The genes encoding the thermostable a-amylases of Bacillus stearothermophilus and Bl. licheniformis were cloned in Escherichia coli, and their DNA sequences were determined. The coding and deduced polypeptide sequences are 59 and 62% homologous to each other, respectively. The B. stearothermophilus protein differs most significantly from that of B. licheniformis in that it possesses a 32-residue COOH-terminal tail. Transformation of E. coli with vectors containing either gene resulted in the synthesis and secretion of active enzymes similar to those produced by the parental organisms. A plasmid was constructed in which the promoter and the NH2-terminal two-thirds of the B. stearothermophilus coding sequence was fused out of frame to the entire mature coding sequence of the B. licheniformis gene. Approximately 1 in 5,000 colonies transformed with this plasmid was found to secrete an active amylase. Hybridization analysis of plasmids isolated from these amylase-positive colonies indicated that the parental coding sequences had recombined by homologous recombination. DNA sequeince analysis of selected hybrid genes revealed symmetrical, nonraudom distribution of loci at which the crossovers had resolved. Several purified hybrid ec-amylases were characterized and found to differ with respect to thermostability and specific activity.The ax-amylases secreted by a variety of Bacillus species have been intensively studied. Ihterest has been focused on their mode of secretion, regulation of synthesis, protein structure, and industrial applications. In recent years, the amylase genes of B. coagulans (4) The mesophile B. licheniformis and the thermophile B. stearothermophilus produce amylases which are active at temperatures in excess of 75°C (7). It is therefore of interest to determine their primary structures and compare them with each other and with those known for other amylases to ascertain which sequences are associated with the unusual thermophilicity of these enzymes. In this study we showed that the B. stearothermophilus and B. licheniformis enzymes differ markedly in their specific activities and thermostabilities. Primary structure analysis might also offer clues to these differences.It has been known for some time that the mesophilic amylase of B. amyloliquefaciens has considerable amino acid homology with the B. licheniformis amylase but no homology to the B. subtilis enzyme (21). Recently, comparison of the B. stearothermophilus and B. amyloliquefaciens primary structures has revealed that these proteins are also evolutionarily related (16). In addition, strong similarities between the restriction endonuclease cleavage maps of the amylase genes of B. coagulans and B. licheniformis indicate that these genes may also show homology (21).Since DNA sequence divergence has led to differing chemical properties of the encoded proteins, it is expected that further diversity in this enzyme family might be found in additional natural Bacillus isolates. Alternatively, methods to generate amylase DNA sequence divergence i...
Using synthetic oligonucleotide probes, we cloned genomic DNA sequences encoding an alpha-amylase gene from Aspergillus niger var. awamori (A. awamori) on a 5.8 kb EcoRI fragment. Hybridization experiments, using a portion of this cloned fragment to probe DNA from A. awamori, suggested the presence of two alpha-amylase gene copies which were subsequently cloned as 7 kb (designated as amyA) and 4 kb (amyB) HindIII fragments. DNA sequence analysis of the amyA and amyB genes revealed the following: (1) Both genes are arranged as nine exons and eight introns; (2) The nucleotide sequences of amyA and amyB are identical throughout all but the last few nucleotides of their respective coding regions; (3) The amyA and amyB genes from A. awamori share extensive homology (greater than or equal to 98% identity) with the genes encoding Taka-amylase from A. oryzae. In order to test whether both amyA and amyB were functional in the genome, we constructed vectors containing gene fusions of either amyA and amyB to bovine prochymosin cDNA and used these vectors to transform A. awamori. Transformants which contained either the amyA- or amyB-prochymosin gene fusions produced extracellular chymosin, suggesting that both genes are functional.
The oliC3 gene of Aspergillus niger has been isolated and sequenced. This gene encodes an oligomycin-resistant variant of the mitochondrial ATP synthase subunit 9. In transformation experiments the gene can serve as a semi-dominant selectable marker for A. niger. It was possible to recognize transformants in which oliC3 had integrated at the homologous oliC locus, as opposed to elsewhere in the genome, by observation of phenotypes on medium containing oligomycin. DNA sequencing has allowed comparison of the deduced amino acid sequence with subunit 9 proteins from other species and comparison of 5' untranslated sequences with those from other fungi.
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