International Journal of Biological Macromolecules 72 (2015) 1117–1128

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

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

Purification, characterization, and molecular cloning of an

extracellular chitinase from Bacillus licheniformis stain LHH100
isolated from wastewater samples in Algeria
Hassiba Laribi-Habchi a,c,∗ , Amel Bouanane-Darenfed b , Nadjib Drouiche a,d,∗ ,
André Pauss e , Nabil Mameri a,d
a
Laboratory of Environmental Biotechnology and Process Engineering, Department of Environmental Engineering, Ecole Nationale Polytechnique,
Avenue Pasteur, Hacène Badi, PO Box 182, El Harrach Algiers, 16200, Algeria
b
Laboratory of Cellular and Molecular Biology, Microbiology Team, Faculty of Biological Sciences, University of Science and Technology of Houari
Boumediene, PO Box 32, El Alia, Bab Ezzouar Algiers, 16000, Algeria
c
Department of Industrial Chemistry, Faculty of Engineering Science, University of Saàd Dahlab of Blida, PO Box 270 Blida, 09000, Algeria
d
Centre de Recherche en technologie des Semi-conducteurs pour l’Energétique (CRTSE). 2, Bd Frantz Fanon BP140, Alger – 7 merveilles, 16038, Algeria
e
Chemical Engineering Department, University of Technology of Compiegne PO Box 20.529, 60205 Compiegne Cedex, France

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

Article history: An extracellular chitinase (ChiA-65) was produced and purified from a newly isolated Bacillus licheni-
Received 22 April 2014 formis LHH100. Pure protein was obtained after heat treatment and ammonium sulphate precipitation
Received in revised form 16 October 2014 followed by Sephacryl S-200 chromatography. Based on matrix assisted laser desorption ionization-time
Accepted 17 October 2014
of flight mass spectrometry (MALDI-TOF/MS) analysis, the purified enzyme is a monomer with a molec-
Available online 27 October 2014
ular mass of 65,195.13 Da. The sequence of the 27 N-terminal residues of the mature ChiA-65 showed
high homology with family-18 chitinases. Optimal activity was achieved at pH 4 and 75 ◦ C. Among the
Keywords:
inhibitors and metals tested, p-chloromercuribenzoic acid, N-ethylmaleimide, Hg2+ , and Hg+ completely
Chitinase
Bacillus licheniformis
inhibited enzyme activity. Chitinase activity was high on colloidal chitin, glycol chitin, glycol chitosane,
Purification chitotriose, and chitooligosaccharide. Chitinase activity towards synthetic substrates in the order of p-
NP-(GlcNAc)n (n = 2–4) was p-NP-(GlcNAc)2 > p-NP-(GlcNAc)4 > p-NP-(GlcNAc)3 . Our results suggest that
ChiA-65 preferentially hydrolyzed the second glycosidic link from the non-reducing end of (GlcNAc)n .
ChiA-65 obeyed Michaelis-Menten kinetics, the Km and kcat values being 0.385 mg, colloidal chitin/ml and
5000 s−1 , respectively. The chiA-65 gene encoding ChiA-65 was cloned in Escherichia coli and its sequence
was determined. Above all, ChiA-65 exhibited remarkable biochemical properties suggesting that this
enzyme is suitable for bioconversion of chitin waste.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Chitin, a linear ␤-1, 4-linked N-acetyl-d-glucosamine (GlcNAc)

Abbreviations: MALDI-TOF/MS, matrix assisted laser desorption ionization-time
of flight/mass spectrometry; GlcNAc, ␤-1,4-linked N-acetyl-d-glucosamine; p-NP,
p-nitrophenyl; BSA, bovine serum albumin; DNS, 5-dinitrosalycilic acid; HEPES,
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; SDS-PAGE, Sodium dodecyl
sulphate-polyacrylamide gel electrophoresis; p-CMB, p-chloromercuribenzoic
acid; NEM, N-ethylmaleimide; DTT, dithiothreitol; 2-ME, 2-mercaptoethanol;
TNBS, 2,4,6-trinitrobenzenesulfonic acid; PMSF, phenylmethylsulfonyl fluoride;
EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; DEP, diethylpyrocarbon-
ate; NBS, N-bromosuccinimide; NAI, N-acetylimidazole; EGTA, ethylene glycol-bis
(␤-aminoethyl ether)-N,N,N ,N -tetraacitic acid; MES, 2-(N-morpholino) ethanesul-
fonic acid.
∗ Corresponding authors. Tel: +213 5 51 43 53 87; fax: +213 021 52 29 73.
E-mail addresses: hassibalar@yahoo.fr (H. Laribi-Habchi),
nadjibdrouiche@yahoo.fr (N. Drouiche).
polysaccharide, is the major structural component of fungal cell
walls, insect exoskeletons, and shells of crustaceans. It is one of
the most abundant naturally occurring polysaccharides and has
attracted tremendous attention in the fields of agriculture, phar-
macology and biotechnology. Most of the chitin in nature has either
an ␣- or a ␤-crystalline structure, with a predominance of the ␣-
form. Each year, a vast amount of chitin waste is released from the
aquatic food industry, where crustaceans (prawn, crab, shrimp, and
lobster) constitute one of the main agricultural products. This cre-
ates a serious environmental problem, because chitin is a rotting
protein. This linear polymer can be hydrolysed by bases, acids or
enzymes, such as lysozyme, some glucanases, and chitinase.

