Amino acid sequence of alpha-amylase from Bacillus amyloliquefaciens deduced from the nucleotide sequence of the cloned gene.

Abstract

We have isolated by molecular cloning the gene coding for the alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1.) from Bacillus amyloliquefaciens and determined its complete nucleotide sequence. The gene cloned in the plasmid pUB110 using Bacillus subtilis as a host, was contained in a 2.3-kilobase insert. Starting from an ATG initiator codon, an open reading frame comprising a total of 514 amino acids (1542 base pairs) was found within the cloned DNA fragment. The gene region encoding the COOH terminus of alpha-amylase was located by direct COOH-terminal analysis of the purified exoenzyme. The NH2-terminal portion of the gene encodes a 31 amino acid-long signal peptide (Palva, L., Pettersson, R. F., Kalkkinen, N., Lehtovaara, P., Sarvas, M., Söderlund, H., Takkinen, K., and Kääriäinen, L. (1981) Gene 15, 43-51). Since the signal peptide is correctly cleaved in the new host, as shown here by direct NH2-terminal sequence analysis, the exoamylase consists of 483 amino acid residues, corresponding to a molecular weight of 54,778. The reading frame used to deduce the amino acid sequence was found to be correct by comparison with partial amino acid sequence data published previously (Detera, S. D., and Friedberg, F. (1979) Int. J. Peptide Protein Res. 14, 364-372; Chung, H., and Friedberg, F. (1980) Biochem. J. 185, 387-395). Several differences between the sequence presented here and the partial ones published previously, however, were found. The nucleotide sequences both 5' and 3' to the alpha-amylase gene revealed palindromic structures including a stretch of six T-residues, suggesting transcription termination signals on both sides of the gene. Thus, it appears that alpha-amylase is translated from a monocistronic mRNA.