Molecular cloning, characterization and nucleotide sequence of the gene for secreted alpha-amylase from Xanthomonas campestris pv. campestris.

Abstract

alpha-Amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) of apparent molecular mass 45 kDa was secreted by Xanthomonas campestris pv. campestris grown in medium containing starch or maltose. We isolated its structural gene from a recombinant lambda library and located it on a 2.7 kb DNA fragment. Nucleotide sequencing of the fragment revealed a potential ORF encoding a protein of 475 amino acid residues, including a potential signal sequence of 35 amino acids. The signal processing site was confirmed by N-terminal amino acid sequence analysis of the exported alpha-amylase. The deduced amino acid sequence of the mature protein is very similar to that of the alpha-amylase of Aeromonas hydrophila. It also contains all four amino acid sequences highly conserved in the alpha-amylases from a wide range of organisms. Expression of the amy gene in Escherichia coli was poor from its own promoter, but was enhanced by the upstream promoter on the vector. The alpha-amylase synthesized in E. coli was located in the periplasm.