Complete nucleotide sequence of a gene coding for heat- and pH-stable alpha-amylase of Bacillus licheniformis: comparison of the amino acid sequences of three bacterial liquefying alpha-amylases deduced from the DNA sequences

The gene coding for the heat-stable and pH-stable alpha-amylase of Bacillus licheniformis 584 (ATCC 27811) was cloned in Escherichia coli and the nucleotide sequence of a DNA fragment of 1,948 base pairs containing the entire amylase gene was determined. As inferred from the DNA sequence, the B. licheniformis alpha-amylase had a signal peptide of 29 amino acid residues and the mature enzyme comprised 483 amino acid residues, giving a molecular weight of 55,200. The amino acid sequence of B. licheniformis alpha-amylase showed 65.4% and 80.3% homology with those of heat-stable Bacillus stearothermophilus alpha-amylase and relatively heat-unstable Bacillus amyloliquefaciens alpha-amylase, respectively. Nevertheless, several regions of the alpha-amylases appeared to be clearly distinct from one another when their hydropathy profiles were compared.     

Cloning and expression of an alpha-1,3-glucanase gene from Bacillus circulans KA-304: the enzyme participates in protoplast formation of Schizophyllum commune

A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia, and a combination of alpha-1,3-glucanase and chitinase I, which were isolated from the filtrate, brings about the protoplast-forming activity. The gene of alpha-1,3-glucanase was cloned from B. circulans KA-304. It consists of 3,879 nucleotides, which encodes 1,293 amino acids including a putative signal peptide (31 amino acid residues), and the molecular weight of alpha-1,3-glucanase without the putative signal peptide was calculated to be 132,184. The deduced amino acid sequence of alpha-1,3-glucanase of B. circulans KA-304 showed approximately 80% similarity to that of mutanase (alpha-1,3-glucanase) of Bacillus sp. RM1, but no significant similarity to those of fungal mutanases.The recombinant alpha-1,3-glucanase was expressed in Escherichia coli Rosetta-gami B (DE 3), and significant alpha-1,3-glucanase activity was detected in the cell-free extract of the organism treated with isopropyl-beta-D-thiogalactopyranoside. The recombinant alpha-1,3-glucanase showed protoplast-forming activity when the enzyme was combined with chitinase I.     

Complete nucleotide sequence of a thermophilic alpha-amylase gene: homology between prokaryotic and eukaryotic alpha-amylases at the active sites

The nucleotide sequence of a thermophilic, liquefying alpha-amylase gene cloned from B. stearothermophilus was determined. The NH2-terminal amino acid sequence analysis of the B. stearothermophilus alpha-amylase confirmed that the reading frame of the gene consisted of 1,644 base pairs (548 amino acids). The B. stearothermophilus alpha-amylase had a signal sequence of 34 amino acids, which was cleaved at exactly the same site in E. coli. The mature enzyme contained two cysteine residues, which might play an important role in maintenance of a stable protein conformation. Comparison of the amino acid sequence inferred from the B. stearothermophilus alpha-amylase gene with those inferred from other bacterial liquefying alpha-amylase genes and with the amino acid sequences of eukaryotic alpha-amylases showed three homologous sequences in the enzymatically functional regions.     

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.     

Cloning and expression of a Bacillus circulans KA-304 gene encoding chitinase I, which participates in protoplast formation of Schizophyllum commune

KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune, has an activity to form protoplasts from S. commune mycelia, and a combination of alpha-1,3-glucanase and chitinase I, isolated from KA-prep, brings about the protoplast-forming activity. The gene of chitinase I was cloned from B. circulans KA-304 into pGEM-T Easy vector. The gene consists of 1,239 nucleotides, which encodes 413 amino acids including a putative signal peptide (24 amino acid residues). The molecular weight of 40,510, calculated depending on the open reading frame without the putative signal peptide, coincided with the apparent molecular weight of 41,000 of purified chitinase I estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C-terminal domain of the deduced amino acid sequence showed high similarity to that of family 19 chitinases of actinomycetes and other organisms, indicating that chitinase I is the first example of family 19 chitinase in Bacillus species. Recombinant chitinase I without the putative signal peptide was expressed in Escherichia coli Rosetta-gami B (DE 3). The properties of the purified recombinant enzyme were almost the same as those of chitinase I purified from KA-prep, and showed the protoplast-forming activity when it was combined with alpha-1,3-glucanase from KA-prep. Recombinant chitinase I as well as the native enzyme inhibited hyphal extension of Trichoderma reesei.     

