Cloning and sequencing of a gene encoding acidophilic amylase from Bacillus acidocaldarius.
Two starch-degrading enzymes produced by Bacillus acidocaldarius (renamed as Alicyclobacillus acidocaldarius) were identified. According to SDS-PAGE, the apparent molecular masses of the enzymes were 90 and 160 kDa. Eight peptide fragments and the N-terminal end of the 90 kDa polypeptide were sequenced. An oligonucleotide, based on the amino acid sequence of a peptide fragment of the 90 kDa protein, was used to screen a lambda gt10 bank of B. acidocaldarius, and the region encoding the 90 kDa protein was cloned. Unexpectedly, the ORF continued upstream of the N terminus of the 90 kDa protein. The entire ORF was 1301 amino acids (aa) long (calculated molecular mass 140 kDa) and it was preceded by a putative ribosomal binding site and a promoter. Computer analysis showed that the 1301 aa protein was closely related to an alpha-amylase-pullulanase of Clostridium thermohydrosulfuricum. We suggest that the starch-degrading 160 kDa protein of B. acidocaldarius is an alpha-amylase-pullulanase, and the 90 kDa protein is a cleavage product of the 160 kDa protein. Another ORF, apparently in the same transcription unit, was found downstream from the amylase gene. It encoded a protein that was closely related to the maltose-binding protein of Escherichia coli.
Koivula
T T
TT
Department of Genetics, University of Helsinki, Finland.
Hemilä
H
H
Pakkanen
R
R
Sibakov
M
M
Palva
I
I
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D001412:Bacillus subtilis / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D008320:Maltose / Q000737:chemistry; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins; D016366:Open Reading Frames; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D000516:alpha-Amylases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010455:Peptides; C461599:maltose transport system, E coli; D008320:Maltose; D006026:Glycoside Hydrolases; D000516:alpha-Amylases; C100181:pullulanase