Nucleotide sequence of the G6-amylase gene from alkalophilic Bacillus sp. H-167

The nucleotide sequence of the G₆-amylase gene from alkalophilic Bacillus sp. H-167 was determined. The open reading frame of the gene consisted of 2865 base pairs, encoding 955 amino acids. The NH₂-terminal amino acid sequence analysis of the G₆-amylase indicated that the enzyme had a single peptide of 33 amino acid residues and the mature enzyme was composed of 922 amino acids, giving a molecular mass of 102598. Identity of the NH₂-terminal amino acid sequences among each component of the multiform G₆-amylase suggested the proteolytic processing of the COOH-terminal side of the enzyme. The DNA sequence and the deduced amino acid sequence of the G₆-amylase gene showed no homology with those of other bacterial α-amylases although the consensus amino acid sequences of the active center were well conserved.     

Nucleotide sequence of the beta-lactamase gene of alkalophilic Bacillus sp. strain 170

A gene for the beta-lactamase from alkalophilic Bacillus sp. strain 170 was cloned in a functional state on a 1.0 kb DNA fragment and its nucleotide sequence was determined. The coding sequence showed an open reading frame of 257 amino acids, which represents the beta-lactamase precursor protein. It is considered that the signal peptide consisted of 30 amino acids including 12 hydrophobic amino acids.     

Nucleotide sequence of the maltohexaose-producing amylase gene from an alkalophilic Bacillus sp. #707 and structural similarity to liquefying type alpha-amylases

The nucleotide sequence of the gene for maltohexaose-producing amylase from an alkalophilic Bacillus sp. #707 was determined. Starting at an ATG initiation codon, an open reading frame was composed of 1554 bp (518 amino acids). The NH2-terminal portion encoded a 33 amino acid-long signal peptide. The deduced amino acid sequence of the extracellular mature enzyme was more than 60% homologous to those of the liquefying type alpha-amylases but not to those of the saccharifying type alpha-amylases. The sequence of its signal peptide was completely different from those of other alpha-amylases.     

The nucleotide sequence of the lipo-penicillinase gene of alkalophilic Bacillus sp. strain 170

The lipo-penicillinase (LIPEN) gene from an alkalophilic Bacillus sp. strain 170 was cloned in Escherichia coli using the vector pHSG399. A plasmid, pFAP121, was isolated from an ampicillin resistant transformant and the cloned LIPEN gene was found to be in a 2.2 kb DNA fragment. The nucleotide sequence of a 1.9 kb segment encoding the LIPEN was determined. This segment showed an open reading frame which would encode a polypeptide of 310 amino acids. The amino acid sequence of this LIPEN gene product has strong homology with those of the Bacillus cereus -lactamase III and Bacillus licheniformis penicillinase.     

Molecular cloning and nucleotide sequence of the alkaline cellulase gene from the alkalophilic Bacillus sp. strain 1139

The cellulase gene from the alkalophilic Bacillus sp. strain 1139 was cloned in Escherichia coli using pBR322. Plasmid pFK1 was isolated from transformants producing cellulase, and the cloned cellulase gene was found to be in a 4 X 6 kb HindIII fragment. The cellulase gene was subcloned in a functional state on a 2 X 9 kb DNA fragment and its nucleotide sequence was determined. The coding sequence showed an open reading frame encoding 800 amino acids. The pFK1-encoded cellulase had the same enzymic properties as the extracellular cellulase produced by the alkalophilic Bacillus sp. strain 1139, but its Mr was slightly higher.     

Nucleotide sequence of the beta-cyclodextrin glucanotransferase gene of alkalophilic Bacillus sp. strain 1011 and similarity of its amino acid sequence to those of alpha-amylases.

The nucleotide sequence of the gene for cyclodextrin glucanotransferase of alkalophilic Bacillus sp. strain 1011 was determined. The deduced amino acid sequence at the NH2-terminal side of the enzyme showed a high homology with the sequences of alpha-amylase in the three regions which constitutes the active centers of alpha-amylases.     

Nucleotide sequence of the gene for an alkaline endoglucanase from an alkalophilic Bacillus and its expression in Escherichia coli and Bacillus subtilis.

The gene for an alkaline endoglucanase from the alkalophilic Bacillus sp. KSM-64 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The nucleotide sequence of a 4.1-kb region of the HindIII insert had two open reading frames, ORF-1 and ORF-2. The protein deduced from ORF-1 was composed of 244 amino acids with an M(r) of 27,865. Subcloning analysis proved that the alkaline endoglucanase was encoded by ORF-2 (822 amino acids with an M(r) of 91,040). Upstream from ORF-2, there were three consensus like sequences of the sigma A-type promoter of Bacillus subtilis, a putative Shine-Dalgarno sequence (AGGAGGT), and a catabolite repression operator-like sequence (TGTAAGCGGTTAACC). The HindIII insert was subcloned into a shuttle vector, pHY300PLK, and the encoded alkaline endoglucanase gene was highly expressed both in E. coli and B. subtilis. One of the three promoter-like sequences in ORF-2 could be suitable for high levels of enzyme expression in both host organisms.     

