MOLECULAR-CLONING, CHARACTERIZATION AND NUCLEOTIDE-SEQUENCE OF THE GENE FOR SECRETED ALPHA-AMYLASE FROM XANTHOMONAS-CAMPESTRIS PV CAMPESTRIS

alpha-Amylase (1,4-a-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.