http://dx.doi.org/10.1016/j.ijbiomac.2014.10.035
0141-8130/© 2014 Elsevier B.V. All rights reserved.
1118 H. Laribi-Habchi et al. / International Journal of Biological Macromolecules 72 (2015) 1117–1128
Chitinases are essential glycoside hydrolases that catalyze the
degradation of chitin by cleaving its GlcNAc bond, and endochiti-
nases cleave randomly at internal sites of chitin, generating soluble
low mass multimers of GlcNAc such as chitotetraose, chitotriose,
and chitobiose. Several chitinases have been isolated and charac-
terized from various sources [1–3]. They are abundant in nature,
occurring in plants, animals, viruses, bacteria, fungi, and insects,
and they have various functions, including defense, nutrient diges-
tion, morphogenesis, and pathogenesis [3]. One of the potential
applications of these types of enzymes is for the bioremediation
and bioconversion of chitin wastes from food processing industry
into pharmacological active products, namely N-acetylglucosamine
(NAG) and chito-oligosaccharides. Production of chitin derivatives
with suitable enzyme is more appropriate for a sustainable envi-
ronment than using chemical reactions. In addition, they can be
used as anti-fungal agents and for the preparation of protoplasts
of filamentous fungi [4]. Potential roles of chitinase in bio-control
of insects and mosquitoes and in production of single cell protein
have also been suggested [5]. Thus, there have been many reports
on cloning, expression and characterization of chitinases from var-
ious organisms, including bacteria, fungi, plant and animals [5].
Furthermore, chitinases are essential for the enzymatic production
of (GlcNAc)n and GlcNAc, whose physiological roles are gaining
increasing attention in recent research [6]. Accordingly, research
on chitinases in various organisms should not only clarify these
physiological roles, but should also be of use in the production of
(GlcNAc)n and GlcNAc.
Chitin hydrolyzing enzymes are classified into three categories
(endochitinases [EC 3.2.1.14], exochitinases [EC 3.2.1.29], and N-
acetylglucosaminidases [EC 3.2.1.52]) according to the manner in
which they cleave chitin chains. Endochitinases randomly cleave
␤-1, 4-glycosidic bonds of chitin, whereas exochitinases cleave the
chain from the reducing and non-reducing end to form diacetyl-
chitobiose (GlcNAc2 ). N-acetylglucosaminases hydrolyze GlcNAc2
into GlcNAc or produce GlcNAc from the nonreducing end of N-
acetyl-chitooligosaccharides [7]. To date, various chitinases have
been isolated from some microorganism such as B. cereus [8], B.
licheniformis [9], and Stenotrophomonas maltophilia [10]. The chiti-
nases so far sequenced are classified into two glycosyl hydrolase
families, family 18 and 19, on the basis of the homology of their
amino acid sequences and their catalytic mechanisms [11]. Mem-
bers belonging to chitinase family 18 are widely distributed among
bacteria, fungi, viruses, animals, plants, and other organisms [11].
Family 19 chitinases, on the other hand, are present mainly in
higher-order plants and are reported to have strong antibacte-
rial properties [12]. The chitinases of the two different families do
not share amino acid sequence similarity, have completely differ-
ent three-dimensional (3D) structures and molecular mechanisms,
and are therefore likely to have evolved from different ancestors
[13]. Bacterial chitinases generally consist of multiple functional
domains, such as chitin-binding domain (CBD) and fibronectin type
III-like domain (Fn3 domains), linked to the catalytic domain. The
importance of the CBD in the degradation of insoluble chitin has
been demonstrated for some bacterial chitinase [14]. In addition
to the potential applications of chitinase itself, the (GlcNAc)n have
been found to function as anti-bacterial agents, elicitors, lysozyme
inducers, immuno-enhancers, and natural cancer prevention and
treatment [14]. To prepare chito-oligosaccharides with a specific
degree of polymerization is particularly valuable. Obtaining a chiti-
nase and its corresponding gene is the starting point to pursuing
this goal.
Accordingly, the present study aimed to reports on the purifi-
cation and biochemical characterization of a chitinase enzyme
(ChiA-65) from Bacillus licheniformis strain LHH100. The nucleotide
and amino acid sequences and cloning of the encoding gene (chiA-
65) were also determined. The characterization of its biochemical
properties suggested that this chitinase is appropriate for various
industrial applications, including bioconversion of colloidal chitin
into N-acetyl glucosamine and chitobiose.
2. Materials and methods
2.1. Substrates, enzymes, and chemicals
Chitin from crab shells, chitin azure, glycol chitin, gly-
col chitosan, p-nitrophenyl N-acetylchitooligosaccharides (p-NP-
(GlcNAc)n , n = 1–4), bovine serum albumin (BSA), 5-dinitrosalycilic
acid (DNS), calcofluor white M2R, and Chitinase from Serratia
marcescens (SMChi) were purchased from Sigma Chemical (St.
Louis, MO, USA). Sephacryl S-200 was from Pharmacia (Pharmacia,
Uppsala, Sweden). A protein assay kit was from Bio-Rad Laborato-
ries (Hercules, CA). All of the other chemicals and reagents used
were of analytical grade or the best grade commercially available,
unless otherwise stated.
2.2. Preparation of colloidal chitin
Colloidal chitin was prepared according to the method of
Roberts and Selitrennikoff [15] with some modifications. Briefly,
5 g of chitin from crab shells was gradually added into 100 ml of
cold concentrated HCl with gentle agitation on a magnetic stirrer at
4 ◦ C for 24 h. The mixture was then added to 500 ml of ice-cold 96%
ethanol and left for 24 h with rapid stirring at 4 ◦ C. The precipitate
was harvested by centrifugation at 12,000 g for 25 min at 4 ◦ C and
washed repeatedly with sterile distilled water until the pH reached
6. The colloidal chitin was kept at 4 ◦ C until further use. Approxi-
mately 96 g of colloidal chitin was obtained by this procedure from
5 g of chitin powder.
2.3. Isolation and cultivation of chitinase-producing
microorganisms
Wastewater samples were collected from the aquatic food
industry in Algiers (Algeria) to isolate chitinase-producing microor-
ganisms. The samples were then dispersed in sterile distilled water
and heated 80 ◦ C for 30 min to kill vegetative cells. The heat-treated
samples were then plated onto chitinase-detection agar (CHDA)
plates containing (g/l): peptone, 5; yeast extract, 3; colloidal chitin,
10; and bacteriological agar, 16 at pH 6.5. The plates were incubated
at 37 ◦ C for 72 h to obtain colonial growth. The colonies with a clear
zone formed by the hydrolysis of chitin on the CHDA plate were
evaluated as chitinase producers. Several chitinolytic strains were
isolated, and strain LHH100, which exhibited a large clear zone of
hydrolysis, was selected for further experimental work.
The growth medium used for chitinase production by strain
LHH100 at pH 6.5 consisted of (g/l): chitin colloidal, 10; K2 HPO4 ,
0.65; KH2 PO4 , 1.5; CaCl2 , 0.5; MgSO4 ·7H2 O, 0.12; NaCl, 0.25; NH4 Cl,
0.5; and trace elements 1% (v/v) [composed of (g/l): ZnCl2 , 0.4;
FeSO4 ·7H2 O, 2; H3 BO3 , 0.065; and MoNa2 O4 ·2H2 O, 0.135]. The
Media were autoclaved at 121 ◦ C for 20 min. Cultivations were per-
formed on a rotary shaker (250 rpm) at 37 ◦ C for 48 h and in 500 ml
conical flasks with a working volume of 50 ml. The growth kinetics
was monitored by measuring absorbance at 600 nm. The cell-free
supernatant was recovered by centrifugation (12,000 g, 30 min) at
4 ◦ C, and served as chitinase preparation in subsequent studies.
2.4. Identification of microorganism, DNA sequencing, and
phylogenetic analysis
Analytical profiling index (API) strip tests and 16S rRNA gene
sequencing (ribotyping) were carried out for the identification
of the genus to which the strain belonged. API 50 CH strips
 H. Laribi-Habchi et al. / International Journal of Biological Macromolecules 72 (2015) 1117–1128
(bioMérieux, SA, Marcy-l’Etoile, France) were used to investi-
gate the physiological and biochemical characteristics of strain
LHH100 in accordance with the instructions of the manufacturer.
The 16S rRNA gene was amplified by polymerase chain reac-
tion (PCR) using forward primer, 5 -AGAGTTTGATCCTGGCTCAG-3 ,
and reverse primer, 5 -AAGGAGGTGATCCAAGCC-3 . The genomic
DNA of strain LHH100 was purified by the Wizard® Genomic
DNA Purification Kit (Promega, Madison, WI, USA) and then used
as a template for PCR amplification (35 cycles, 94 ◦ C for 30 s
denaturation, 60 ◦ C for 45 s primer annealing, and 72 ◦ C for 60
s extension). The amplified ∼1.5 kb PCR product was cloned in
the pGEM-T Easy vector (Promega, Madison, WI, USA), leading to
pLHH100-16S plasmid (This study). The E. coli DH5␣ [F− supE44
˚80 ılacZ M15  (lacZYA-argF) U169 endA1 recA1 hsdR17 (rk − ,
mk + ) deoR thi-1 − gyrA96 relA1] (Invitrogen, Carlsbad, CA, USA)
was used as a host strain. All recombinant clones of E. coli were
grown in Lauria-Bertani (LB) media with the addition of ampi-
cillin, isopropyl-thio-␤-d-galactopyranoside (IPTG), and X-gal for
screening. DNA electrophoresis, DNA purification, restriction, liga-
tion, and transformation were all performed according to the
method previously described by Sambrook et al. [16].
The nucleotide sequence of both strands of the cloned 16S rRNA
gene sequence was determined using the BigDye® Terminator v3.1
Cycle Sequencing Kit and the automated DNA sequencer ABI Prism®
3100-Avant Genetic Analyzer (Applied Biosystems, Foster City, CA,
USA). Sequence comparisons were performed using the BLAST
program (NCBI, USA). Phylogenetic and molecular evolutionary
analyses were conducted via the molecular evolutionary genet-
ics analysis (MEGA) software version 4.1. Distances and clustering
were calculated using the neighbor-joining method. Bootstrap