Structural genes encoding the thermophilic alpha-amylases of Bacillus stearothermophilus and Bacillus licheniformis.

The genes encoding the thermostable alpha-amylases of Bacillus stearothermophilus and B. 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 sequence analysis of selected hybrid genes revealed symmetrical, nonrandom distribution of loci at which the crossovers had resolved. Several purified hybrid alpha-amylases were characterized and found to differ with respect to thermostability and specific activity.     

Cloning, nucleotide sequence, and expression in Escherichia coli of the Bacillus stearothermophilus peroxidase gene (perA).

The gene encoding a thermostable peroxidase was cloned from the chromosomal DNA of Bacillus stearothermophilus IAM11001 in Escherichia coli. The nucleotide sequence of the 3.1-kilobase EcoRI fragment containing the peroxidase gene (perA) and its flanking region was determined. A 2,193-base-pair open reading frame encoding a peroxidase of 731 amino acid residues (Mr, 82,963) was observed. A Shine-Dalgarno sequence was found 9 base pairs upstream from the translational starting site. The deduced amino acid sequence coincides with those of the amino terminus and four peptides derived from the purified peroxidase of B. stearothermophilus IAM11001. E. coli harboring a recombinant plasmid containing perA produced a large amount of thermostable peroxidase which comigrated on polyacrylamide gel electrophoresis with the B. stearothermophilus peroxidase. The peroxidase of B. stearothermophilus showed 48% homology in the amino acid sequence to the catalase-peroxidase of E. coli.     

Cyclization characteristics of cyclodextrin glucanotransferase are conferred by the NH2-terminal region of the enzyme.

Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) is produced mainly by Bacillus strains. CGTase from Bacillus macerans IFO3490 produces alpha-cyclodextrin as the major hydrolysis product from starch, whereas thermostable CGTase from Bacillus stearothermophilus NO2 produces alpha- and beta-cyclodextrins. To analyze the cyclization characteristics of CGTase, we cloned different types of CGTase genes and constructed chimeric genes. CGTase genes from these two strains were cloned in Bacillus subtilis NA-1 by using pTB523 as a vector plasmid, and their nucleotide sequences were determined. Three CGTase genes (cgt-1, cgt-5, and cgt-232) were isolated from B. stearothermophilus NO2. Nucleotide sequence analysis revealed that the three CGTase genes have different nucleotide sequences encoding the same amino acid sequence. Base substitutions were found at the third letter of five codons among the three genes. Each open reading frame was composed of 2,133 bases, encoding 711 amino acids containing 31 amino acids as a signal sequence. The molecular weight of the mature enzyme was estimated to be 75,374. The CGTase gene (cgtM) of B. macerans IFO3490 was composed of 2,142 bases, encoding 714 amino acids containing 27 residues as a signal sequence. The molecular weight of the mature enzyme was estimated to be 74,008. The sequence determined in this work was quite different from that reported previously by other workers. From data on the three-dimensional structure of a CGTase, seven kinds of chimeric CGTase genes were constructed by using cgt-1 from B. stearothermophilus NO2 and cgtM from B. macerans IFO3490. We examined the characteristics of these chimeric enzymes on cyclodextrin production and thermostability. It was found that the cyclization reaction was conferred by the NH2-terminal region of CGTase and that the thermostability of some chimeric enzymes was lower than that of the parental CGTases.     

Cyclization characteristics of cyclodextrin glucanotransferase are conferred by the NH2-terminal region of the enzyme.

Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) is produced mainly by Bacillus strains. CGTase from Bacillus macerans IFO3490 produces alpha-cyclodextrin as the major hydrolysis product from starch, whereas thermostable CGTase from Bacillus stearothermophilus NO2 produces alpha- and beta-cyclodextrins. To analyze the cyclization characteristics of CGTase, we cloned different types of CGTase genes and constructed chimeric genes. CGTase genes from these two strains were cloned in Bacillus subtilis NA-1 by using pTB523 as a vector plasmid, and their nucleotide sequences were determined. Three CGTase genes (cgt-1, cgt-5, and cgt-232) were isolated from B. stearothermophilus NO2. Nucleotide sequence analysis revealed that the three CGTase genes have different nucleotide sequences encoding the same amino acid sequence. Base substitutions were found at the third letter of five codons among the three genes. Each open reading frame was composed of 2,133 bases, encoding 711 amino acids containing 31 amino acids as a signal sequence. The molecular weight of the mature enzyme was estimated to be 75,374. The CGTase gene (cgtM) of B. macerans IFO3490 was composed of 2,142 bases, encoding 714 amino acids containing 27 residues as a signal sequence. The molecular weight of the mature enzyme was estimated to be 74,008. The sequence determined in this work was quite different from that reported previously by other workers. From data on the three-dimensional structure of a CGTase, seven kinds of chimeric CGTase genes were constructed by using cgt-1 from B. stearothermophilus NO2 and cgtM from B. macerans IFO3490. We examined the characteristics of these chimeric enzymes on cyclodextrin production and thermostability. It was found that the cyclization reaction was conferred by the NH2-terminal region of CGTase and that the thermostability of some chimeric enzymes was lower than that of the parental CGTases.     

Cloning and expression of chitinase A gene from Streptomyces cyaneus SP-27: the enzyme participates in protoplast formation of Schizophyllum commune

Chitinase A of Streptomyces cyaneus SP-27 or chitinase I of Bacillus circulans KA-304 showed the protoplast-forming activity when combined with alpha-1,3-glucanase of B. circulans KA-304. The gene of chitinase A was cloned. It consisted of 903 nucleotides encoding 301 amino acid residues, including a putative signal peptide (35 amino acid residues). The deduced N-terminal moiety of chitinase A showed sequence homology with the chitin-binding domain of chitinase F from Streptomyces coelicolor and chitinase 30 from Streptomyces olivaceoviridisis. The C-terminal moiety also showed high sequence similarity to the catalytic domain of several Streptomyces family 19 chitinases as well as that of chitinase I of B. circulans KA-304. Recombinant chitinase A was expressed in Escherichia coli Rosetta-gami B (DE 3). The properties of the recombinant enzyme were almost the same as those of chitinase A purified from a culture filtrate of S. cyaneus SP-27. The recombinant enzyme was superior to B. circulans KA-304 chitinase I not only in respect to protoplast forming activity in a mixture containing alpha-1,3-glucanase, but also to antifungal activity and powder chitin-hydrolyzing activity.     

Nucleotide sequence and promoter region for the neutral protease gene from Bacillus stearothermophilus.

The thermostable neutral protease gene nprT of Bacillus stearothermophilus was sequenced. The DNA sequence revealed only one large open reading frame, composed of 1,644 bases and 548 amino acid residues. A Shine-Dalgarno sequence was found 9 bases upstream from the translation start site (ATG), and the deduced amino acid sequence contained a signal sequence in its amino-terminal region. The sequence of the first 14 amino acids of purified extracellular protease completely matched that deduced from the DNA sequence starting at GTC (Val), 687 bases (229 amino acids) downstream from ATG. This suggests that the protease is translated as a longer polypeptide. The amino acid sequence of the extracellular form of this protease (319 amino acids) was highly homologous to that of the thermostable neutral protease from Bacillus thermoproteolyticus but less homologous to the thermolabile neutral protease from Bacillus subtilis. A promoter region determined by S1 nuclease mapping (TTTTCC for the -35 region and TATTTT for the -10 region) was different from the conserved promoter sequences recognized by the known or factors in bacilli. However, it was very homologous to the promoter sequence of the spo0B gene from B. subtilis. The guanine-plus-cytosine content of the coding region of the nprT gene was 58 mol%, while that of the third letter of the codons was much higher (72 mol%).     

