Highly thermostable chitinase from pineapple: Cloning, expression, and enzymatic properties

Abstract

A complementary DNA encoding the thermostable chitinase of pineapple (Ananas comosus), designated PLChiA, was cloned, sequenced, and expressed in Escherichia coli cells. The determined nucleotide sequence of the gene revealed an 882-bp open reading frame that encoded a putative signal sequence (25 amino acids) and a mature protein (268 amino acids, 27,757Da). Based on the amino acid sequence homology, PLChiA belongs to the class III chitinases in glycoside hydrolase family 18. The recombinant enzyme was purified to homogeneity by a single-step column chromatography. The purified enzyme displayed optimal catalytic activity at pH 3.0 and 70°C and had a molecular mass of 27.8kDa on SDS gel. Strikingly, the half-life was more than 5.2 days when heated at pH 7.0 and 75°C. The half-lives at pH 7.0 were 4.5h at 80°C and 27min at 85°C. Even at 80°C, the half-life was 65min at pH 4.0. Hence, PLChiA is the most thermostable chitinase reported to date from land plants.