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Studies on Exo-Chitinase Production from Trichoderma asperellum UTP-16 and Its Characterization.

ABSTRACT: The growth conditions for chitinase production by Trichoderma asperellum UTP-16 in solid state fermentation was optimized using response surface methodology based on central composite design. The chitinase production was optimized, using one-factor at a time approach, with six independent variables (temperature, pH, NaCl, incubation period, nitrogen and carbon sources) and 3.31 Units per gram dry substrate (U gds(-1)) exo-chitinase yield was obtained. A 21.15% increase was recorded in chitinase activity (4.01 U gds(-1)) through surface response methodology, indicates that it is a powerful and rapid tool for optimization of physical and nutritional variables. Further, efficiency of crude enzyme was evaluated against phytopathogenic Fusarium spp. and a mycelial growth inhibition up to 3.5-6.5 mm was achieved in well diffusion assay. These results could be supplemented as basic information for the development of enzyme based formulation of T. asperellum UTP-16 and its use as a biocontrol agent.

SUBMITTER: Kumar DP

PROVIDER: S-EPMC3460120 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Studies on Exo-Chitinase Production from Trichoderma asperellum UTP-16 and Its Characterization.

Kumar D Praveen DP Singh Rajesh Kumar RK Anupama P D PD Solanki Manoj Kumar MK Kumar Sudheer S Srivastava Alok K AK Singhal Pradeep K PK Arora Dilip K DK

Indian journal of microbiology 20111014 3

The growth conditions for chitinase production by Trichoderma asperellum UTP-16 in solid state fermentation was optimized using response surface methodology based on central composite design. The chitinase production was optimized, using one-factor at a time approach, with six independent variables (temperature, pH, NaCl, incubation period, nitrogen and carbon sources) and 3.31 Units per gram dry substrate (U gds(-1)) exo-chitinase yield was obtained. A 21.15% increase was recorded in chitinase ...[more]

PMID: 23997329

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Project description:Anopheline larvicidal property of T. asperellum has been found recently in medical science. The mechanism of actions exhibited by T. asperellum to infect mosquito larvae is the pivotal context of our present study. To infect an insect, entomopathogens must undergo some events of pathogenesis. We performed some experiments to find out the mechanisms of action of T. asperellum against anopheline larvae and compared its actions with other two well recognized entomopathogens like Metarhizium anisopliae and Beauveria bassiana. The methodology adopted for this includes Compound light and SE Microscopic study of host-pathogen interaction, detection of fungal spore adhesion on larval surface (Mucilage assay), detection of cuticle degrading enzymes (Spore bound pr1, chitinase and protease) by spectro-photometric method, Quantitative estimation of chitinase and protease enzymes, and determination of nuclear degeneration of hemocyte cells of ME (methanolic extract) treated larvae by T. asperellum under fluorescence microscope. Compound light microscopic studies showed spore attachment, appressorium and germ tube formation, invasion and proliferated hyphal growth of T. asperellum on epicuticle and inside of dead larvae. SEM study also supported them. After 3 h of interaction, spores were found to be attached on larval surface exhibiting pink colored outer layer at the site of attachment indicating the presence of mucilage surrounding the attached spores. The enzymatic cleavage of the 4-nitroanilide substrate yields 4-nitroaniline which indicates the presence of spore-bound PR1 protein (Pathogenecity Related 1 Protein) and it was highest (absorbance 1.298 ± 0.002) for T. asperellum in comparison with control and other two entomopathogens. T. asperellum exhibited highest enzymatic index values for both chitinase (5.20) and protease (2.77) among three entomopathogens. Quantitative experiment showed that chitinase enzyme concentration of T. asperellum (245 µg mL-1) was better than other two M. anisopliae (134.59 µg mL-1) and B. bassiana (128.65 µg mL-1). Similarly protease enzyme concentration of this fungus was best (298.652 µg mL-1) among three entomopathogens. Here we have detected and estimated fragmentized nuclei of hemocyte cells by fluorescence microscopy in treated larvae with different ME doses of T. asperellum, and also observed that mosquito larvae exposed to 0.1 mg mL-1 dose of ME showed maximum (100%) nuclear fragmentations of hemocytes and while 20, 45, 70 and 85% of nuclear deformities were recorded at 0.02, 0.04, 0.06 and 0.08 mg mL-1 concentrations of ME. The knowledge of this work certainly will help in understanding of mechanism of action of T. asperellum for anopheline larval killing and consequently in eradication of malaria vector.

Dataset Information

Studies on Exo-Chitinase Production from Trichoderma asperellum UTP-16 and Its Characterization.

Publications

Studies on Exo-Chitinase Production from Trichoderma asperellum UTP-16 and Its Characterization.

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