analysis was used to evaluate the tree topology of the neighbor-
joining data by performing 100 re-samplings.
2.5. Standard assay of ChiA-65 activity
Chitinase activity was measured colorimetrically by detecting
the amount of GlcNAc released from colloidal chitin substrate [17].
Unless otherwise stated, suitably diluted enzyme solution (500 ␮l)
was mixed with 500 ␮l, 100 mM citrate buffer supplemented with
5 mM CaCl2 at pH 4 (Buffer A) containing 10 mg/ml, colloidal chitin,
and this was incubated for 1 h at 70 ◦ C. The mixture was boiled
for 10 min, chilled, and centrifuged to remove insoluble chitin. The
resulting products of reducing sugars were measured by the DNS
method [18]. Readings were compared with a standard curve pre-
pared with a series of dilutions of GlcNAc (from 0 to 10 mg/ml).
One unit of chitinase activity was defined as the amount of enzyme
required to produce 1 ␮mol of GlcNAc from colloidal chitin per min
under the specified assay conditions.
When using p-NP-(GlcNAc)n (n = 1–4) as substrate, enzyme
activity was measured by the method of Ohtakara [19]. Unless oth-
erwise stated, 250 ␮l of a suitably diluted enzyme solution and
250 ␮l of 1 mg/ml p-NP-(GlcNAc)n were added to 250 ␮l of buffer A,
and this was incubated at 70 ◦ C for 30 min. After incubation, 250 ␮l
of 200 mM sodium carbonate was added, and the absorbance of the
p-nitrophenol released was measured spectrophotometerically at
420 nm. One unit of chitinase activity was defined as the amount
of enzyme releasing 1 ␮mol of p-nitrophenol per min under the
specified assay conditions.
2.6. ChiA-65 purification procedure
Five hundred ml of a 48-h culture of B. licheniformis strain
LHH100 was centrifuged for 30 min at 8000 g to remove micro-
bial cells. The supernatant containing extracellular chitinase was
used as the crude enzyme preparation and was submitted to the
following purification steps. The supernatant was incubated for
1119
30 min at 70 ◦ C, and insoluble material was removed by centrifuga-
tion at 12,000 g for 25 min. The clear supernatant was precipitated
between 30 and 60% ammonium sulfate saturation. The precipi-
tate was then recovered by centrifugation at 12,000 g for 30 min,
resuspended in a minimal volume of buffer A containing 10 mM
NaCl (Buffer B), and dialyzed overnight against repeated changes
of buffer B. Insoluble material was removed by centrifugation at
12,000 g for 30 min. The supernatant was loaded and applied to gel
filtration on a Sephacryl S-200 column (2.5 × 150 cm) equilibrated
with buffer B, pre-equilibrated with 100 mM HEPES buffer supple-
mented with 5 mM CaCl2 and 50 mM NaCl at pH 7.5 (Buffer C).
Proteins were separated by isocratic elution at a flow rate of 30 ml/h
with buffer C and detected using a UV–VIS Spectrophotometric
detector (Knauer, Berlin, Germany) at 280 nm. Fractions of 5 ml
each were collected at a flow rate of 30 ml/h and analyzed for
chitinolytic activity and protein concentration. Pooled fractions
containing chitinase activity were concentrated in centrifugal
micro-concentrators (Amicon Inc., Beverly, MA, USA) with 30-kDa
cut-off membranes and were stored at −20 ◦ C in a 20% glycerol (v/v)
solution for further analysis.
2.7. Protein quantification, electrophoresis, and mass
spectrometry
The protein concentration was determined by the method of
Bradford [20] using a Dc protein assay kit purchased from Bio-Rad
Laboratories, with BSA as standard.
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE) was performed using 10% (w/v) acrylamide in gels,
as described by Laemmli [21]. Protein bands were visualized with
Coomassie Brilliant Blue R-250 (Bio-Rad Laboratories, Inc., Her-
cules, CA, USA) staining. The molecular weight markers were from
Amersham Pharmacia Biotech (Amersham Biosciences, Piscataway,
NJ, USA).
Discontinuous substrate SDS-PAGE (Zymogram analysis) was
performed with a 4% stacking gel, except that 1 mg/ml of heat-
denatured chitin azure was incorporated into the 10% separation
gel. Electrophoresis was performed at a constant current of 25 mA.
After electrophoresis, the gel was immersed with 100 ml of refol-
ding buffer (Buffer A, 1% Triton X-100) overnight at 70 ◦ C to replace
the SDS and separation buffer in the gel. The gel was washed with
distilled water and then stained with 0.01% (w/v) calcofluor white
M2R in 50 mM Tris-HCl (pH 8). After 5 min, the brightener solution
was removed and the gel was washed with distilled water. Lytic
zones were visualized by placing the gels on a UV-transilluminator
[22].
The molecular mass of the purified chitinase was analyzed in
linear mode by MALDI-TOF/MS using a Voyager DE-RP instru-
ment (Applied Biosystems/PerSeptive Biosystems, Framingham,
MA, USA). Data were collected with a Tektronix TDS 520 numeric
oscillograph and analyzed using GRAMS/386 software (Galactic
Industries Corporation, Salem, NH).
2.8. Amino acid sequencing
To determine the N-terminal sequence of the purified chiti-
nase ChiA-65, a protein band from the SDS gel was transferred
to a ProBlott membrane (Applied Biosystems, Foster City, CA,
USA). Automated Edman’s protein degradation was performed
with a protein sequencer (Applied Biosystems Protein sequencer
ABI Procise 492/610A). The sequence was compared to those
in the Swiss-Prot/TrEMBL database by BLAST homology search
(www.ncbi.nlm.nih.gov/blast).
1120 H. Laribi-Habchi et al. / International Journal of Biological Macromolecules 72 (2015) 1117–1128
2.9. Effects of chemical reagent and heavy metallic ions on the
activity of ChiA-65
Chemical reagents, p-chloromercuribenzoic acid (p-CMB),
N-ethylmaleimide (NEM), dithiothreitol (DTT), 2-mercaptoethanol
(2-ME), 2,4,6-trinitrobenzenesulfonic acid (TNBS), phenylmethyl-
sulfonyl fluoride (PMSF), 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC), diethylpyrocarbonate (DEP), N-bromo-
succinimide (NBS), and N-acetylimidazole (NAI), were investigated
at various concentrations for their effects on enzyme activity.
The effects of several metallic ions assayed at concentrations of
5 mM were also investigated by adding divalent [Ca2+ (CaCl2 ),
Mn2+ (MnSO4 ), Mg2+ (MgSO4 ), Co2+ (CoSO4 ), Cu2+ (CuSO4 ), Zn2+
(ZnSO4 ), Ba2+ (BaSO4 ), Fe2+ (FeSO4 ), Ag2+ (AgNO3 ), Al2+ (AlSO4 ),
Cd2+ (CdCl2 ), and Hg2+ (HgCl2 )], as well as monovalent [Hg+
(HgNO3 ), K+ (KCl), and Li+ (LiSO4 )] metallic ions to the reaction
mixture. The effect of the calcium concentration (from 0.5 to
15 mM) on the purified chitinase activity was also measured. The
remaining chitinase activity was measured after pre-incubation of
the purified enzyme with each of the various chemical reagents
at 60 ◦ C for 30 min. Activity was expressed as a percentage of the
activity level in the absence of reagent or metallic ions. Chitinase
activities measured, using colloidal chitin as substrate, in the
absence of any reagent or metallic ions supplemented with 2 mM
ethylene glycol-bis (␤-aminoethyl ether)-N,N,N ,N -tetraacitic acid
(EGTA), were taken as control (100%).
2.10. Effects of pH on the activity and stability of ChiA-65
The effects of pH were determined using colloidal chitin as sub-
strate. Chitinase activity was assayed over a pH range of 2–13 at
70 ◦ C. With regard to measurement of pH stability, the enzyme
was pre-incubated in buffers of different pH values in a range
of 2– 9 for 72 h at 70 ◦ C. Aliquots were withdrawn, and resid-
ual enzymatic activities were under standard assay conditions.
The following buffer systems, supplemented with 5 mM CaCl2 ,
were used at 100 mM: glycine-HCl for pH 2–3, citrate for pH
3–5, 2-(N-morpholino) ethanesulfonic acid (MES) for pH 5– 6,
HEPES for pH 6–8, Tris-HCl for pH 8–9, glycine-NaOH for pH 9–11,
bicarbonate-NaOH for pH 11–11.5, Na2 HPO4 -NaOH for pH 11.5–12,
and KCl-NaOH for pH 12–13.
2.11. Optimum temperature and thermal stability of ChiA-65
The effects of temperature on ChiA-65 activity were studied over
temperatures ranging from 40 to 100 ◦ C using colloidal chitin as
substrate for 1 h in 100 mM citrate buffer (pH 4). The thermosta-
bility of the purified chitinase was determined by incubating the
enzyme for 14 h at various temperatures in the absence and the
presence of 5 mM CaCl2 . Aliquots were withdrawn at set time inter-
vals to test the remaining activity under standard conditions. The
non-heated enzyme was used as control (100%).
2.12. Substrate specificity of ChiA-65
The substrate specificity of the purified chitinase ChiA-65 was
determined using natural and synthetic substrates at various con-
centrations under standard assay conditions. The natural substrates
were used at concentrations, and the reaction was carried out at
60 ◦ C for up to 72 h. The amount of reducing sugar was quantified
colorimetrically, as described above for the standard assay.
2.13. Kinetic measurements
Kinetic parameters were calculated from the initial rate activ-
ities of the purified bacterial enzymes (ChiA-65 and SMChi) using
natural (colloidal chitin) and synthetic [p-NP-(Glc-NAc)2 ] sub-
strates at concentrations ranging from 0.10 to 30 mg/ml at 60 ◦ C
for 5 min in assay buffer A supplemented with 5% (v/v) dimethyl
sulphoxide and 0.1% (v/v) Triton X-100 at pH 5. Assays were
carried out in triplicate and kinetic parameters were estimated
by Lineweaver–Burk plots. Kinetic constants, Michaelis–Menten
constant (Km ) and maximal reaction velocity (Vmax ) values, were
calculated using the Hyper32 software package developed at
Liverpool University (http://hompage.ntlword.com/john.easterby/
hyper32.html). The value of the turnover number (kcat ) was calcu-
lated by the following equation:
Vmax
kcat =
[E]
where [E] refers to the active enzyme concentration and Vmax to
the maximal velocity.
2.14. Molecular cloning of the chiA-65 gene
The preparation of plasmid DNA, digestion with restriction
endonucleases, and separation of fragments by agarose gel elec-
