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

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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

Article history: Received 22 April 2014 Received in revised form 16 October 2014 Accepted 17 October 2014 Available online 27 October 2014 Keywords: Chitinase Bacillus licheniformis Purification

a b s t r a c t An extracellular chitinase (ChiA-65) was produced and purified from a newly isolated Bacillus licheniformis LHH100. Pure protein was obtained after heat treatment and ammonium sulphate precipitation followed by Sephacryl S-200 chromatography. Based on matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis, the purified enzyme is a monomer with a molecular 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 inhibitors and metals tested, p-chloromercuribenzoic acid, N-ethylmaleimide, Hg2+ , and Hg+ completely inhibited enzyme activity. Chitinase activity was high on colloidal chitin, glycol chitin, glycol chitosane, chitotriose, and chitooligosaccharide. Chitinase activity towards synthetic substrates in the order of pNP-(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

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, diethylpyrocarbonate; NBS, N-bromosuccinimide; NAI, N-acetylimidazole; EGTA, ethylene glycol-bis (␤-aminoethyl ether)-N,N,N ,N -tetraacitic acid; MES, 2-(N-morpholino) ethanesulfonic acid. ∗ Corresponding authors. Tel: +213 5 51 43 53 87; fax: +213 021 52 29 73. E-mail addresses: [email protected] (H. Laribi-Habchi), [email protected] (N. Drouiche). http://dx.doi.org/10.1016/j.ijbiomac.2014.10.035 0141-8130/© 2014 Elsevier B.V. All rights reserved.

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

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Chitinases are essential glycoside hydrolases that catalyze the degradation of chitin by cleaving its GlcNAc bond, and endochitinases 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 characterized 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 digestion, 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 environment 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 various 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 Nacetylglucosaminidases [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 diacetylchitobiose (GlcNAc2 ). N-acetylglucosaminases hydrolyze GlcNAc2 into GlcNAc or produce GlcNAc from the nonreducing end of Nacetyl-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 chitinases 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]. Members 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 antibacterial properties [12]. The chitinases of the two different families do not share amino acid sequence similarity, have completely different 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 chitinase and its corresponding gene is the starting point to pursuing this goal. Accordingly, the present study aimed to reports on the purification 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 (chiA65) 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, glycol 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 Laboratories (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. Approximately 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 microorganisms. 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 performed 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

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(bioMérieux, SA, Marcy-l’Etoile, France) were used to investigate 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 reaction (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 ampicillin, isopropyl-thio-␤-d-galactopyranoside (IPTG), and X-gal for screening. DNA electrophoresis, DNA purification, restriction, ligation, 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 genetics 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 neighborjoining 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 prepared 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 otherwise 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 microbial 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

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30 min at 70 ◦ C, and insoluble material was removed by centrifugation at 12,000 g for 25 min. The clear supernatant was precipitated between 30 and 60% ammonium sulfate saturation. The precipitate 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 supplemented 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., Hercules, 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 heatdenatured 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 refolding 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 instrument (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 chitinase 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).

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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), phenylmethylsulfonyl fluoride (PMSF), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), diethylpyrocarbonate (DEP), N-bromosuccinimide (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 substrate. 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 residual 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 thermostability 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 intervals 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 concentrations 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 activities of the purified bacterial enzymes (ChiA-65 and SMChi) using

natural (colloidal chitin) and synthetic [p-NP-(Glc-NAc)2 ] 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. 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 calculated by the following equation: kcat =

Vmax [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 electrophoresis were performed using general molecular biology techniques as described by Sambrook et al. [16]. Two oligonucleotides 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 flanking regions were amplified using the forward primer F-ChiA (5 -ATGAAAATCGTGTTGATCAAC-3 ) and the reverse primer RChiA (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 amplification was carried out using the Gene Amp® PCR System 2700 (Applied Biosystems, Foster City, CA, USA). The amplification reaction 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 lacUV5T7 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 Sequencing Kit and the automated DNA sequencer ABI Prism® 3100-Avant Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Multiple nucleotide sequence alignment was performed with BioEdit version 7.0.2 software program. The amino acid sequence homology 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.

