Cloning and nucleotide sequence of an extracellular  -amylase gene from Aeromonas hydrophila MCC-1

Abstract

A gene encoding the extracellular a-amylase of Aevomonas hydrophila MCC-1 was cloned and expressed using its own promoter on the recombinant plasmid pCA101. Subcellular fractionation of Eschevichia coli JA221 carrying pCAlOl revealed that approximately 60 YO of the amylase activity was localized in the periplasmic space. The extracellular amylase was purified to homogeneity, identified as an a-type and its amino-terminal sequence was determined. Nucleotide sequence analysis predicted a 443 amino acid ORF and 24 amino acids at the amino terminus of the sequence that are not found in the secreted protein. This 24 amino acid sequence has many of the characteristics common to known signal peptides. The predicted amino acid sequence has considerable similarity with mammalian, invertebrate and Streptomycete a-amylases. Most of the amino acid residues that are involved in catalytic activity, substrate binding and calcium binding in several a-amylases were also present in A . hydrophila a-amylase at the corresponding positions.

The cloning and characterization of a second alpha-amylase of A. hydrophila JMP636

“…There was no further significant homology between amyA and amyB. There was similarity between amyB and amyMCC (the amylase coned from A. hydrophila strain MCC-1 (Chang et al 1993)). The level of similarity between the cloned and sequenced amylases from different A. hydrophila strains is shown in Table 2.…”

“…Both amyA and amyB differ significantly from this amylase. AmyA shows little sequence homology even though protein size and codon usage are similar (Gobius and Pemberton 1988;Chang et al 1993). Comparison at nucleotide level between amyB and the MCC-1 amylase is close (homology of about 80%) with less similarity when comparing amino acid sequences (60%).…”

“…Cloning of an amylase of 46 000 Da from a different A. hydrophila strain, MCC-1, has been reported (Chang et al 1993). Both amyA and amyB differ significantly from this amylase.…”

“…Comparison at nucleotide level between amyB and the MCC-1 amylase is close (homology of about 80%) with less similarity when comparing amino acid sequences (60%). AmyB is a protein of 72 000 Da compared to the MCC-1 amylase which is 46 000 Da (Chang et al 1993). Whether there are two amylases in the MCC-1 strain is unknown.…”

“…Many of such enzymes, such as amylases, chitinase, nucleases and lipases, are well known to science and of considerable importance in medicine, agriculture, industry and the environment. Already many genes encoding these enzymes have been cloned from Aeromonas; these include chitinases, lipases, a phospholipase C (Ingham and Pemberton 1995), nucleases (Dodd and Pemberton 1996) and amylases (Gobius and Pemberton 1988;Chang et al 1993). One of the definitive characteristics of A. hydrophila is its amylolytic activity.…”