Purification and properties of two chitinolytic enzymes of Serratia plymuthica HRO-C48

Abstract.

The chitinolytic rhizobacterium Serratia plymuthica HRO-C48 was previously selected as a biocontrol agent of phytopathogenic fungi. One endochitinase (E.C. 3.2.1.14), CHIT60, and one N-acetyl-β-1,4-D-hexosaminidase (E.C. 3.2.1.52), CHIT100, were purified and characterized. The endochitinase CHIT60, with an apparent molecular mass of 60.5 kDa, had a N-terminal amino acid sequence highly similar to that of chitinases A from Serratia liquefaciens and Serratia marcescens. The enzyme activity had its peak at 55 °C and pH 5.4, and increased by more than 20% in the presence of 10 mM Ca²⁺, Co²⁺ or Mn²⁺. Activity was inhibited by 80% in the presence of 10 mM Cu²⁺. CHIT100 appeared to be a monomeric enzyme with a molecular mass of 95.6 kDa and a pI of 6.8. Optimal activity was obtained at 43 °C and pH 6.6, and decreased by more than 90 % in the presence of 10 mM Co²⁺ or Cu²⁺. CHIT100 (100 µg ml^–1) inhibited spore germination and germ tube elongation of the phytopathogenic fungus Botrytis cinerea by 28 % and 31.6 %, respectively. With CHIT60 (100 µg ml^–1), the effect was more pronounced: 78 % inhibition of of germination and 63.9 % inhibition of germ tube elongation.