trophoresis were performed using general molecular biology
techniques as described by Sambrook et al. [16]. Two oligonu-
cleotides were synthesized, based on the high degree of sequence
homology published for the chitinase chiA-65 gene of Bacillus
strains, and used for the isolation and determination of the
chitinase gene sequence. The complete chiA-65 gene and its flank-
ing regions were amplified using the forward primer F-ChiA
(5 -ATGAAAATCGTGTTGATCAAC-3 ) and the reverse primer R-
ChiA (5 -CGGCTGATCGGAGGCTGCGAATAA-3 ) [4] to generate an
approximately 1.8 kb PCR fragment using genomic DNA from B.
licheniformis strain LHH100 as a template and DNA polymerase
from Pyrococcus furiosus (Pfu) (Biotools, Madrid, Spain). DNA ampli-
fication was carried out using the Gene Amp® PCR System 2700
(Applied Biosystems, Foster City, CA, USA). The amplification reac-
tion mixtures (100 ␮l) contained 25 pg of each primer, 300 ng of
DNA template, amplification buffer, and 2 U of Pfu DNA polymerase.
The cycling parameters used were 95 ◦ C for 2 min, followed by 30
cycles of 95 ◦ C for 45 s denaturation, 56 ◦ C for 60 s primer annealing,
and 72 ◦ C for 120 s extension, flowed by final extension step at 72 ◦ C
for 10 min. The PCR products were then purified using an agarose
gel extraction kit (Jena Bioscience, GmbH, Germany). The purified
1.8 kb PCR fragment was cloned in pCR-Blunt cloning vector into
E. coli BL21 [F− ompT gal dcm lon hsdSB(rB- mB-) (DE3 [lacI lacUV5-
T7 gene 1 ind1 sam7 nin5] (Invitrogen, Carlsbad, CA, USA) host strain.
Recombinant clones of E. coli were grown in LB broth media with
the addition of ampicillin (100 ␮g/ml), IPTG (160 ␮g/ml), and X-gal
(360 ␮g/ml). A clone noted to harbor a plasmid called pHL1 was
used for further study.
2.15. DNA sequencing and amino acid sequence alignment
The nucleotide sequence of the chiA-65 gene was determined
on both strands by the BigDye® Terminator v3.1 Cycle Sequenc-
ing Kit and the automated DNA sequencer ABI Prism® 3100-Avant
Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Mul-
tiple nucleotide sequence alignment was performed with BioEdit
version 7.0.2 software program. The amino acid sequence homol-
ogy was analyzed using BLASTP available at the NCBI server.
2.16. Statistical analysis
The data represent the mean of three independent replicates
with their standard deviation (mean ± SD) using Microsoft Excel.
The results were considered statistically significant for P values of
less than or equal to 0.05.
 H. Laribi-Habchi et al. / International Journal of Biological Macromolecules 72 (2015) 1117–1128
2.17. Nucleotide sequences accession numbers
The nucleotide sequence data of 16S rRNA and chiA-65
genes reported in this paper has been submitted to the
DDBJ/EMBL/GenBank databases under accession nos KJ017975 and
KJ017976, respectively.
3. Results and discussion
3.1. Screening of chitinase-producing strains
About fifty bacterial strains that were isolated from the aquatic
food industry in Algiers (Algeria) wastewater samples were identi-
fied as chitinase producers on the basis of their clear zone formation
on CHDA plate at pH 6.5. The ratio of the diameter of the clear zone
and that of the colony served as an index for the selection of strains
with high chitinase production ability. Ten isolates were noted to
exhibit a ratio that was higher than 3, with the highest ratio being 5.
Strain LHH100 exhibited the highest extracellular chitinase activ-
ity (about 900 U/ml) after 48 h incubation in an optimized medium
and was, therefore, retained for all subsequent studies.
3.2. Identification and molecular phylogeny of the microorganism
The identification of the newly isolated bacterium (LHH100)
was based on both catabolic and molecular methods. Morphologi-
cal, biochemical and physiological characteristics, according to the
methods described in Bergey’s Manual of Systematic Bacteriology,
showed that the LHH100 isolated strain appears in a bacillus form,
aerobic, endospore-forming, Gram-positive, catalase+, oxydase+,
motile and aerobic rod-shaped bacterium. Furthermore, finding
from API 50 CH gallery test showed that this isolate metabolize
matose, d-xylose, l-arabinose, d-tagatose, starch, ribose, mannitol
besides other simple sugars. All the data obtained with regards to
the physiological and biochemical properties of the isolate, there-
fore, strongly confirmed that the LHH100 strain belonged to the
Bacillus genus.
In order to establish further support for the identification of
the LHH100 isolate, a 1535 bp fragment of the 16S rRNA gene
was amplified from the genomic DNA of the isolate, cloned in
the pGEM-T Easy vector, and sequenced on both strands. The 16S
rRNA gene sequence obtained was subjected to GenBank BLAST
search analyses, which yielded a strong homology with those of
several cultivated strains of Bacillus, reaching a maximal of 99%
sequence identity. The nearest Bacillus strains identified by the
BLAST analysis were the B. licheniformis strain DSM 13T (Gen-
Bank accession n◦ X68416) and B. licheniformis strain NCDO 1772T
(GenBank accession n◦ X60623). Those sequences were imported
into MEGA software package version 4.1 and aligned. Phylogenetic
trees were then constructed (Fig. 1) and the findings further con-
firmed that the LHH100 strain (GenBank accession n◦ KJ017975)
was closely related to those of the Bacillus strains. In a nutshell, all
the results obtained strongly suggested that this isolate should be
assigned as B. licheniformis strain LHH100.
3.3. ChiA-65 purification
The protein elution profile obtained at the final purification step
indicated that the chitinase was eluted at a void volume of ∼35 ml
(1.9 V0 ) (Fig. 2a). The results of the purification procedure are sum-
marized in Table 1. The purity of the enzyme was estimated to be
about 15.9-fold greater than that of the crude extract. The purified
enzyme preparation contained about 40% of the total activity of the
crude, and showed a specific activity of 7869.5 U/mg when colloidal
chitin was used as substrate.
1121
3.4. Molecular weights determination and zymography analysis
of ChiA-65
The purified enzyme exhibited a unique protein band on native
PAGE and single symmetrical elution peaks corresponding to a
protein of nearly 65 kDa according to gel filtration chromatogra-
phy (Fig. 2a). The homogeneity of the purified chitinase was also
checked by SDS-PAGE under reducing conditions and by protein
staining analysis. A unique protein band was obtained for the
purified enzyme. The purified ChiA-65 enzyme had a molecular
weight of approximately 65 kDa (Fig. 2b) and clear chitinase activity
(Fig. 2c) similar to those of the bacterial chitinases, which had MWs
of 30–72 kDa (Table 2). A similar result was obtained when ChiA-
65 was denatured by SDS in the absence of DTT or 2-ME (data not
shown). MALDI-TOF/MS yielded a molecular mass of 65,195 kDa
(Fig. 2d).
These observations strongly suggest that ChiA-65 from B.
licheniformis strain LHH100, like chitinase Chi72 from B. licheni-
formis strain SK1, is a monomeric protein comparable to other
Bacillus chitinases (Table 2).
3.5. N-terminal amino acid sequences of ChiA-65
The first 27 N-terminal amino acid residues of the ChiA-65
chitinase from B. licheniformis LHH100 were determined to be
ADSGKNYKIIGYYPSWGAYGRDFQVWD. This sequence showed uni-
formity, indicating that it was isolated in pure form. It shared
greatest homology with other sequences of family-18 chitinases,
ChiA from B. licheniformis DSM13 and DSM8785 [4] (100% iden-
tity), ChiA-67 from B. licheniformis MB-2 (100% identity), ChiD from
B. circulans WL-12 (73% identity), and ScChi50 from the stomach
of red scorpionfish Scorpaena scrofa (50% identity). These findings
strongly suggest that ChiA-65 is closely related to family 18 glycosyl
hydrolase.
3.6. Chemical modification and effect of metallic ions on the
activity of ChiA-65
When ChiA-65 was incubated with various group-specific
reagents for amino acid modification, its activity was found to be
inhibited in the presence of p-CMB and NEM. Partial activity loss
was observed when it was incubated with DTT and 2-ME (Table 3).
This indicates the presence of sulfyhydryl groups on active site
of the enzyme, as confirmed by total inhibition observed in the
presence of mercuric ion. An S-S bridge has been reported in the
chitin binding domain of Alteromonas sp. strain O-70 [23]. EDC did
not inhibit the activity of the enzyme, suggesting that the glu-
tamic acid residue in the active site was not accessible to EDC.
This behavior was similar to that observed for the purified chiti-
nase ScChi50 from the stomach of red scorpionfish Scorpaena scrofa
already described by the authors [24]. Chitinase activity was, how-
ever, strongly inhibited by Ag2+ and by Cd2+ , completely inhibited
by Hg2+ and by Hg+ , and moderately inhibited by Fe2+ . The enzyme
was activated by Ca2+ , K+ , Li+ , Zn2+ , Mn2+ , Mg2+ , Co2+ , Al2+ , and Cu2+ ,
and was found to undergo no significant inhibition in the presence
of Ba2+ . The activity of chitinase in the presence of metallic ions is a
highly valued property with regard to potential industrial applica-
tions. The chitinase purified from B. cereus TKU030 was significantly
inhibited by Mn2+ and EDTA but activated by Cu2+ , Fe2+ and Ca2+ [8].
The addition of CaCl2 at 5 mM was found to enhance the activ-
ity of the enzyme, which increased by 295% as compared to that
attained without Ca2+ (Fig. 3). This result indicates that the enzyme
requires Ca2+ for optimal activity. The particular sensitivity of the B.
licheniformis strain LHH100 chitinase to cobalt might be related to
the importance of the carboxylic groups, mainly aspartic and glu-
tamic acid residues, in chitinases, where these amino acids have
1122 H. Laribi-Habchi et al. / International Journal of Biological Macromolecules 72 (2015) 1117–1128