Nucleotide sequence of the extracellular glucoamylase gene STA1 in the yeast Saccharomyces diastaticus.

The complete nucleotide sequence of the extracellular glucoamylase gene STA1 from the yeast Saccharomyces diastaticus has been determined. A single open reading frame codes for a 778-amino-acid protein which contains 13 potential N-glycosylation sites. In the 5'- and 3'-flanking regions of the gene, there are striking sequence homologies to the corresponding regions of ADH1 for alcohol dehydrogenase and MAT alpha 2 for mating type control in the yeast Saccharomyces cerevisiae. The putative precursor begins with a hydrophobic segment that presumably acts as a signal sequence for secretion. The presumptive signal sequence showed a significant homology to that of Bacillus subtilis alpha-amylase precursor. The next segment, of ca. 320 amino acids, contains a threonine-rich tract in which direct repeat sequences of 35 amino acids exist, and is bordered by a pair of basic amino acid residues (Lys-Lys) which may be a proteolytic processing signal. The carboxy-terminal half of the precursor is a presumptive glucoamylase which contains several peptide segments showing a high degree of homology with alpha-amylases from widely diverse organisms including a procaryote (B. subtilis) and eucaryotes (Aspergillus oryzae and mouse). Analysis of both the nucleotide sequence of the STA1 gene and the amino acid composition of the purified glucoamylase suggested that the putative precursor is processed to yield subunits H and Y of mature enzyme by both trypsin-like and chymotrypsin-like cleavages.     

Cloning, expression, and purification of Bacillus stearothermophilus DNA primase and crystallization of the zinc-binding domain

The dnaG gene encoding DNA primase has been isolated from chromosomal DNA of Bacillus stearothermophilus and its entire nucleotide sequence determined. The deduced amino acid sequence comprised 597 amino acid residues and the molecular mass was calculated to be 67068 Da. B. stearothermophilus primase was overexpressed in Escherichia coli and purified to homogeneity. The N-terminal 12 kDa zinc-binding domain has been crystallized. The crystals are of the monoclinic space group P21 with cell dimensions a=36 A, b=59 A, c=46 A, beta=91.8 degrees and diffract to 1.7 A resolution.     

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.     

Cloning, sequencing and expression of the gene encoding the extracellular neutral protease, vibriolysin, of Vibrio proteolyticus.

The gene (nprV), encoding the extracellular neutral protease, vibriolysin (NprV), of the Gram- marine microorganism, Vibrio proteolyticus, was isolated from a V. proteolyticus DNA library constructed in Escherichia coli. The recombinant E. coli produced a protease that co-migrated with purified neutral protease from V. proteolyticus on non-denaturing polyacrylamide gels, and that demonstrated enzymatic specificity towards the neutral protease substrate N-[3-(2-furyl)acryloyl]-L-alanylphenylalanine amide. The nucleotide (nt) sequence of the cloned nprV gene revealed an open reading frame encoding 609 amino acids (aa) including a putative signal peptide sequence followed by a long 'pro' sequence consisting of 172 aa. The N-terminal aa sequence of NprV purified from cultures of V. proteolyticus, identified the beginning of the mature protein within the aa sequence deduced from the nt sequence. Comparative analysis of mature NprV to the sequences of the neutral proteases from Bacillus thermoproteolyticus (thermolysin) and Bacillus stearothermophilus identified extensive regions of conserved aa homology, particularly with respect to active-site residues, zinc-binding residues, and calcium-binding sites. NprV was overproduced in Bacillus subtilis by placing the DNA encoding the 'pro' and mature enzyme downstream from a Bacillus promoter and signal sequence.