ISOLATION AND CHARACTERIZATION OF THE 54-KDA AND 22-KDA CHITINASE GENES OF SERRATIA-MARCESCENS KCTC2172

A DNA fragment (pCHI5422) containing two genes encoding a 54-kDa and a 22-kDa chitinase was isolated from a cosmid DNA library of Serratia m arcescens KCTC2172. The complete nucleotide sequence of pCHI5422 consi sting of 4581 bp was determined. The nucleotide sequence of the 22-kDa chitinase consists of 681 bp of open reading frame encoding 227 amino acids and is located 1422 bp downstream of the translation terminatio n codon of the 54-kDa chitinase sequence. The 54-kDa chitinase gene co nsisted of 1497 bp in a single open reading frame encoding 499 amino a cids. The genes encoding the 54-kDa and 22-kDa chitinase were separate ly subcloned in Escherichia coli and the individual chitinases were ex pressed and purified from the culture broth using chitin affinity chro matography. When chitohexaose was used as substrate, the major product of the enzymatic reaction of both the 54-kDa and 22-kDa chitinases wa s a (GlcNAc)(2) dimer with a minor amount of monomer. The specific act ivity of the 54-kDa and 22-kDa chitinases were 300 mu M (min)(-1) mg(- 1) and 17 mu M (min)(-1) mg(-1) on the natural swollen chitin, respect ively.