activity. This, combined with partial inhibition
of the enzyme with iodoacetic acid, suggests that AbCP is a cysteine
protease. Cysteine proteases catalyze hydrolysis through reaction
of the cysteine thiol with the amide carbonyl of the substrate. The
thiol is rendered nucleophilic through acid/base coordination with
a histidine imidazole ring [35]. The active site of cysteine proteases
is readily oxidized, hence these enzymes are most active in the presence of reducing agents [36]. Iodoacetic acid irreversibly inhibits
cysteine protease by alkylation of the catalytic cysteine residue.
Bacterial cysteine proteases described are important virulence factors in pathogens where they are known to degrade a variety of host
proteins [3740]. These cysteine proteases generally have acidic
and neutral pH optima. A cysteine protease from an environmental isolate Bacillus sp. 17N-1 was described. However, it was only
slightly alkaliphilic showing activity over the pH range 6.58.5 with
maximal activity at pH 7.0 [41].
AbCP exhibited remarkable stability in the presence of surfactants and different metal ions; retaining full activity and in some
cases being stimulated by denaturants, oxidizing agents and surfactants. AbCP was also stable in the presence of different alcohols.
Activity and stability at high temperature, alkaline pH values, a
broad range of sodium chloride concentrations in addition to high
concentrations of denaturants, detergents, oxidizing agents and
metal ions may be distinct features of this enzyme. The cysteine

protease from Bacillus sp. 17N-1 was found to be active in the presence of dimethylsulfoxide and acetonitrile. However, this enzyme
was active only over the temperature range of 1433 C and the
NaCl range 0.20.8 M [41].
Proteases are the largest selling industrial enzymes, with
current applications in detergent formulations, and anticipated
applications in protein processing and peptide synthesis. However, the above applications require that the enzymes be stable in
salts and organic solvents [42]. Other industrial applications such
as food processing and environmental bioremediation also necessitate proteases that are not only salt stable, but also tolerant to
high temperatures and stable in the presence of organic solvents
[43]. In the food industry, salt and alkaline stable proteases have
potential applications in fermentation. During the processing of
soy sauce, proteolytic modication of soy proteins by proteases
results in production of soluble hydrolysates with low bitterness
[44]. Industries which include processes such as pickling as well
as oil and gas recovery processes lead to the production of wastewater brines [45]. Enzymes that retain activity in the presence of
high salt concentrations as well as a wide range of pH values can
facilitate remediation of these wastewaters in an environmentally
sound manner. Aerobic treatment systems for biological treatment
of industrial wastewaters with salt concentration up to 1.5 M have
been developed, but only produce satisfactory results at salt concentrations around 0.5 M [45,46]. Addition of enzyme preparations
that are both salt stable and temperature tolerant could increase
the efcacy of such bioreactors.

5. Conclusions
To conclude, the present study describes purication and
characterization of a new protease, named AbCP, produced by Alkalibacillus sp. NM-Fa4. AbCP shows many differences from other
described cysteine proteases in terms of maximal pH, temperature and salt concentration for activity, in addition to stability
in the presence of denaturants, surfactants, oxidizing agents and
metal ions. Activity of AbCP over a wide range of sodium chloride
concentrations, temperatures and alkaline pH values makes the
enzyme a potential efcient choice in chemical, pharmaceutical and
biotechnological industries where resistance to harsh conditions is
required.

Author's personal copy

N.M. Mesbah, J. Wiegel / Journal of Molecular Catalysis B: Enzymatic 105 (2014) 7481

Acknowledgements
This work was supported by the US-Egypt Science and Technology Joint Fund in cooperation with the Suez Canal University
(Egypt) under project number 1841 and the University of Georgia
(USA) under project number NSF-OISE-1132412.
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