Nucleotide sequence of the neopullulanase gene from Bacillus stearothermophilus

The gene (nplT) for a new type of pullulan-hydrolysing enzyme, neopullulanase, from Bacillus stearothermophilus TRS40 was sequenced. The DNA sequence revealed only one large open reading frame, composed of 1764 bases and 588 amino acid residues (Mr 69144). Although the thermostable neopullulanase contained eight cysteine residues, they did not provide conformational stability by disulphide bonds. A comparison was made of the amino acid sequences of alpha-amylase, neopullulanase, isoamylase, pullulanase and cyclodextrin glucanotransferase. All the enzymes examined contained four highly conserved regions which probably constitute the active centres of the enzymes. The amino acid residues required for the specificity of neopullulanase are compared with those of alpha-amylase and other amylolytic enzymes.     

Nucleotide sequence of the Bacillus subtilis phoR gene.

The nucleotide sequence of phoR, the positive and negative regulatory gene for alkaline phosphatase and phosphodiesterase formation in Bacillus subtilis, was determined. The sequence data predicted an open reading frame of 1,740 base pairs (579 amino acids) which overlaps the 5 base pairs of the preceding phoP coding sequence. The deduced amino acid sequence was significantly homologous with that of the Escherichia coli phoR gene product, which is the sensory element for the pho regulon.     

Nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene.

The nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene and its flanking regions was determined. An open reading frame was found, comprising a total of 1,647 base pairs (549 amino acids) and starting from a GUG codon as methionine. It was shown by NH2-terminal amino acid sequence analysis that the extracellular amylase consisted of 515 amino acid residues, which corresponded to a molecular weight of 58,779. Thus the NH2-terminal portion of the gene encodes 34 amino acid residues as a signal peptide. The amino acid sequence deduced from the alpha-amylase gene was fairly homologous (61%) with that of another thermostable amylase from Bacillus amyloliquefaciens.     

Nucleotide sequence of the gene encoding NADH dehydrogenase from an alkalophile, Bacillus sp. strain YN-1

The gene encoding NADH dehydrogenase from an alkalophile, Bacillus sp., was cloned and sequenced. The cloned DNA fragment contained an open reading frame of 1,557 nucleotides which encodes a polypeptide composed of 519 amino acid residues (Mr 55,830). The predicted amino acid sequence was consistent with the partial amino acid sequences including the N-terminal and C-terminal sequences determined in a previous study. Sequence comparison with other flavoenzymes revealed high homology between the present dehydrogenase and Escherichia coli thioredoxin reductase.     

Purification and properties of cyclomaltodextrinase from alkalophilic Bacillus sp.

An alkalophilic strain isolated from soil produced intracellular cyclomaltodextrinase on the culture medium at an initial pH of 10.6. The strain was identified as closely resembling Bacillus circulans. The enzyme was purified 252-fold from the cell extract by chitosan treatment, ammonium sulfate fractionation, DEAE-Toyopearl column chromatography, and gel filtration. The pH and temperature optima of the purified enzyme were 6.0 and 50°C. The molecular weight of the enzyme was 126,000, with two subunits of 67,000. The isoelectric point was pH 4.2. Enzyme activity was inhibited by Ag⁺, Hg²⁺, Cu²⁺, and p-chloromercuribenzoate. The enzyme hydrolyzed α-, β-, and γ-cyclodextrins, as well as linear maltodextrins, to yield maltooligosaccharides. Starch and maltose were not degraded by the enzyme.     

Nucleotide sequences of two cellulase genes from alkalophilic Bacillus sp. strain N-4 and their strong homology.

Two genes for cellulases of alkalophilic Bacillus sp. strain N-4 (ATCC 21833) have been sequenced. From the DNA sequences the cellulases encoded in the plasmids pNK1 and pNK2 consist of 488 and 409 amino acids, respectively. The DNA and protein sequences of the pNK1-encoded cellulase are related to those of the pNK2-encoded cellulase. The pNK2-encoded cellulase lacks the direct repeat sequence of a stretch of 60 amino acids near the C-terminal end of the pNK1-encoded cellulase. The duplication of the cellulase genes and the formation of the direct repeat in the pNK1-encoded cellulase occurred at almost the same time.