Purification and characterization of chitinases from transformed callus suspension cultures of Trichosanthes kirilowii Maxim

Three extracellular basic chitinases designated as TKC 15, TKC 28-I, and TKC 28-II were purified from Agrobacterium rhizogenes A4 transformed Trichosanthes kirilowii Maxim. callus suspension cultures using PerSeptive HS M cation exchange and Sephadex G 75 S gel filtration chromatography. These chitinases exhibited maximal activity at pH 6 and temperature at 40–45°C. N-terminal analysis suggests that two chitinases (TKC 28-I and TKC 28-II) with indistinguishable molecular masses (28 kDa) belonged to the Class III chitinase family. Another basic protein with a molecular mass of 15 kDa (TKC 15) also possesses chitinase activity. Chitobiose was the major end product from chitin digested by TKC 28-I and TKC 28-II whereas TKC 15 released a mix of tetramers, trimers and dimers from chitin. Slow cleavage of chitotriose by TKC 28-I and 28-II and no cleavage of tetramers and trimers by TKC 15 were observed. TKC 28-I cleaved tetramer faster than trimers, 1.1 × 10 −4 M·h −1·μg −1 and 1.5 × 10 −6 M·h −1·μg −1 respectively. All chitinases showed inhibitory ability in a cell-free protein translation system but were far less potent than trichosanthin, a ribosome inactivating protein, found in the storage root tuber of T. kirilowii. The purified T. kirilowii chitinases did not show antifungal activity against Aspergillus flavus or Trichoderma viride.