Cloning and sequencing of a gene encoding acidophilic amylase from Bacillus acidocaldarius

Abstract

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.

A Lipid‐Anchored Binding Protein is a Component of an ATP‐Dependent Cellobiose/Cellotriose‐Transport System from the Cellulose Degrader Streptomyces reticuli

“…Thus, in addition to those described above, other solute-binding proteins from grampositive bacteria, i.e. for ribose (Woodson and Devine, 1994), maltose (Gilson et a]., 1988;Koivula et al, 1993), dipeptides (Mathiopoulos et al, 1991), oligopeptides (Alloing et al, 1994) and iron hydroxamate (Schneider and Hantke, 1993), are encoded by genes, the deduced products of which contain the consensus amino acid residues for the lipoprotein-cleavage site.…”

Maltose and Maltodextrin Transport in the Thermoacidophilic Gram-Positive Bacterium Alicyclobacillus acidocaldarius Is Mediated by a High-Affinity Transport System That Includes a Maltose Binding Protein Tolerant to Low pH

“…The sequence of the N-terminal 20 amino acids of the purified protein was found to be almost identical to that of a peptide fragment derived from an incomplete open reading frame (ORF2) downstream of the amyA gene (32). The ORF2 product displays homology to the maltose binding protein (MalE) of Escherichia coli (32,54).…”

“…The sequence of the N-terminal 20 amino acids of the purified protein was found to be almost identical to that of a peptide fragment derived from an incomplete open reading frame (ORF2) downstream of the amyA gene (32). The ORF2 product displays homology to the maltose binding protein (MalE) of Escherichia coli (32,54). Interestingly, when compared to the translated nucleotide sequence, the purified protein lacked 23 amino acids from the amino terminus (24), most likely due to the action of an extracellular protease (49).…”

“…The organism grows best at pH 3.6 and 57°C and is further characterized by the presence of -alicyclic fatty acids in the cytoplasmic membrane (60). A. acidocaldarius can utilize a variety of organic compounds as sole sources of carbon and energy, including sugars and polysaccharides, such as starch and xylan (32,49; U. Eckert, S. Wilken, E. Bakker, and E. Schneider, unpublished data). Since polysaccharides cannot penetrate the cell membrane, the bacteria excrete specific hydrolases that degrade the macromolecules into soluble oligomers and monomers that serve as substrates for the transport proteins.…”

Identification and Characterization of Lipolytic Enzymes from a Peat-Swamp Forest Soil Metagenome

“…The K cat =K m ratio for p-nitrophenyl butyrate was higher than that for p-nitrophenyl acetate, suggesting that p-nitrophenyl butyrate (C 4 ) was the most favored substrate among the p-nitrophenyl esters tested here. EstPS2 exhibits preference for short-chained substrates with chain The accession numbers of the aligned sequences are as follows: Alicyclobacillus acidocaldarius (X62835), 20) uncultured bacterium (ACL67849), 21) uncultured bacterium (ACL67845), 21) uncultured bacterium (AAX37296). The sequence in square boxes is highly conserved in family IV of lipases/esterases.…”