Ann Microbiol (2012) 62:835–841 DOI 10.1007/s13213-011-0324-4 ORIGINAL ARTICLE Characterization of a novel chitinase from a moderately halophilic bacterium, Virgibacillus marismortui strain M3-23 Badiâa Essghaier & Abdeljabbar Hedi & Mohamed Bejji & Haϊssam Jijakli & Abdellatif Boudabous & Najla Sadfi-Zouaoui Received: 16 September 2010 /Accepted: 13 July 2011 /Published online: 5 August 2011 # Springer-Verlag and the University of Milan 2011 Abstract A new chitinase produced by the moderately structural component of the shells of crustaceans, insect halophilic bacterium Virgibacillus marismortui strain M3- exoskeletons and other arthropods, as well as a component of 23 was identified and characterized. Distinguishable charac- the cell walls of most fungi and some algae (Patil et al. teristics of high activity and stability at different pH, 2000). Chitin is degraded by chitinases, which are produced temperatures and salinity of M3-23 chitinase are reported. by a large variety of chitin-degrading organisms including Analysis of the catalytic domain sequence from the enzyme bacteria, fungi, insects and plants. Chitinolytic microorganisms highlighted its relationship to glycosyl hydrolase family 18. or chitinolytic enzymes have shown immense potential for Comparison of the deduced chitinase sequence from strain application in the biological control of plant pathogenic fungi M3-23 to known chitinases from Bacillus species showed low and insects in agricultural or environmental applications similarity (82%), suggested its novelty. This is the first report (Freeman et al. 2004;Jungetal.2005;Chuan2006). of the characterization of chitinase from the species V. Several bacteria produce enzymes that degrade chitin, marismortui. The halo- and thermo-tolerant nature of the including many Bacillus species, e.g., B. circulans (Watanabe chitinolytic enzyme allows its potential use in agricultural et al. 1990), B. licheniformis (Trachuck et al. 1996), B. and industrial applications. cereus (Pleban et al. 1997), B. subtilis and B. pumilus (Wang et al. 2006; Ahmadian et al. 2007), Streptomyces species Keywords Chitinase . Halotolerant . Thermotolerant . (Tsujibo et al. 1993a)andAlteromonas sp. (Tsujibo et al. Virgibacillus marismortui . Catalytic domain 1993b)—all these cited species have been found to be potential biocontrol agents. However, our laboratory was the first to take an interest in the study of antifungal chitinase Introduction from moderately halophilic bacteria such as Virgibacillus marismortui, Terribacillus halophilus and Planococcus rifi- Chitin, a homopolymer of N-acetyl-D-glucosamine (GlcNAc) toensis. These moderately halophilic bacteria were isolated residues linked by β-1,4 bonds, is the second most abundant previously from shallow salt lakes in Tunisia and selected as polymer in nature after cellulose. It is widely distributed as a strong antagonists of Botrytis cinerea—the causal agent of grey mold disease on strawberries and tomatoes under the commercial standard conditions applied in Tunisia (Essghaier : : : B. Essghaier A. Hedi A. Boudabous N. Sadfi-Zouaoui (*) et al. 2009; Sadfi-Zouaoui et al. 2008). Laboratoire Microorganismes et Biomolécules Actives, Faculté It should be noted that, on the one hand, the use of the des Sciences de Tunis, Université de Tunis El Manar, present enzyme toolbox is limited as supply is insufficient Campus Universitaire, 2092, Tunis, Tunisia to meet most industrial demands. Therefore, efforts are still e-mail: [email protected] being made to find newer sources of enzymes, higher- : : yielding production techniques and novel applications of B. Essghaier M. Bejji H. Jijakli these enzymes in unexplored fields. In view of these Unité de Phytopathologie, Faculté Universitaire des Sciences Agronomiques de Gembloux, restrictions, researchers have diverted their attention to the Gembloux, Belgium isolation and characterization of enzymes from extremo- 836 Ann Microbiol (2012) 62:835–841 philic organisms, including halophiles (Rothschild and medium is nutrient broth medium (Difco, Detroit, MI) Mancinelli 2001). supplemented with yeast extract, 0.3% (w/v) and 0.5% (w/ On the other hand, in Tunisia, large yield losses are caused v) colloidal chitin prepared according to Rodriguez-Kabana by variations in temperature and high salinity. The presence of et al. (1983). Cultures were incubated at 37°C for 5 days on high salt concentrations characterizes some Tunisian soils, a rotary shaker (150 rpm). After centrifugation at 8,000 rpm especially in Sahel regions due to the extensive use of a heavy for 10 min, the cell-free supernatant from culture was used dressing of manure and irrigation with brackish waters for measurement of chitinase activity. containing high concentrations (3–4g/l)ofNaCl(Messaïet The effect of salinity on chitinase production was al. 2006). Consequently, many investigations based on evaluated using a series of media containing various biotechnological approaches (Liu and Li 1991;Messaïet concentrations of salt (0, 5, 10, 15, 20, 25 and 30% NaCl al. 2006) have been directed towards