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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 identified 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 activity (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. Morphological, 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, therefore, 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 (GenBank 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 confirmed 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 summarized 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.

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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 chromatography (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 ChiA65 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. licheniformis 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 uniformity, 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% identity), 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 glutamic acid residue in the active site was not accessible to EDC. This behavior was similar to that observed for the purified chitinase ScChi50 from the stomach of red scorpionfish Scorpaena scrofa already described by the authors [24]. Chitinase activity was, however, 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 applications. 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 activity 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 glutamic acid residues, in chitinases, where these amino acids have

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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

Bacillus licheniformis strain LHH100 (KJ017975)

79

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)

91

Bacillus aerophilus strain 28KT (AJ831844) Bacillus altitudinis strain 41KF2b T (AJ831842)

Bacillus acidicola strain 105-2T (AF547209)

92

Bacillus shackletonii strain LMG 18435T (AJ250318) Bacillus methanolicus strain NCIMB 13113T (AB112727) Bacillus seohaeanensis strain BH724T (AY667495)

58

Bacillus marisflavi strain TF-11 T(AF483624)

87

Bacillus vietnamensis strain 15-1T (AB099708)

91 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. licheniformis 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 (mg)b,d

Specific activity (U/mg of protein)b

Activity recovery rate (%)

Purification factor (-fold)

Crude extract Heat treatment (70 ◦ C, 30 min) (NH4 )2 SO4 fractionation (30–60%) Sephacryl S-200

450 ± 4.5 380 ± 3.1 319 ± 2.1 181 ± 0.9

910 ± 9.8 397 ± 2.5 125 ± 1.8 23 ± 0.4

494.5 ± 1.3 957.1 ± 2.4 2,552.0 ± 7.6 7,869.5 ± 7.6

100 84 70 40

1 1.9 5.1 15.9

a b c d

From 500 ml of a 48 h culture of B. licheniformis strain LHH100. The experiments were conducted 3 times and ± standard errors are reported. 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. Amounts of protein were estimated by the Bradford method [20].

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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 ChiA-65 Chi Chi72 Chi Chi-L Chi-M Chi-S Chi Chi30 Chi-F1 Chi-F2 SsChi50 a

Microorganism B. licheniformis LHH100 B. licheniformis MB-2 B. licheniformis SK1 Bacillus sp. NTCU2 Bacillus MH-1 Bacillus MH-1 Bacillus MH-1 Streptomyces RC1071 S. thermoviolaceus OPC-520 P. aeruginosa K-187 P. aeruginosa K-187 Scorpaena scrofa

MW, molecular weight.

pH opt. 4 6 6-8 7 6.5 5.5 5.5 8 4 8 7 4

Optima temp. (◦ C) 75 70 55 60 75 65 75 40 60 50 40 75

Half-life time (min) ◦

240 (90 C) 80 (60 ◦ C) 90 (60 ◦ C) 30 (60 ◦ C) 10 (80 ◦ C) 10 (70 ◦ C) 10 (80 ◦ C) 60 (60 ◦ C) 30 (60 ◦ C) 50 (10 ◦ C) 50 (10 ◦ C) 480 (90 ◦ C)

MW (kDa)a

Reference

65 67 72 36.5 71 62 53 70 30 30 30 50

This study [9] [25] [27] [31] [31] [31] [32] [33] [34] [34] [24]

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3.8. Effects of temperature on the activity and stability of ChiA-65 The optimum temperature recorded for the activity of the purified 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. licheniformis 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 thermostability of the enzyme. Maximal thermostability was achieved with 5 mM Ca2+ (data not shown). As shown in Fig. 4d, the half-life of the purified chitinase at 70, 80, and 90 ◦ C increased to 9, 6, and 4 h in the presence 5 mM CaCl2 . Most chitinases have been reported to be significantly stabilized by the addition of Ca2+ at higher temperatures [9,25]. The improvement of enzyme thermostability against thermal inactivation in the presence of CaCl2 can be attributed to strengthening of the interactions inside the protein molecules and to the binding of Ca2+ to the active site. Further studies are necessary in order to understand this upgrading process.