56 Bacillus methylotrophicus strain CBMB205T (EU194897)

47 Bacillus subtilis subsp. subtilis strain NBRC 13719T (AB271744)

Bacillus subtilis subsp. spizizenii NRRL B-23049T (AF074970)

95
66 Bacillus vallismortis strain 15-1T (AB021198)
91 Bacillus amyloliquefaciens strain ATCC 23350T (X60605)
Bacillus licheniformis strain NCDO 1772T (X60623)
T
100 Bacillus licheniformis strain DSM 13 (X68416)
99 79 Bacillus licheniformis strain LHH100 (KJ017975)
88 Bacillus aerius strain 24KT (AJ831843)

97 Bacillus safensis strain FO-036b T (AF234854)

Bacillus pumilus strain DSMZ27T (AY456263)

T
100 Bacillus stratosphericus strain 41KF2a (AJ831841)
Bacillus aerophilus strain 28KT (AJ831844)
91
Bacillus altitudinis strain 41KF2b T (AJ831842)
92 Bacillus acidicola strain 105-2T (AF547209)
Bacillus shackletonii strain LMG 18435T (AJ250318)
Bacillus methanolicus strain NCIMB 13113T (AB112727)
58 Bacillus seohaeanensis strain BH724T (AY667495)

87 Bacillus marisflavi strain TF-11 T(AF483624)

91 Bacillus vietnamensis strain 15-1T (AB099708)

99 Bacillus aquaemaris strain TF-12T (AF483625)
Micrococcus luteus strain DSM 20030T (AJ536198)

0.02

Fig. 1. Phylogenetic tree based on 16S rRNA gene sequences showing the position of strain LHH100 within the radiation of the genus Bacillus. The sequence of Micrococcus
luteus strain DSM 20030T (GenBank accession n◦ AJ536198) was chosen arbitrarily as an outgroup. Bar, 0.02 nt substitutions per base. Numbers at nodes (>50%) indicate
support for the internal branches within the tree obtained by bootstrap analysis (percentages of 100 bootstraps). NCBI accession numbers are presented in parentheses.

been found to bind divalent cations e.g., Ca2+ , Co2+ or Mg2+ , with
the active sites and to confer better accessibility on the substrate,
causing activation of the enzyme.
3.7. Effects of pH on the activity and stability of ChiA-65
As shown in Fig. 4a, ChiA-65 displayed activity over a broad
range of pH, with an optimum pH at 4. The relative activities at
pH 3 and 9 were 75%. The optimum pH values reported for Chi
from B. licheniformis strain DSM13 [9] and Chi72 from B. licheni-
formis strain SK-1 [25] are about 6 and 6–8, respectively. The pH
stability profile indicated that ChiA-65 was highly stable over a
broad range of pH, maintaining 100% of its original activity at
pH values between 4 and 8 after incubation for 72 h at 70 ◦ C
(Fig. 4b). The high activity and stability exhibited by ChiA-65 in an
acidic environment (pH 3–7) makes it suitable for biotechnological

Table 1
Flow sheet for the purification of ChiA-65 chitinase from B. licheniformis strain LHH100.

Purification step Total activity (units)a,b,c × 103 Total protein Specific activity Activity recovery Purification
(mg)b,d (U/mg of protein)b rate (%) factor (-fold)

Crude extract 450 ± 4.5 910 ± 9.8 494.5 ± 1.3 100 1

Heat treatment (70 ◦ C, 30 min) 380 ± 3.1 397 ± 2.5 957.1 ± 2.4 84 1.9
(NH4 )2 SO4 fractionation (30–60%) 319 ± 2.1 125 ± 1.8 2,552.0 ± 7.6 70 5.1
Sephacryl S-200 181 ± 0.9 23 ± 0.4 7,869.5 ± 7.6 40 15.9
a
From 500 ml of a 48 h culture of B. licheniformis strain LHH100.
b
The experiments were conducted 3 times and ± standard errors are reported.
c
One unit (U) of chitinase activity was defined as the amount of enzyme releasing 1 ␮mol of Glc-NAc per min at 70 ◦ C with colloidal chitin as substrate.
d
Amounts of protein were estimated by the Bradford method [20].
 H. Laribi-Habchi et al. / International Journal of Biological Macromolecules 72 (2015) 1117–1128 1123

Fig. 2. (a) Size exclusion chromatography of the chitinase from B. licheniformis strain LHH100 on Sephacryl S-200. The column (2.5 × 150 cm) was equilibrated with buffer B.
The elution of proteins was performed with the same buffer at a rate of 30 ml/h and 5 ml by fraction. ChiA-65 activity was measured as described in Section 2 using colloidal
chitin as substrate. (b) SDS-PAGE of the purified chitinase. Lane 1, protein markers. Lane 2, total cell extract. Lane 3, sample after heat treatment (30 min at 70 ◦ C). Lane
4, sample after ammonium sulphate fractionation (30-60%). Lane 5, purified ChiA-65 (20 ␮g) obtained after gel filtration chromatography (fractions 32–41), (c) Zymogram
activity staining of the purified chitinase, and (d) MALDI-TOF spectrum of 10 pmol purified ChiA-65 chitinase from B. licheniformis strain LHH100. The mass spectrum shows
a series of multiply protonated molecular ions. The molecular mass of the enzyme was found to be 65195.13 Da.

