Purification and Characterization of a Thermophilic Fungal Recombinant Chitinase, Overexpressed in Saccharomyces cerevisiae–Suitable For Bioconversion of Chitin Wastes

Muthu Prasad; Peramachi Palanivelu

Volume : IV, Issue : XI, November - 2014

Purification and Characterization of a Thermophilic Fungal Recombinant Chitinase, Overexpressed in Saccharomyces cerevisiae–Suitable For Bioconversion of Chitin Wastes

Muthu Prasad, Peramachi Palanivelu

Abstract :

A chitinase gene of the thermophilic fungus, T. lanuginosus, was cloned and overexpressed in Saccharomyces cerevisiae. The overexpressed recombinant chitinase was purified by ammonium sulfate precipitation, ion exchange and hydrophobic interaction column chromatographic techniques. The enzyme was purified 20&amp;amp;ndash; fold with a specific activity of 69817. The recombinant chitinase was homogeneous as judged by SDS&amp;amp;ndash;PAGE and the molecular mass of the protein was ~ 39 kDa. The purified chitinase was optimally active at pH 6.0 and at 60 &amp;Euml;&amp;scaron;C. KM and Vmax of the purified recombinant were found to be 0.66 mM and 81.6 moles/min/mg protein, respectively. The enzyme was found to be highly thermostable and retained about 80% of the activity even after 3 h at 50 &amp;Euml;&amp;scaron;C. The enzyme was found to be glycosylated. MALDI&amp;amp;ndash;MS analysis confirmed that the purified enzyme belongs to an extracellular, family 18 chitinase type. Atomic force microscopic data clearly indicate the degradation of the native chitin polymer by the recombinant purified chitinase to much smaller units. Efficient hydrolysis of native chitins by the recombinant chitinase suggests potential industrial applications for this enzyme requiring chitin processing at elevated temperatures. CD spectra of the native and denatured recombinant chitinase suggest that the beta structures play an important role on the stability of the enzyme at high temperatures.