This paper reports the isolation and identification of chitinase-producing Bacillus from chitin-containing wastes, production of a thermostable and alkaline chitinasese, and enzyme characterization. Bacillus thuringiensis subsp. kurstaki HBK-51 was isolated from soil and was identified. Chitinase was obtained from supernatant of B. thuringiensis HBK-51 strain and showed its optimum activity at 110°C and at pH 9.0. Following 3 hours of incubation period, the enzyme showed a high level of activity at 110°C (96% remaining activity) and between pH 9.0 and 12.0 (98% remaining activity). Considering these characteristics, the enzyme was described as hyperthermophile-thermostable and highly alkaline. Two bands of the enzyme weighing 50 and 125？kDa were obtained following 12% SDS-PAGE analyses. Among the metal ions and chemicals used, Ni2+ (32%), K+ (44%), and Cu2+ (56%) increased the enzyme activity while EDTA (7%), SDS (7%), Hg2+ (11%), and ethyl-acetimidate (20%) decreased the activity of the enzyme. Bacillus thuringiensis subsp. kurstaki HBK-51 is an important strain which can be used in several biotechnological applications as a chitinase producer. 1. Introduction Chitin, a linear β-(1,4)-linked N-acetylglucosamine (GlcNAc) polysaccharide, is the main structure component of the fungal cell wall and the exoskeletons of invertebrates, such as insects and crustaceans. Chitinase plays a variety of important roles in these organisms ranging from nutrition to defense and control of ecdysis in insects. The importance of chitinases in many biological processes makes their inhibitors important targets for potential antifungal and insecticidal agents as well as antimalarial agents [1]. It is one of the most abundant naturally occurring polysaccharides and has attracted tremendous attention in the fields of agriculture, pharmacology, and biotechnology. Chitin is the second most abundant component of biomass on earth [2]. This linear polymer can be hydrolyzed by bases, acids or enzymes, such as lysozyme, some glucanases, and chitinase. Chitinases (EC 3.2.1.14), essential enzymes catalyzing the conversion of chitin to its monomeric or oligomeric components (low-molecular-weight products), have been found in a wide range of organisms, including bacteria, plants, fungi, insects, and crustaceans. Because chitin is not found in vertebrates, it has been suggested that inhibition of chitinases may be used for the treatment of fungal infections and human parasitosis [3]. Biological control, using microorganisms to repress plant disease, offers an alternative environmentally
References
[1]
J. N. Tabudravu, V. G. H. Eijsink, G. W. Gooday et al., “Psammaplin A, a chitinase inhibitor isolated from the Fijian marine sponge Aplysinella rhax,” Bioorganic and Medicinal Chemistry, vol. 10, no. 4, pp. 1123–1128, 2002.
[2]
H. S. Lee, D. S. Han, S. J. Choi et al., “Purification, characterization, and primary structure of a chitinase from Pseudomonas sp. YHS-A2,” Applied Microbiology and Biotechnology, vol. 54, no. 3, pp. 397–405, 2000.
[3]
C. M. Wen, C. S. Tseng, C. Y. Cheng, and Y. K. Li, “Purification, characterization and cloning of a chitinase from Bacillus sp. NCTU2,” Biotechnology and Applied Biochemistry, vol. 35, no. 3, pp. 213–219, 2002.
[4]
W. T. Chang, Y. C. Chen, and C. L. Jao, “Antifungal activity and enhancement of plant growth by Bacillus cereus grown on shellfish chitin wastes,” Bioresource Technology, vol. 98, no. 6, pp. 1224–1230, 2007.
[5]
S. Kudan and R. Pichyangkura, “Purification and characterization of thermostable chitinase from Bacillus licheniformis SK-1,” Applied Biochemistry and Biotechnology, vol. 157, no. 1, pp. 23–35, 2009.
[6]
S. A. Shaikh and M. V. Deshpande, “Chitinolytic enzymes: their contribution to basic and applied research,” World Journal of Microbiology and Biotechnology, vol. 9, no. 4, pp. 468–475, 1993.
[7]
J. D. Holt, Manual of Determinative Bacteriology, Williams & Wilkins, Baltimore, Md, USA, 1993.
[8]
R. M. Cody, N. D. Davis, J. Lin, and D. Shaw, “Screening microorganisms for chitin hydrolysis and production of ethanol from amino sugars,” Biomass, vol. 21, no. 4, pp. 285–295, 1990.
[9]
J. Frankowski, M. Lorito, F. Scala, R. Schmid, G. Berg, and H. Bahl, “Purification and properties of two chitinolytic enzymes of Serratia plymuthica HRO-C48,” Archives of Microbiology, vol. 176, no. 6, pp. 421–426, 2001.
[10]
R. A. K. Srivastava, “Purification and chemical characterization of thermostable amylases produced by Bacillus stearothermophilus,” Enzyme and Microbial Technology, vol. 9, no. 12, pp. 749–754, 1987.
[11]
J. Folders, J. Tommassen, L. C. van Loon, and W. Bitter, “Identification of a chitin-binding protein secreted by Pseudomonas aeruginosa,” Journal of Bacteriology, vol. 182, no. 5, pp. 1257–1263, 2000.
