Biochemical and genetic analysis of a maltopentaose-producing amylase from an alkaliphilic gram-positive bacterium.
Two amylases have been purified from the culture fluid of an alkaliphilic bacterium. Amylase A-60 consists of a single type of polypeptide chain of 60 kDa and exhibits an alpha-amylase-type of starch cleavage. Amylase A-180 is approximately 180 kDa in size, represents the largest exoenzyme so far identified in prokaryotes and in the initial enzyme reaction cleaves starch exclusively to maltopentaose. A-60 and A-180 are immunologically unrelated enzymes. The structural gene for amylase A-180 has been cloned and its nucleotide sequence was determined. An open reading frame was identified for a putative protein of 182 kDa whose amino-terminal sequence, deduced from the nucleotide sequence, was identical in 23 out of 25 positions to that determined for the protein. The amino-terminus of the mature protein, at the gene level, is preceded by a sequence segment showing all the characteristics of a signal peptide from Gram-positive bacteria. Analysis of the deduced amino acid sequence revealed that the 70-kDa N-terminal part is similar to classical alpha-amylases. The C-terminal part contains three repeated sequence blocks of 99 amino acid residues each which are also present in two bacterial beta-amylases. It appears, therefore, that A-180 has arisen by gene fusion events.
Candussio
A
A
Lehrstuhl für Mikrobiologie der Universität München, Federal Republic of Germany.
Schmid
G
G
Böck
A
A
D000595:Amino Acid Sequence; D000681:Amylases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004586:Electrophoresis / Q000379:methods; D005798:Genes, Bacterial; D006094:Gram-Positive Bacteria / Q000201:enzymology*; D015151:Immunoblotting / Q000379:methods; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D015183:Restriction Mapping
D009844:Oligosaccharides; D011994:Recombinant Proteins; C019193:maltopentaose; D000681:Amylases