امکان‌سنجی تولید کیتیناز از جدایه بومی Beauveria bassiana
محورهای موضوعی : آلودگی های محیط زیست (آب، خاک و هوا)معصومه کردی 1 , الناز فهیمی 2 , ناصر فرَخی 3
1 - دانشجوی دکتری بیوتکنولوژی کشاورزی، گروه علوم و زیست‌فناوری گیاهی، دانشکده علوم و فناوری زیستی، دانشگاه شهید بهشتی، تهران، ایران.
2 - کارشناسی ارشد بیوتکنولوژی کشاورزی، گروه زراعت و اصلاح نباتات، دانشکده کشاورزی، دانشگاه شاهرود، شاهرود، ایران.
3 - استادیار گروه علوم سلولی و مولکولی، دانشکده علوم و فناوری زیستی، دانشگاه شهید بهشتی، تهران، ایران. *(مسوول مکاتبات)
کلید واژه: Beauveria spp, کیتین, آنزیم کیتیناز, کنترل بیولوژیک,
چکیده مقاله :
کنترل بیولوژیک روش جایگزین مناسبی برای روش های شیمیایی مبارزه با آفات می باشد. قارچ بیمارگر حشرات Beauveria spp. میزبان هدف را از طریق نفوذ در کوتیکول، با ترشح آنزیم های تجزیه کننده کوتیکول از قبیل کیتینازها مورد حمله قرار می دهد. کیتینازها قادرند کیتین را هیدرولیز نمایند و در زمینه های مختلفی از جمله کنترل بیولوژیک، اصلاح زیستی آب دریا، تولید حشره کش ها و مواد دارویی استفاده می شود. در این تحقیق فعالیت های آنزیم کیتیناز در قارچ Beauveria bassiana که در سه محیط مختلف کشت داده شده بود به روش رنگ سنجی مورد مطالعه قرار گرفت. گونه قارچی بومی استفاده شده از مینودشت با قدرت بیماری زایی بالا جداسازی و شناسایی گردید، نتایج نشان داد که بیش ترین غلظت آنزیم کل مربوط به محیط کشت P(7) می باشد.بیش ترین فعالیت آنزیم در محلول رویی محیط کشت Pontecorvo با 7 = pH ، در دمای 50 درجه سانتی گراد به عنوان نمونه آنزیمی، در روز سوم کشت و در حضور کیتین کلوئیدی 75/0 درصد اندازه گیری شد، که نتایج حاصله می تواند در تولید بیشتر آنزیم کیتناز بکار گرفته شود و بدین ترتیب شاهد کاربرد آن در کنترل بیولوژیک به منظور حفظ سلامت انسان و محیط زیست باشیم.
Biological control is a suitable substitute technique for chemical battle with pests. Enthomopathogenic fungus Beauveria spp. attacks its target host by penetrating into cuticle through secretion of hydrolytic enzymes such as chitinases. Chitinases are capable of hydrolyzing chitins with many other applications in biocontrol, sea bioremediation, and development of biopesticides, pharmaceuticals. In this research, chitinase enzyme activities from Beauveria bassiana fungus, cultured in 3 culture media, were measured through a colorimetric assay. A native isolate with high mortality rate isolated and identified from Minoodasht. The highest chitinase activity was achieved at day 3 when incubated at 50 ˚C in Pontecorvo medium containing 0.75% colloidal chitin (pH = 7). Our results might be useful for higher production of chitinase that can be implemented in biological control and further elevate human health and environmental biosafety.
1- Kim, J. S. & Je, Y. H. 2010. A novel biopesticide production: attagel-mediated precipitation of chitinase from Beauveria bassiana SFB-205 supernatant for thermotolerance. Applied Microbiology and Biotechnology, VOL. 87, PP. 1639-1648.
2- Pathan, A. A., Devi, K. U., Vogel, H. & Peineke, A. 2007. Analysis of differential gene expression in the generalist entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin grown on different insect cuticular extracts and synthetic medium through cDNA-AFLPs. Fungal Genetics and Biology, VOL. 44, PP. 1231-1241.