regenerating salt- w/v) (Essghaier et al. 2010). tolerant plants adapted to the conditions of high salinity found in Tunisia. Finding a broad antifungal chitinase able to Chitinase assay function under such environmental conditions is relevant for biocontrol purposes. Chitinase activity was determined according to the method of The present work is a continuation of a project focused Gomez Ramirez et al. (2004) as previously detailed on discovering new biological control agents isolated from (Essghaier et al. 2009, 2010). A 1:1 mixture (v/v) of cell extreme saline soil from Tunisia that are able to reduce grey free supernatant and 10% (w/v) colloidal chitin in 0.2 M mold disease on strawberries and tomatoes. Previous phosphate buffer pH 7 was incubated for 1 h at 50°C. The research also aimed to identify the mode of action of the reaction was stopped by adding 1 ml 1% NaOH and shaking. most successful isolates as this information may help The concentration of reaction products was determined by optimize their biocontrol efficiency in the field. Under- 3,5-dinitrosalicylic acid assay, and the absorbance was standing the mechanisms involved in biological control measured at 535 nm. The chitinase activity was defined as may enable efficacy to be enhanced (Sadfi-Zouaoui et al. theamountofenzymerequiredtoproduce1μmol N- 2008; Essghaier et al. 2009). We reported previously the acetylglucosamine (Sigma, St. Louis, MO) per hour per efficiency of the moderately halophilic Virgibacillus maris- milliliter of supernatant (Roja-Avelizapa et al. 1999). All mortui strain M3-23 in biological control, and its high experiments were carried out in triplicate. production of chitinase. The purpose of this study was to evaluate the effect of three variables (pH, temperature and Influence of pH and temperature on chitinase activity salt concentration) on the activity and stability of the and stability chitinase produced by strain M3-23. The partially purified chitinase was also characterized biochemically and the gene The optimal temperature for enzyme activity was determined encoding it was sequenced. by monitoring activity at pH 8 at various temperatures ranging from 40 to 90°C. Heat stability was analyzed by measuring the residual activity after preincubation of the enzyme solution Materials and methods (cell free supernatants) for 30 min at various temperatures in the interval between 40 and 90°C (Smaali et al. 2003). Screening and taxonomic analysis of strain M3-23 The pH optimum of enzyme was determined by applying substrate solution at different pH values (5–12), and activity A new strain of moderately halophilic bacterium Virgiba- was measured at optimum temperature (70°C). pH values cillus marismortui strain M3-23 was isolated from a were adjusted using the following buffers: 0.2 M phosphate Tunisian shallow salt lake (Essghaier et al. 2009). The buffer (pH 5.0–6.0), 0.2 M Tris-HCl buffer (pH 7.0–8.0), morphological, physiological and molecular characteristics 0.2 M H3BO3-NaOH buffers (pH 9.0–10.0) and 0.2 M of this strain were previously reported and its nucleotide Na2HPO4-NaOH (pH 12.0). The pH stability was examined sequence of 16S rDNA has been deposited in the GenBank by incubating enzyme solution in the above buffers for 1 h database under the accession number GQ2825501 as at 4°C before adding the substrate. The remaining activities previously described (Essghaier et al. 2009). (%) were subsequently determined (Ellouze et al. 2007). Media composition and culture conditions for chitinase Crude enzyme preparation production Bacterial strain M3-23 was grown in 500-ml flasks Bacterial growth was carried out on Mc medium as described containing 200 ml Mc medium supplemented with 10 % by Leelasuphakul et al. (2006), with minor modifications. Mc NaCl (w/v) at 37°C for 5 days with stirring at 150 rpm. The Ann Microbiol (2012) 62:835–841 837 culture fluid was centrifuged at 8,000 rpm at 4°C for Jolla, CA). Cells carrying the recombinant plasmids were 10 min. The supernatant was subjected to precipitation with screened and cultured individually in Luria-Bertani (LB) ammonium sulphate to 80% saturation at 4°C with constant medium supplemented with 50 μg/ml ampicillin. After stirring overnight. The precipitate was collected by centri- growth on a rotary shaker at 37°C for 20 h, cells were fugation at 9,000 rpm for 30 min at 4°C, dissolved in an harvested by centrifugation (8,000 rpm, 10 min). The pellet appropriate volume of 0.2 M phosphate buffer (pH 9), and obtained was retained for the extraction and purification of dialysed extensively against the same buffer. The resultant recombinants plasmids using the Gene jet TM plasmid dialysate was used as chitinase crude extract, and was miniprep Kit (Fermentas); plasmid DNA was eluted in sterilized by filtration through a 0.2 μm pore size filter (Life distilled water. Sciences, PALL, Ann Arbor, MI, Acrodisc 32 mm syringe DNA sequencing and analysis were performed on an filter with 0.2 μm Supor membrane) and stored at −20°C automated system (GATC Biotech, Germany). DNA and until further use for electrophoresis.