Table 2
Comparison between the properties of ChiA-65 chitinase from B. licheniformis strain LHH100 and those of other common mackerel chitinases and glycosyl hydrolase family
18 chitinases.

Chitinase Microorganism pH opt. Optima temp. (◦ C) Half-life time (min) MW (kDa)a Reference
◦
ChiA-65 B. licheniformis LHH100 4 75 240 (90 C) 65 This study
Chi B. licheniformis MB-2 6 70 80 (60 ◦ C) 67 [9]
Chi72 B. licheniformis SK1 6-8 55 90 (60 ◦ C) 72 [25]
Chi Bacillus sp. NTCU2 7 60 30 (60 ◦ C) 36.5 [27]
Chi-L Bacillus MH-1 6.5 75 10 (80 ◦ C) 71 [31]
Chi-M Bacillus MH-1 5.5 65 10 (70 ◦ C) 62 [31]
Chi-S Bacillus MH-1 5.5 75 10 (80 ◦ C) 53 [31]
Chi Streptomyces RC1071 8 40 60 (60 ◦ C) 70 [32]
Chi30 S. thermoviolaceus OPC-520 4 60 30 (60 ◦ C) 30 [33]
Chi-F1 P. aeruginosa K-187 8 50 50 (10 ◦ C) 30 [34]
Chi-F2 P. aeruginosa K-187 7 40 50 (10 ◦ C) 30 [34]
SsChi50 Scorpaena scrofa 4 75 480 (90 ◦ C) 50 [24]
a
MW, molecular weight.
1124 H. Laribi-Habchi et al. / International Journal of Biological Macromolecules 72 (2015) 1117–1128

3.8. Effects of temperature on the activity and stability of ChiA-65

The optimum temperature recorded for the activity of the puri-

fied chitinase at pH 4 was 70 ◦ C in the absence of CaCl2 and 75 ◦ C
in the presence of 5 mM Ca2+ , using colloidal chitin as substrate
(Fig. 4c). The optimal temperature for the ChiA-65 chitinase from
B. licheniformis strain LHH100 was higher than those previously
reported for chitinases Chi72 and Chi [25] and [27], respectively.
The half-lives of the purified ChiA-65 chitinase from B. licheni-
formis strain LHH100 in the absence of any of the additives used
were 8, 5, and 3 h at 70, 80, and 90 ◦ C, respectively. The addition
of concentrations of CaCl2 (0.5–15 mM) enhanced the thermosta-
bility of the enzyme. Maximal thermostability was achieved with
5 mM Ca2+ (data not shown). As shown in Fig. 4d, the half-life of
Fig. 3. Effect of the calcium concentration (from 0.5 to 15 mM) on the purified chiti- the purified chitinase at 70, 80, and 90 ◦ C increased to 9, 6, and 4 h
nase activity. Chitinase activity measured, using colloidal chitin as substrate, in the
in the presence 5 mM CaCl2 . Most chitinases have been reported to
absence of calcium supplemented with 2 mM EGTA, was taken as control (100%).
Each point represents the mean (n = 3) ± standard deviation. be significantly stabilized by the addition of Ca2+ at higher temper-
atures [9,25]. The improvement of enzyme thermostability against
applications involving the bioconversion of chitin waste, a highly thermal inactivation in the presence of CaCl2 can be attributed
resistant and abundant biopolymer from crustacean food industry, to strengthening of the interactions inside the protein molecules
into glucosamine and chito-oligosaccharide value-added products and to the binding of Ca2+ to the active site. Further studies are
[26]. necessary in order to understand this upgrading process.

(a) (b)
125 125
Residual chitinase activity (%)
Relative chitinase activity (%)

100 100

75 75

50 50 .

25 25

0 0
2 3 4 5 6 7 8 9 10 11 12 13 3 4 5 6 7 8 9
pH pH

(c) (d)
250 150
0 mM Ca2+ 70 °C (0 m M Ca2+) 70 °C (5 m M Ca2+)
Residual chitinase activity (%)
Relative chitinase activity (%)

5 mM Ca2+ 80 °C (0 m M Ca2+) 80 °C 5 m M Ca2+)

125
200 90 °C (0 m M Ca2+) 90 °C (5 m M Ca2+)

100
150
75

100
50

50
25

0 0
40 45 50 55 60 65 70 75 80 85 90 95 100 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Temperature (°C) Time (h)

Fig. 4. Effects of pH on the activity (a) and stability (b) of the purified ChiA-65 Enzyme. Chitinase activity was evaluated in a pH range of 2–13 using buffers of pH values with
colloidal chitin as substrate. The pH stability of the enzyme was determined by incubating the chitinase in buffers ranging from 3–9 for 72 h at 60 ◦ C, and residual activity
was measured colorimetrically using standard procedures. Effect of temperature on the activity (c) and stability (d) of the purified ChiA-65 enzyme. Temperature profiles
were determined by assaying chitinase activity at temperatures ranging from 40 to 90 ◦ C using colloidal chitin for 1 h in buffer A. Effect on the thermostability of the purified
chitinase ChiA-65 (b). The enzyme was pre-incubated in the absence and the presence of CaCl2 at various temperatures: 70 ◦ C without Ca2+ (×); 70 ◦ C with 5 mM Ca2+ (+);
80 ◦ C without Ca2+ (); 80 ◦ C with 5 mM Ca2+ (䊉); 90 ◦ C without Ca2+ (); and 90 ◦ C with 5 mM Ca2+ (). Aliquots were withdrawn at set time intervals to test remaining
activity under standard assay conditions. Non-heated chitinase was taken to be 100%. Each point represents the mean (n = 3) ± standard deviation.
 H. Laribi-Habchi et al. / International Journal of Biological Macromolecules 72 (2015) 1117–1128 1125