3- Martin-Gil, F., Leal, J. A., Gomez-Miranda, B., Martin-Gil, J., Prieto, A. and Ramos-Sanchez, M. C. 1992. Low temperature thermal behaviour of chitins and chitin-glucans. Thermochimica Acta, VOL. 211, PP. 241-254.
4- De León, A., Jimenez-islas, H., Gonzalez-cuevas, M. & Rosa, A. P. 2004. Analysis of the expression of the Trichoderma harzianum ech42 gene in two isogenic clones of Escherichia coli by surface response methodology. Process Biochemistry, VOL. 39, PP. 2173-2178.
5- Raaijmakers, J. M., Leeman, M. & Oorschot, V. 1995. Dose-response relationships in biological control of Fusarium wilt of radish by Pseudomonas spp. Phytopathology, VOL. 85(10), PP. 1075-1081.
6- Bierbaum, S., Nickel, R., Koch, A., Lau, S., Deichmann, K., Wahn, U., Superti-Furga, A. & Heinzmann, A. 2005. Polymorphisms and haplotypes of acid mammalian chitinase are associated with bronchial asthma. American Journal of Respiratory and Critical Care Medicine, VOL. 172, PP. 1505-1512.
7- Binod, P., Pusztahelyi, T., Nagy, V., Sandhya, C., Szakacs, G., Pocsi, I. & Pandey, A. 2005. Production and purification of extracellular chitinases fromPenicilliumaculeatum NRRL 2129 under solid-state fermentation. Enzyme and Microbial Technology, VOL. 36, PP. 880-887.
8- Smith, R. J. & Grula, E. A. 1986. Chitinase is an inducible enzyme in Beauveria bassiana. Journal of Invertebrate Pathology, VOL. 42, PP. 319-326.
9- Coudron, T. A., Kroha, M. J. & Ignoffo, C. M. 1984. Levels of chitinolytic activity during development of three entomopathogenic fungi. Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology, VOL. 3, PP. 339-348.
10- Havukkala, I., Mitamura, C., Hara, S., Hirayae, K., Nishizawa, Y. & Hibi, T. 1993. Induction and Purification of Beauveria bassiana Chitinolytic Enzymes. Plos pathogens, VOL. 61, PP. 97-102.
11- Suresh, P. V. & Chandrasekaran, M. 1998. Utilization of prawn waste for chitinase production by the marine fungus Beauveria bassiana by solid state fermentation. World Journal of Microbiology & Biotechnology, VOL.14, PP. 655-660.
12- Patidar, P., Agrawal, D., Banerjee, T. & Patil, S. 2005. Chitinase production by Beauveria felina RD 101: optimization of parameters under solid substrate fermentation conditions. World Journal of Microbiology & Biotechnology, VOL. 21, PP. 93-95.
13- 13-Pontecorvo, G. 1953. The genetic of Aspergillus nidulans. Department of Genetics, the university, Glasgow Scotland.Advances in Genetics, VOL. 5, PP. 141–239.
14- 14-Roberts, W. K. & Selitrennikoff, C. P. 1998. Plant and bacterial chitinases differ in antifungal activity. Journal of General Microbiology, VOL. 134, PP. 426-428.
15- Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein -dye binding. Analytical Biochemistry, VOL. 72, PP. 248-252.
16- Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing suger. Analytical Chemistry, VOL. 3, PP. 426-428.
17- 17-Leopold, J. & Samsinakova, A. 1970. Quantitative estimation of chitinase and Severalother enzymes in the fungus Beuuveria bassiana. Journal of Invertebrate pathology, VOL. 15, PP. 34-42.
18- 18-Coudron, T. A., Kroha, M. J. & Ignoffo, C. M. 1984. Levels of chitinolytic activity during development of three entomopathogenic fungi. Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology, VOL. 3, PP. 339-348.
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1- Kim, J. S. & Je, Y. H. 2010. A novel biopesticide production: attagel-mediated precipitation of chitinase from Beauveria bassiana SFB-205 supernatant for thermotolerance. Applied Microbiology and Biotechnology, VOL. 87, PP. 1639-1648.
