alpha-Amylase of Clostridium thermosulfurogenes EM1: nucleotide sequence of the gene, processing of the enzyme, and comparison of other alpha-amylases

Abstract

The nucleotide sequence of the a-amylase gene (amyA) from Clostridium thermosulfurogenes EM1 cloned in Escherichia coli was determined. The reading frame of the gene consisted of 2,121 bp. Comparison of the DNA sequence data with the amino acid sequence of the N terminus of the purified secreted protein of C. thermosulfurogenes EM1 suggested that the oe-amylase is translated from mRNA as a secretory precursor with a signal peptide of 27 amino acid residues. The deduced amino acid sequence of the mature aL-amylase contained 679 residues, resulting in a protein with a molecular mass of 75,112 Da. In E. coli the enzyme was transported to the periplasmic space and the signal peptide was cleaved at exactly the same site between two alanine residues. Comparison of the amino acid sequence of the C. thermosulfurogenes EM1 a-amylase with those from other bacterial and eucaryotic a-amylases showed several homologous regions, probably in the enzymatically functioning regions. The tentative Ca2+-binding site (consensus region I) of this Ca2+independent enzyme showed only limited homology. The deduced amino acid sequence of a second obviously truncated open reading frame showed significant homology to the malG gene product of E. coli. Comparison of the L-amylase gene region of C. thermosulfurogenes EM1 (DSM3896) with the 0-amylase gene region of C. thermosulfurogenes (ATCC 33743) indicated that both genes have been exchanged with each other at identical sites in the chromosomes of these strains.

Raw-starch-digesting and thermostable α-amylase from the yeast Cryptococcus sp. S-2: purification, characterization, cloning and sequencing

“…In addition to being notable for its raw-starch-digesting ability, AMY-CS2 is also notable for its thermostability. The thermostability of α-amylases from B. stearothermophilus and Clostridium thermosulfurogenes [9] have been studied because of the industrial importance of these enzymes. The distance between the α-amylase consensus region I and region II, the numbers of cysteine residues, and the aliphatic index have been discussed as possible factors that make the α-amylases thermostable.…”

“…It is interesting that almost all of these α-amylases are from thermophilic bacteria, such as B. stearothermophilus, Cl. thermosulfurogenes [9], Streptomyces thermo iolaceus [10] and Thermomonospora cur ata [11] and that they are all thermostable. However, these α-amylases have not been shown to have rawstarch-binding and raw-starch-digesting activities because they have not been studied in this respect.…”

“…In contrast, thermostable α-amylases are of interest because of their potential industrial applications. Several thermostable α-amylases have been purified from thermophilic bacteria [9][10][11][12] and possible factors in their thermostability have been investigated. However, these factors remain unclear.…”

Cyclodextrin formation by the thermostable alpha-amylase of Thermoanaerobacterium thermosulfurigenes EM1 and reclassification of the enzyme as a cyclodextrin glycosyltransferase

“…The same enzyme has been characterized previously as an ␣-amylase (30). The gene encoding this ''␣-amylase'' has been cloned and expressed in Escherichia coli, and its primary structure has been determined (1,8). We will show that the amino acid sequence of this ''␣-amylase'' in fact is highly homologous to known bacterial CGTase sequences.…”

New conserved amino acid region of α-amylases in the third loop of their (β/α)8-barrel domains

“…1). The sequences (taken from the literature) have been either determined by direct amino acid sequencing for Aspergillus oryzae [11] and porcine pancreatic [12] a-amylases or deduced from nucleotide sequences for a-amylases from barley [13], Bacillus subtilis [14], Bacillus stearothermophilus [15], Drosophila melanogaster [16], Streptomyces limosus [17], and Clostridium thermosulfurogenes [18]. This set of amino acid sequences was constructed on the basis of low sequence similarities, i.e.…”