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Year : 2010  |  Volume : 2  |  Issue : 1  |  Page : 38-43

Isolation and purification of fungal pathogen (Macrophomina phaseolina) induced chitinase from moth beans (Phaseolus aconitifolius)

1 Banasthali University, Rajasthan - 304022, India
2 Jamia Hamdard, Hamdard University, New Delhi-110 062, India

Correspondence Address:
Himanshu Gupta
Jamia Hamdard, Hamdard University, New Delhi-110 062
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0975-7406.62708

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Objective : Chitinase (EC is one of the major pathogenesis-related proteins, which is a polypeptide that accumulates extracellularly in infected plant tissue. An attempt was made to isolate and purify the chitanase enzyme using moth beans as an enzyme source. Materials and Method : The enzyme was isolated and purified from moth beans against the fungal pathogen Macrophomina phaseolina strain 2165. The isolation and purification was done in both in vitro and in vivo conditions. Purification of chitinase was carried out to obtain three fractions, viz. 50C heated, ammonium sulfate precipitated and sephadex G-25 column-eluted fractions. The molecular mass of Chitinase was directly estimated by sodium dodecyl sulfate-polyacryamide gel electroresis (SDS-PAGE). Result : The yield is sufficient for initial characterization studies of the enzyme. The molecular study of the enzyme shows the possibility of generating the defense mechanism in plants in which it cannot occur. Chitinase was purified by gel filtration chromatography with 20.75-fold and 32.78-fold purification in the in vitro and in vivo conditions, respectively. The enzyme shows a maximum activity after 90 min with 0.1 ml of colloidal chitin as a substrate and 0.4 ml of crude chitinase extract. The optimum pH of 5.0 and an optimum temperature of 40C was found for maximal activity. The molecular weight of purified chitinase was estimated to be 30 kDa by SDS-PAGE. Conclusion : The chitinase isolated in both in vitro and in vivo conditions is stable andactive.

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