2- Pathan, A. A., Devi, K. U., Vogel, H. & Peineke, A. 2007. Analysis of differential gene expression in the generalist entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin grown on different insect cuticular extracts and synthetic medium through cDNA-AFLPs. Fungal Genetics and Biology, VOL. 44, PP. 1231-1241.
3- Martin-Gil, F., Leal, J. A., Gomez-Miranda, B., Martin-Gil, J., Prieto, A. and Ramos-Sanchez, M. C. 1992. Low temperature thermal behaviour of chitins and chitin-glucans. Thermochimica Acta, VOL. 211, PP. 241-254.
4- De León, A., Jimenez-islas, H., Gonzalez-cuevas, M. & Rosa, A. P. 2004. Analysis of the expression of the Trichoderma harzianum ech42 gene in two isogenic clones of Escherichia coli by surface response methodology. Process Biochemistry, VOL. 39, PP. 2173-2178.
5- Raaijmakers, J. M., Leeman, M. & Oorschot, V. 1995. Dose-response relationships in biological control of Fusarium wilt of radish by Pseudomonas spp. Phytopathology, VOL. 85(10), PP. 1075-1081.
6- Bierbaum, S., Nickel, R., Koch, A., Lau, S., Deichmann, K., Wahn, U., Superti-Furga, A. & Heinzmann, A. 2005. Polymorphisms and haplotypes of acid mammalian chitinase are associated with bronchial asthma. American Journal of Respiratory and Critical Care Medicine, VOL. 172, PP. 1505-1512.
7- Binod, P., Pusztahelyi, T., Nagy, V., Sandhya, C., Szakacs, G., Pocsi, I. & Pandey, A. 2005. Production and purification of extracellular chitinases fromPenicilliumaculeatum NRRL 2129 under solid-state fermentation. Enzyme and Microbial Technology, VOL. 36, PP. 880-887.
8- Smith, R. J. & Grula, E. A. 1986. Chitinase is an inducible enzyme in Beauveria bassiana. Journal of Invertebrate Pathology, VOL. 42, PP. 319-326.
9- Coudron, T. A., Kroha, M. J. & Ignoffo, C. M. 1984. Levels of chitinolytic activity during development of three entomopathogenic fungi. Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology, VOL. 3, PP. 339-348.
10- Havukkala, I., Mitamura, C., Hara, S., Hirayae, K., Nishizawa, Y. & Hibi, T. 1993. Induction and Purification of Beauveria bassiana Chitinolytic Enzymes. Plos pathogens, VOL. 61, PP. 97-102.
11- Suresh, P. V. & Chandrasekaran, M. 1998. Utilization of prawn waste for chitinase production by the marine fungus Beauveria bassiana by solid state fermentation. World Journal of Microbiology & Biotechnology, VOL.14, PP. 655-660.
12- Patidar, P., Agrawal, D., Banerjee, T. & Patil, S. 2005. Chitinase production by Beauveria felina RD 101: optimization of parameters under solid substrate fermentation conditions. World Journal of Microbiology & Biotechnology, VOL. 21, PP. 93-95.
13- 13-Pontecorvo, G. 1953. The genetic of Aspergillus nidulans. Department of Genetics, the university, Glasgow Scotland.Advances in Genetics, VOL. 5, PP. 141–239.
14- 14-Roberts, W. K. & Selitrennikoff, C. P. 1998. Plant and bacterial chitinases differ in antifungal activity. Journal of General Microbiology, VOL. 134, PP. 426-428.
15- Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein -dye binding. Analytical Biochemistry, VOL. 72, PP. 248-252.
16- Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing suger. Analytical Chemistry, VOL. 3, PP. 426-428.
17- 17-Leopold, J. & Samsinakova, A. 1970. Quantitative estimation of chitinase and Severalother enzymes in the fungus Beuuveria bassiana. Journal of Invertebrate pathology, VOL. 15, PP. 34-42.
18- 18-Coudron, T. A., Kroha, M. J. & Ignoffo, C. M. 1984. Levels of chitinolytic activity during development of three entomopathogenic fungi. Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology, VOL. 3, PP. 339-348.