Extracellular collagenase from Rhizoctonia solani: Production, purification and characterization

Abstract

Potentiality of R. solani grown on Sabouraud-glucose-collagen medium to produce glycosylated metallo-proteinase with collagenolytic activity was optimized and maximum production (212.33 U/mL) was recorded after 108 h of submerged incubation (175 rpm) at pH 5.5 and 30ºC of temperature. Two-step column chromatography technique on DEAE-cellulose and Sephadex G₁₅₀ was adopted to purify the partially purified enzyme produced by ammonium sulfate (40%, w/v) precipitation. Yield of purification was 60.49% of the original activity with specific activity of 18064.7 × 10³ U/mg protein and 18.72-folds of purification. The purified enzyme showed maximum activity at 40ºC and pH 5 ,which was stimulated by ions of Ca, Co, Cu, K, Mg, Na or Zn and inhibited by ions of Fe and Hg. Metal composition of the purified enzyme revealed that it contains Ca²⁺ and Zn²⁺. Tm (midpoint of thermal inactivation) was recorded at 65ºC and 55ºC after 1 and 6 h of exposure, respectively and T₁/₂ was found to be 7 and 5 weeks at ‒15ºC and 4ºC, respectively. Molecular mass, Kmg/mL, Vmax of the enzyme were found to be 66 ± 4 kDa, 0.033 kDa and 0.28 U/mg min⁻¹, respectively. Activities toward gelatin and casein were also detected.