MOLECULAR-CLONING, NUCLEOTIDE SEQUENCING, AND REGULATION OF THE CHIA GENE ENCODING ONE OF CHITINASES FROM ENTEROBACTER SP. G-1

The chiA gene encoding a chitinase of 60-kDa has been cloned from Ente robacter sp. G-1 by PCR using synthetic oligonucleotides corresponding to the amino acid sequences of the purified enzyme and subsequently g enomic library screening was performed. The products of the positive c lones were found to degrade water-insoluble chitin. The primary struct ure of the chiA gene consisted of 1,686-bp encoding 562 amino acid res idues. Comparison of the deduced amino acid sequence of the cloned chi tinase gene product (chiA) with other chitinases revealed a high homol ogy (95.7% identity) with chitinase A from Serratia marcescens QMB1466 . The coding region of the chiA gene for higher expression in Escheric hia coli was identified using deletion and sequence analysis. The expr ession of the chiA gene in Enterobacter sp. G-1 was controlled by pres ence of chitin, as determined by Northern blotting hybridization analy sis. We found that the expression of the chiA gene in E. coli was cont rolled by an inverted repeat sequence located in the upstream region f rom a promoter region.