Table 3 kinetic experiments were performed via reducing sugar assays

Effects of various reagents on purified chitinase ChiA-65 chitinase from B. licheni-
using natural substrates. The results are summarized in Table 4. The
formis strain LHH100 with colloidal chitin as substrate.
findings indicate that maximum chitinase activity was obtained at
Chemical Amino acid Concentration Relative 24 h for the natural substrates, presumably due to the low solubil-
reagent/metallic involved/material (mM) activity (%)b
ity and viscosity of the biopolymers. The most preferred natural
ionsa
substrate was colloidal chitin, followed by glycol chitin, glycol
None – – 100 ± 2.50 chitosane, chitotriose, and chitooligosaccharide. No activity was
p-CMB Cysteine 2 0
observed toward chitobiose or any other substrate that consisted
NEM Cysteine 2 0
DTT Cysteine 1 10 ± 0.50 of a glucosidic linkage. This indicates the absence of chitobiase and
2-ME Cysteine 5 21 ± 0.65 glycosidases other than chitinases.
TNBS Lysine 2 98 ± 2.20 Kinetics experiments with synthetic substrates carrying the p-
PMSF Serine 5 104 ± 2.52
nitrophenol group were performed by standard activity assay. The
EDC Carboxylic 2 112 ± 3.05
amino acids results indicate that maximum chitinase activity was obtained at
DEP Histidine 1 99 ± 2.50 6 h for the synthetic substrates and clearly showed that ChiA-65
NBS Typtophane 2 101 ± 2.55 cleaved specific ␤-glycosidic bonds (Table 4). Although chitinase
NAI Tyrosine 5 98 ± 2.30 ChiA-65 released p-NP from p-NP-(Glc-NAc)n (n = 2–4), it probably
Ca2+ CaCl2 5 295 ± 5.05
did not exhibit any activity toward p-NP-(Glc-NAc). This suggests
Mn2+ MnSO4 5 127 ± 2.65
Mg2+ MgSO4 5 115 ± 2.66 that the enzyme, like other Bacillus chitinases, has a preference
Co2+ CoSO4 5 112 ± 3.09 for glycosidic bonds, which are second from the non-reducing end
Cu2+ CuSO4 5 105 ± 2.56 [9,25]. Although the p-NP-releasing activity of ChiA-65 showed
Zn2+ ZnSO4 5 131 ± 3.01
high activity (15,900 U/mg) toward p-NP-(Glc-NAc)2 , its activ-
Ba2+ BaSO4 5 86 ± 1.85
Fe2+ FeSO4 5 95 ± 2.23
ity toward p-NP-(Glc-NAc)n (n = 3, 4) was found to decrease
Ag2+ AgNO3 5 10 ± 0.80 markedly.
Al2+ AlSO4 5 113 ± 3.06
Cd2+ CdCl2 5 35 ± 0.96
Hg2+ HgCl2 5 0 3.10. Determination of the kinetic parameters of ChiA-65
Hg+ HgNO3 5 0
K+ KCl 5 145 ± 4.01
Li+ LiSO4 5 132 ± 3.48
ChiA-65 and SMChi exhibited the classical kinetics of Michaelis-
Menten for the two substrates used. Kinetic parameters were
a
Incubation with 25 ␮g of purified enzyme at 60 ◦ C at pH 4 for 30 min.
obtained from Michaelis-Menten plots (Fig. 5). The order of the
b
Values represent means of 3 replicates, and ± standard errors are reported.
catalytic efficiency (kcat /Km ) values of each enzyme was almost the
same, i.e., colloidal chitin < p-NP-(Glc-NAc) 2 (Fig. 5, Table 5). When
Overall, the thermocativity (a temperature optimum of 75 ◦ C) colloidal chitin was used as a protein substrate, the kcat /Km exhib-
and the thermostability (a half-life of 4 h at 90 ◦ C) measured for ited by ChiA-65 was 2.75 times higher than that of SMChi. When
the ChiA-65 chitinase from B. licheniformis strain LHH100 were p-NP-(Glc-NAc) 2 was used as a synthetic substrate, ChiA-65 was
higher than those previously reported for other bacterial chitinases. also noted to exhibit kcat /Km values that was 2.22 times higher than
Accordingly, the high temperature optimum and the thermal sta- that of SMChi (Fig. 5, Table 5).
bility of the chitinase from B. licheniformis strain LHH100 provide
further confirmation of its potential industrial application in the
recycling of chitin wastes [26]. 3.11. Cloning and sequencing of the chiA-65 gene

3.9. Substrate specificity of ChiA-65 Using the chitinase gene sequences of Bacillus strains, two
primers, called F-ChiA and R-ChiA, from B. licheniformis DSM13
The ability to hydrolyze several carbohydrates substrates is an and DSM8785 [4] were designed and used to amplify a frag-
important criterion for the effectiveness of chitinases. Accordingly, ment of about 1.8 kb that couldcontain the chiA-65 gene from

Table 4
Substrate specificities of ChiA-65 chitinase from B. licheniformis strain LHH100 for various substrates.

Substrate Concentration (mg/ml) Monitoring wavelength (nm) Specific activity (U/mg of protein)a Relative activity (%)b

Colloidal chitin 40 550 7,800 ± 7.5 100 ± 2.50

Glycol chitin 40 550 7,488 ± 7.1 96 ± 2.12
Glycol chitosan 40 550 7,332 ± 6.4 94 ± 2.10
Chitibiose 50 550 0 0
Chitotriose 50 55 7,146 ± 6.1 92 ± 2.00
Chitooligosaccharide c 50 550 6,846 ± 5.8 88 ± 1.90
Amylose 40 550 0 0
Carboxymethyl cellulose 40 550 Nd Nd
Cellobiose 50 550 0 0
Laminarin 50 550 0 0
p-NP-(GlcNAc) (G-P) 5 420 Nd Nd
p-NP-(GlcNAc)2 (G-G-P) 5 420 15,900 ± 9.4 100 ± 2.50
p-NP-(GlcNAc)3 (G-G-G-P) 5 420 13,515 ± 8.2 85 ± 1.72
p-NP-(GlcNAc)4 (G-G-G-G-P) 5 420 11,282 ± 9.4 71 ± 1.04
a
All measurements were carried out in buffer A (pH 4) at 60 ◦ C. Values represent maximum activity obtained after 24 h of incubation for the natural substrates and 6 h for
the synthetic substrates, and the optimum substrate concentration.
b
Activity is expressed as a percentage of enzyme activity under standard conditions using colloidal chitin or p-NP-(Glc-NAc)2 . Values represent means of 3 replicates,
and ± standard errors are reported.
c
Chitooligosaccharides are a mixture of chitotetraose, chitopentaose, and chitohexaose.
G, GlcNAc. P, p-NP. Nd, Not detected.
1126 H. Laribi-Habchi et al. / International Journal of Biological Macromolecules 72 (2015) 1117–1128

Fig. 5. Michaelis–Menten plots of the purified bacterial chitinases ChiA-65 and SMChi toward natural [colloidal chitin (a, b)] and synthetic [p-NP-(Glc-NAc)2 (c, d)] substrates.
Kinetic parameters were calculated from the initial rate activities of the purified enzymes using substrates at concentrations ranging from 0.10 to 30 mg/ml at 60 ◦ C for 5 min
in assay buffer A supplemented with 5% (v/v) dimethyl sulphoxide and 0.1% (v/v) Triton X-100 at pH 5.

B. licheniformis strain LHH100. This PCR fragment was purified and
cloned in a pCR-Blunt cloning vector using an E. coli BL21 host strain,
thus leading to pHL1.
The complete deduced amino acid sequence of the chiA-65 gene
is shown in Fig. 6. The analysis of the nucleotide sequence of the
chiA-65 gene and its flanking DNA regions revealed the presence of
an open reading frame (ORF) of 1,802-bp that encoded an enzyme
consisting of 598 aa with a predicted molecular weight and pI
of 66347.74 Da and 5.30, respectively. This ORF started with an
ATG codon at nucleotide position 1 and terminated with a TAA
stop codon. This ORF was confirmed as the gene encoding ChiA-
65 since, as determined by the Edman degradation method, the
deduced amino acid sequence was noted to include the 27 N-
terminal amino acids sequence of the purified mature ChiA-65. This
sequence was identical to those of chitinases from other Bacillus
strains.
3.12. Amino acid sequence inspection
Compared to the latest GenBank nucleotide sequence database
B. licheniformis strain LHH100 was 99% homologous to the chitinase
genes (GenBank accession n◦ ) FJ606837, CP000002, FJ465148, and
AY205293 of B. licheniformis CBFO-03, B. licheniformis DSM13,
B. licheniformis DSM8785, and B. licheniformis PR-1, respectively
and 84% homologous to the chitinase gene B. subtilis CHU26.
Such high homologous sequence similarity was found within B.
licheniformis and B. subtilis chitinase genes [28], only 382 mis-
matched nucleotides, and these differences made only some
amino acids variation between these chitinases. It means that, B.
licheniformis ChiA chitinases and B. subtilis CHU26 CHI18 has a
moderately high taxonomic relationship. Besides, the N-terminus
of the deduced ChiA-65 show a typical attributes of a signal pep-
tide, containing a positively charged region, a hydrophilic basic
region (lysine, K) followed by a hydrophobic region [29], and a
signal sequence cleavage site between Ala-33 and Ser-35 (Fig. 6).
These results demonstrate that the chitinase ChiA-65 from B.
licheniformis strain LHH100 was really a complete functional pro-
tein.
A previous study demonstrated that Glu-204 and Asp-200 of B.
circulans WL-12 chiA were critical for the hydrolysis of chitin [30].
Our results revealed that the region from Phe-190 to Leu-205 of
ChiA-65 was completely identical to the same region of the other
chitinases catalytic domain (Fig. 6). It means that ChiA-65 was really
a chitinase gene.

Table 5
Kinetic parameters of purified chitinases ChiA-65 and SMChi toward natural and synthetic substrates.

