Molecular cloning, nucleotide sequencing, and regulation of the chiA gene encoding one of chitinases from Enterobacter sp. G-1

Abstract

The chiA gene encoding a chitinase of 60-kDa has been cloned from Enterobacter sp. G-1 by PCR using synthetic oligonucleotides corresponding to the amino acid sequences of the purified enzyme and subsequently genomic library screening was performed. The products of the positive clones were found to degrade water-insoluble chitin. The primary structure of the chiA gene consisted of 1,686-bp encoding 562 amino acid residues. Comparison of the deduced amino acid sequence of the cloned chitinase gene product (chiA) with other chitinases revealed a high homology (95.7% identity) with chitinase A from Serratia marcescens QMB1466. The coding region of the chiA gene for higher expression in Escherichia coli was identified using deletion and sequence analysis. The expression of the chiA gene in Enterobacter sp. G-1 was controlled by presence of chitin, as determined by Northern blotting hybridization analysis. We found that the expression of the chiA gene in E. coli was controlled by an inverted repeat sequence located in the upstream region from a promoter region.