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-beta -1,4-D-hexosaminidase (E.C. 3.2.1.52), CHIT100, were purified and characterized. The endochitinase CHIT60, with an ent 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 degreesC 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 pl of 6.8. Optimal activity was obtained at 43 degreesC and pH 6.6, and decreased by more than 90 % in the presence of 10 MM Co2+ or Cu2+. CHIT100 (100 mug ml(-1)) inhibited spore germination and germ tube elongation of the phytopathogenic fungus Botrytis cinerea by 28 % and 31.6 %, respectively. With CHIT60 (100 mug ml(-1)), the effect was more pronounced: 78 % inhibition of of germination and 63.9 % inhibition of germ tube elongation.