Substrate Enzyme Km (mg/ml) Vmax (U/mg) kcat (s−1 ) kcat /Km (ml mg s−1 ) Catalytic efficiency relative to ChiA-65

Colloidal chitin ChiA-65 0.385 7,500 5,000 12,987 ± 6.50 1.00

SMChi 0.516 3,651 2,434 4,717 ± 5.60 0.36
p-NP-(GlcNAc)2 (G-G-P) ChiA-65 0.646 36,752 24,501 37,927 ± 9.45 1.00
SMChi 0.819 27,955 13,977 17,065 ± 7.15 0.44

Kinetic parameters of purified chitinases ChiA-65 and SMChi toward natural and synthetic substrates.
Values represent mean of 3 replicates, and ±standard errors are reported.
 H. Laribi-Habchi et al. / International Journal of Biological Macromolecules 72 (2015) 1117–1128 1127

Fig. 6. Chitinase amino acid sequence alignment of B. licheniformis LHH100 (GenBank accession n◦ KJ017976), B. licheniformis CBFOD-03 (GenBank accession n◦ ACM46020), B.
licheniformis DSM133 GenBank accession n◦ (AAU21943), B. licheniformis DSM8785 (GenBank accession n◦ ACK44109), B. licheniformis PR-1 (GenBank accession n◦ AAO22144),
B. subtilis CHU26 (GenBank accession n◦ AAC23715), and B. circulans WL-12 (GenBank accession n◦ AAA81528). Translation starts at a nucleotide position 1. Numbers written
on both sides of the lines indicate the positions of amino acids. The black box indicates the N-terminal amino acid sequence of the purified ChiA-65. The conserved amino
acids and non-coding ones were shown in box and dashed line, respectively.
1128 H. Laribi-Habchi et al. / International Journal of Biological Macromolecules 72 (2015) 1117–1128
4. Conclusions
The extracellular chitinase (called ChiA-65) from B. licheniformis
strain LHH100 was purified and biochemically characterized. The
nucleotide sequence of chiA-65 gene and its flanking regions were
determined. The lasB gene consisted of 1802 bp, encoding a protein
of 598 aa. Overall, the findings indicate that ChiA-65 is endowed
with a number of promising properties that are highly valued for
bioconversion of chitin waste and other biotechnological applica-
tions. This would require further studies on the expression and
structure-functions relationship of the enzyme using site-directed
mutagenesis and 3-D structure modeling.
Acknowledgments
This work was financially supported by the Algerian Ministry
of Higher Education and Scientific Research. We wish to express
our gratitude to Professor Fatima Laraba-Djebari (LCMB, Faculty
of Biological Sciences, University of Science and Technology Houari
Boumediene, Algiers) for here constructive discussion and valuable
help during the preparation of this study.
References
[1] N.S. Patil, S.R. Waghmare, J.P. Jadhav, Process Biochem. 48 (2013)
176–183.
[2] G.C. Pradeep, Y.H. Choi, Y.S. Choi, S.E. Suh, J.H. Seong, S.S. Cho, M.-S. Bae, J.C.
Yoo, Process Biochem. 49 (2014) 223–229.
[3] T. Fukamizo, Curr. Protein Pept. Sci. 1 (2000) 105–124.
[4] C. Songsiriritthigul, S. Lapboonrueng, P. Pechsrichuang, P. Pesatcha, M. Yamab-
hai, Bioresour. Technol. 101 (2010) 4096–4103.
[5] N. Dahiya, R. Tewari, G.S. Hoondal, Appl. Microbiol. Biotechnol. 71 (2006)
773–782.
[6] Z. Wang, L. Zheng, S. Yang, R. Niu, E. Chu, X. Lin, Biochem. Biophys. Res. Commun.
357 (2007) 26–31.
[7] T. Tanaka, T. Fukui, T. Imanaka, J. Biol. Chem. 276 (2001) 35629–35635.
[8] T.-W. Liang, Y.-Y. Chen, P.-S. Pan, S.-L. Wang, Inter. J. Biol. Macromol. 63 (2014)
8–14.
[9] H.A. Nguyen, T.H. Nguyen, T.T. Nguyen, C.K. Peterbauer, G. Mathiesen, D. Hal-
trich, Protein Expr. Purif. 81 (2012) 166–174.
[10] K. Suma, A.R. Podile, Bioresour. Technol. 133C (2013) 213–220.
[11] B. Henrissat, A. Bairoch, Biochem. J. 293 (1993) 781–788.
[12] S. Karasuda, S. Tanaka, H. Kajihara, Y. Yamamoto, D. Koga, Biosci. Biotechnol.
Biochem. 67 (2003) 221–224.
[13] A. Perrakis, I. Tews, Z. Dauter, A.B. Oppenheim, I. Chet, K.S. Wilson, C.E. Vorgias,
Structure 2 (1994) 1169–1180.
[14] F. Uni, S. Lee, R. Yatsunami, T. Fukui, S. Nakamura, Nucleic Acids Symp. Ser.
(Oxf) (2009) 311–312.
[15] W.K. Roberts, C.P. Selitrennikoff, J. Gen. Microbiol. 134 (1988) 169–176.
[16] J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A laboratory Manual,
2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA,
1989, pp. 23–38.
[17] S. Thamthiankul, S. Suan-Ngay, S. Tantimavanich, W. Panbangred, Appl. Micro-
biol. Biotechnol. 56 (2001) 395–401.
[18] G.L. Miller, Anal. Chem. 31 (1959) 426–428.
[19] A. Ohtakara, Method in Enzymology, Academic Press, New York, 1988, pp.
462–470.
[20] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254.
[21] U.K. Laemmli, Nature 227 (1970) 680–685.
[22] J. Trudel, A. Asselin, Anal. Biochem. 178 (1989) 362–366.
[23] K. Hayashi, S. Sato, R. Takano, H. Tsujibo, H. Orikoshi, C. Imada, Y. Okami, Y.
Inamori, S. Hara, Biosci. Biotechnol. Biochem. 59 (1995) 1981–1982.
[24] H. Laribi-Habchi, M. Dziril, A. Badis, S. Mouhoub, N. Mameri, Biosci. Biotechnol.
Biochem. 76 (2012) 1733–1740.
[25] S. Kudan, R. Pichyangkura, Appl. Biochem. Biotechnol. 157 (2009) 23–35.
[26] S.R. Waghmare, J.S. Ghosh, Carbohydr. Res. 345 (2010) 2630–2635.
[27] C.M. Wen, C.S. Tseng, C.Y. Cheng, Y.K. Li, Biotechnol. Appl. Biochem. 35 (2002)
213–219.
[28] M.W. Rey, P. Ramaiya, B.A. Nelson, S.D. Brody-Karpin, E.J. Zaretsky, M. Tang,
A. Lopez de Leon, H. Xiang, V. Gusti, I.G. Clausen, P.B. Olsen, M.D. Rasmussen,
J.T. Andersen, P.L. Jorgensen, T.S. Larsen, A. Sorokin, A. Bolotin, A. Lapidus, N.
Galleron, S.D. Ehrlich, R.M. Berka, Genome Biol. 5 (2004) R77.
[29] J.D. Bendtsen, H. Nielsen, G. von Heijne, S. Brunak, J. Mol. Biol. 340 (2004)
783–795.
[30] T. Watanabe, K. Kobori, K. Miyashita, T. Fujii, H. Sakai, M. Uchida, H. Tanaka, J.
Biol. Chem. 268 (1993) 18567–18572.
[31] K. Sakai, A. Yokota, H. Kurokawa, M. Wakayama, M. Moriguchi, Appl. Environ.
Microbiol. 64 (1998) 3397–3402.
[32] R.C. Gomes, L.T. Semedo, R.M. Soares, L.F. Linhares, C.J. Ulhoa, C.S. Alviano, R.R.
Coelho, J. Appl. Microbiol. 90 (2001) 653–661.
[33] H. Tsujibo, N. Hatano, H. Endo, K. Miyamoto, Y. Inamori, Biosci. Biotechnol.
Biochem. 64 (2000) 96–102.
[34] S.L. Wang, W.T. Chang, Appl. Environ. Microbiol. 63 (1997) 380–386.