Purification and characterization of a novel antifungal endo-type chitosanase from Anabaena fertilissima

Abstract

A novel antifungal chitosanase from Anabaena fertilissima, strain RPAN1, was characterized as a prelude to its use in biocontrol. The culture grown at 8:16 h L:D photoperiod showed highest chitosanase/antifungal activity under environmental and nutritional conditions of 43 μM of P level, pH 9.0 and temperature of 27°C. The transcriptional level of chitosanase encoding gene (cho) measured using quantitative real-time PCR (qRT-PCR) also indicated increased expression levels under the same optimized conditions. Under these conditions, cho encoding chitosanase was purified which exhibited a specific activity of 822 U/mg. The chitosanase activity measured using different substrates showed the highest activity against colloidal chitosan. HPLC profile of the products of enzyme activity with different chitosan oligosaccharides revealed the production of dimer units (GlcN)2 or more, confirming the endo-type nature of the purified chitosanase. The optimum pH and temperature of the purified enzyme was 7.5 and 27°C, respectively. Further, the enzyme was stable in the pH range of 5.5-9.0 up to 12 h and temperature between 27 and 50°C up to 3 h. The enzyme was strongly inhibited by Ag+, Fe 3+ and Hg2+ and stimulated by Cu+2 and Zn 2+. The investigation revealed significant features regarding the stability of the chitosanase enzyme from A. fertilissima under a broad range of pH and temperature which can help in its effective use in biocontrol. © Springer-Verlag and the University of Milan 2011.