[12]
M. G. Ramírez, L. I. R. Avelizapa, N. G. R. Avelizapa, and R. C. Camarillo, “Colloidal chitin stained with Remazol Brilliant Blue R, a useful substrate to select chitinolytic microorganisms and to evaluate chitinases,” Journal of Microbiological Methods, vol. 56, no. 2, pp. 213–219, 2004.
[13]
N. J. R. Shih and K. A. McDonald, “Purification and characterization of chitinases from transformed callus suspension cultures of Trichosanthes kirilowii Maxim,” Journal of Fermentation and Bioengineering, vol. 84, no. 1, pp. 28–34, 1997.
[14]
H. S. Kim, K. N. Timmis, and P. N. Golyshin, “Characterization of a chitinolytic enzyme from Serratia sp. KCK isolated from kimchi juice,” Applied Microbiology and Biotechnology, vol. 75, no. 6, pp. 1275–1283, 2007.
[15]
J. Favaloro, R. Treisman, R. Kamen, L. Grosman, and K. Moldave, Methods in Enzymology, Academic Pres, New York, NY, USA, 1980.
[16]
K. G. Hardy, Plasmids, The Practical Approach Serie, 1993.
[17]
J. F. MacFaddin, Biochemical Tests For Identification of Medical Bacteria, Williams & Wilkins, Baltimore, Md, USA, 2000.
[18]
W. P. Chen and T. T. Kuo, “A simple and rapid method for the preparation of gram-negative bacterial genomic DNA,” Nucleic Acids Research, vol. 21, no. 9, p. 2260, 1993.
[19]
A. A. Denizci, D. Kazan, E. C. A. Abeln, and A. Erarslan, “Newly isolated Bacillus clausii GMBAE 42: an alkaline protease producer capable to grow under higly alkaline conditions,” Journal of Applied Microbiology, vol. 96, no. 2, pp. 320–327, 2004.
[20]
S. F. Altschul, W. Gish, W. Miller, E. W. Myers, and D. J. Lipman, “Basic local alignment search tool,” Journal of Molecular Biology, vol. 215, no. 3, pp. 403–410, 1990.
[21]
N. N. Nawani and B. P. Kapadnis, “Production dynamics and characterization of chitinolytic system of Streptomyces sp. NK1057, a well equipped chitin degrader,” World Journal of Microbiology and Biotechnology, vol. 20, no. 5, pp. 487–494, 2004.
[22]
D. M. Bollag, S. J. Edelstein, and M. D. Rozycki, Protein Methods, Villey-Liss Pres, 1996.
[23]
P. Siwayaprahm, M. Audtho, K. Ohmiya, and C. Wiwat, “Purification and characterization of a Bacillus circulans No. 4.1 chitinase expressed in Escherichia coli,” World Journal of Microbiology and Biotechnology, vol. 22, no. 4, pp. 331–335, 2006.
[24]
Y. S. Lee, I. H. Park, J. S. Yoo et al., “Cloning, purification, and characterization of chitinase from Bacillus sp. DAU101,” Bioresource Technology, vol. 98, no. 14, pp. 2734–2741, 2007.
[25]
J. G. Li, Z. Q. Jiang, L. P. Xu, F. F. Sun, and J. H. Guo, “Characterization of chitinase secreted by Bacillus cereus strain CH2 and evaluation of its efficacy against Verticillium wilt of eggplant,” BioControl, vol. 53, no. 6, pp. 931–944, 2008.
[26]
R. F. Guo, B. S. Shi, D. C. Li, W. Ma, and Q. Wei, “Purification and characterization of a novel thermostable chitinase from Thermomyces lanuginosus SY2 and cloning of its encoding gene,” Agricultural Sciences in China, vol. 7, no. 12, pp. 1458–1465, 2008.
[27]
P. E. Yuli, M. T. Suhartono, Y. Rukayadi, J. K. Hwang, and Y. R. Pyun, “Characteristics of thermostable chitinase enzymes from the indonesian Bacillus sp.13.26,” Enzyme and Microbial Technology, vol. 35, no. 2-3, pp. 147–153, 2004.
[28]
M. Ueda, A. Fujiwara, T. Kawaguchi, and M. Arai, “Purification and some properties of six chitinases from Aeromonas sp. No. 10S-24,” Bioscience, Biotechnology, and Biochemistry, vol. 59, no. 11, pp. 2162–2164, 1995.
[29]
K. Sakai, A. Yokota, H. Kurokawa, M. Wakayama, and M. Moriguchi, “Purification and characterization of three thermostable endochitinases of Bacillus noble strain MH-1 isolated from chitin containing compost,” Applied and Environmental Microbiology, vol. 64, no. 9, pp. 3340–3497, 1998.
[30]
R. C. Gomes, L. T. A. S. Semêdo, R. M. A. Soares, C. S. Alviano, L. F. Linhares, and R. R. R. Coelho, “Chitinolytic activity of Actinomycetes from a cerrado soil and their potential in biocontrol,” Letters in Applied Microbiology, vol. 30, no. 2, pp. 146–150, 2000.