Fig. 3. Effect of the calcium concentration (from 0.5 to 15 mM) on the purified chitinase activity. Chitinase activity measured, using colloidal chitin as substrate, in the absence of calcium supplemented with 2 mM EGTA, was taken as control (100%). Each point represents the mean (n = 3) ± standard deviation.

applications involving the bioconversion of chitin waste, a highly resistant and abundant biopolymer from crustacean food industry, into glucosamine and chito-oligosaccharide value-added products [26].

(a)

(b) 125

Residual chitinase activity (%)

Relative chitinase activity (%)

125

100

75

50

25

100

75

50

.

25

0

0 2

3

4

5

6

7

8

9

10

11

12

3

13

4

5

6

(c)

8

9

(d) 150 0 mM Ca2+ 5 mM Ca2+

200

150

100

50

Residual chitinase activity (%)

250

Relative chitinase activity (%)

7

pH

pH

125

70 °C (0 m M Ca2+)

70 °C (5 m M Ca2+)

80 °C (0 m M Ca2+)

80 °C 5 m M Ca2+)

90 °C (0 m M Ca2+)

90 °C (5 m M Ca2+)

100 75 50 25 0

0 40 45 50 55 60 65 70 75 80 85 90 95 100

Temperature (°C)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

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 Table 3 Effects of various reagents on purified chitinase ChiA-65 chitinase from B. licheniformis strain LHH100 with colloidal chitin as substrate. Chemical reagent/metallic ionsa

Amino acid involved/material

Concentration (mM)

Relative activity (%)b

None p-CMB NEM DTT 2-ME TNBS PMSF EDC

– Cysteine Cysteine Cysteine Cysteine Lysine Serine Carboxylic amino acids Histidine Typtophane Tyrosine CaCl2 MnSO4 MgSO4 CoSO4 CuSO4 ZnSO4 BaSO4 FeSO4 AgNO3 AlSO4 CdCl2 HgCl2 HgNO3 KCl LiSO4

– 2 2 1 5 2 5 2

100 ± 2.50 0 0 10 ± 0.50 21 ± 0.65 98 ± 2.20 104 ± 2.52 112 ± 3.05

1 2 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

99 ± 2.50 101 ± 2.55 98 ± 2.30 295 ± 5.05 127 ± 2.65 115 ± 2.66 112 ± 3.09 105 ± 2.56 131 ± 3.01 86 ± 1.85 95 ± 2.23 10 ± 0.80 113 ± 3.06 35 ± 0.96 0 0 145 ± 4.01 132 ± 3.48

DEP NBS NAI Ca2+ Mn2+ Mg2+ Co2+ Cu2+ Zn2+ Ba2+ Fe2+ Ag2+ Al2+ Cd2+ Hg2+ Hg+ K+ Li+ a b

Incubation with 25 ␮g of purified enzyme at 60 ◦ C at pH 4 for 30 min. Values represent means of 3 replicates, and ± standard errors are reported.

Overall, the thermocativity (a temperature optimum of 75 ◦ C) and the thermostability (a half-life of 4 h at 90 ◦ C) measured for the ChiA-65 chitinase from B. licheniformis strain LHH100 were higher than those previously reported for other bacterial chitinases. Accordingly, the high temperature optimum and the thermal stability of the chitinase from B. licheniformis strain LHH100 provide further confirmation of its potential industrial application in the recycling of chitin wastes [26]. 3.9. Substrate specificity of ChiA-65 The ability to hydrolyze several carbohydrates substrates is an important criterion for the effectiveness of chitinases. Accordingly,

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kinetic experiments were performed via reducing sugar assays using natural substrates. The results are summarized in Table 4. The findings indicate that maximum chitinase activity was obtained at 24 h for the natural substrates, presumably due to the low solubility and viscosity of the biopolymers. The most preferred natural substrate was colloidal chitin, followed by glycol chitin, glycol chitosane, chitotriose, and chitooligosaccharide. No activity was observed toward chitobiose or any other substrate that consisted of a glucosidic linkage. This indicates the absence of chitobiase and glycosidases other than chitinases. Kinetics experiments with synthetic substrates carrying the pnitrophenol group were performed by standard activity assay. The results indicate that maximum chitinase activity was obtained at 6 h for the synthetic substrates and clearly showed that ChiA-65 cleaved specific ␤-glycosidic bonds (Table 4). Although chitinase ChiA-65 released p-NP from p-NP-(Glc-NAc)n (n = 2–4), it probably did not exhibit any activity toward p-NP-(Glc-NAc). This suggests that the enzyme, like other Bacillus chitinases, has a preference for glycosidic bonds, which are second from the non-reducing end [9,25]. Although the p-NP-releasing activity of ChiA-65 showed high activity (15,900 U/mg) toward p-NP-(Glc-NAc)2 , its activity toward p-NP-(Glc-NAc)n (n = 3, 4) was found to decrease markedly.

3.10. Determination of the kinetic parameters of ChiA-65 ChiA-65 and SMChi exhibited the classical kinetics of MichaelisMenten for the two substrates used. Kinetic parameters were obtained from Michaelis-Menten plots (Fig. 5). The order of the 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 colloidal chitin was used as a protein substrate, the kcat /Km exhibited by ChiA-65 was 2.75 times higher than that of SMChi. When p-NP-(Glc-NAc) 2 was used as a synthetic substrate, ChiA-65 was also noted to exhibit kcat /Km values that was 2.22 times higher than that of SMChi (Fig. 5, Table 5).

3.11. Cloning and sequencing of the chiA-65 gene Using the chitinase gene sequences of Bacillus strains, two primers, called F-ChiA and R-ChiA, from B. licheniformis DSM13 and DSM8785 [4] were designed and used to amplify a fragment 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 Glycol chitin Glycol chitosan Chitibiose Chitotriose Chitooligosaccharide c Amylose Carboxymethyl cellulose Cellobiose Laminarin p-NP-(GlcNAc) (G-P) p-NP-(GlcNAc)2 (G-G-P) p-NP-(GlcNAc)3 (G-G-G-P) p-NP-(GlcNAc)4 (G-G-G-G-P)

40 40 40 50 50 50 40 40 50 50 5 5 5 5

550 550 550 550 55 550 550 550 550 550 420 420 420 420

7,800 ± 7.5 7,488 ± 7.1 7,332 ± 6.4 0 7,146 ± 6.1 6,846 ± 5.8 0 Nd 0 0 Nd 15,900 ± 9.4 13,515 ± 8.2 11,282 ± 9.4

100 ± 2.50 96 ± 2.12 94 ± 2.10 0 92 ± 2.00 88 ± 1.90 0 Nd 0 0 Nd 100 ± 2.50 85 ± 1.72 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.

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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 ChiA65 since, as determined by the Edman degradation method, the deduced amino acid sequence was noted to include the 27 Nterminal 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 mismatched 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 peptide, 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 protein. 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)

Colloidal chitin

ChiA-65 SMChi ChiA-65 SMChi

0.385 0.516 0.646 0.819

p-NP-(GlcNAc)2 (G-G-P)

Vmax (U/mg) 7,500 3,651 36,752 27,955

kcat (s−1 ) 5,000 2,434 24,501 13,977

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.

kcat /Km (ml mg s−1 )

Catalytic efficiency relative to ChiA-65

12,987 ± 6.50 4,717 ± 5.60 37,927 ± 9.45 17,065 ± 7.15

1.00 0.36 1.00 0.44

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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.

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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 applications. 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. Yamabhai, 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.

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