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S Udaka

Publications and source records attributed to S Udaka.

At least 73 records · Page 4Linked to original sources
PubMed1991

Proteases involved in generation of beta- and alpha-amylases from a large amylase precursor in Bacillus polymyxa.

The genes for extracellular neutral protease (Npr) and intracellular serine protease (Isp) were cloned from Bacillus polymyxa in order to elucidate the process involved in the generation of multiple beta-amylases and an alpha-amylase from a large amylase precursor. The npr gene was composed of 1,770 bp and 570 amino acids, while the isp gene was composed of 978 bp and 326 amino acids. Both proteases produced by E. coli cleaved the amylase precursor to generate beta- and alpha-amylases. Furthermore, several other proteases produced the same products from the precursor. A 130-kDa amylase precursor has two large domain structures responsible for the generation of beta- and alpha-amylases. The junction region of approximately 200 amino acids may be exposed on the surface of the molecule and susceptible to proteolytic enzymes, which results in the formation of multiple amylases.

Amino Acid Sequence↗
PubMed1991

Analysis by deletion and site-directed mutagenesis of promoters of the cell wall protein gene operon in Bacillus brevis 47.

The cell wall protein gene operon of Bacillus brevis 47 has multiple and tandem promoters. The precise locations of the two major promoters (P2 and P3) of the operon were determined by deletion analysis. This together with results of oligonucleotide-directed mutagenesis of the promoter regions confirmed that the -35 and -10 sequences of the two promoters proposed previously are essential for their promoter activity. A G + C-rich sequence upstream of the -35 region of P2 and an A-rich sequence upstream of the -35 region of P3 facilitate the transcription from P2 and P3, respectively.

Bacillus↗
PubMed1991

Xylose (glucose) isomerase gene from the thermophile Clostridium thermohydrosulfuricum; cloning, sequencing, and expression in Escherichia coli.

The xylose isomerase gene from the thermophile Clostridium thermohydrosulfuricum has been cloned, using a fragment of the Bacillus subtilis gene as a probe. The complete nucleotide sequence of the gene was analyzed. C. thermohydrosulfuricum is the most thermophilic organism from which a xylose isomerase gene has been cloned and characterized. Comparison with amino acid sequences from other xylose isomerases showed that amino acids involved in substrate binding and isomerization are well conserved. Purification of the enzyme produced in E. coli was done by heating a cell-free extract at 85 degrees C for 10 min, giving a 20-fold purified enzyme. The native enzyme is a homomeric tetramer with a molecular weight of 200,000.

Aldose-Ketose Isomerases↗
PubMed1991

Efficient production of Taka-amylase A by Trichoderma viride.

An efficient heterologous protein production system was developed in Trichoderma viride, a very efficient cellulase producer. An expression vector containing the Taka-amylase A gene from Aspergillus oryzae, which was fused to the strong promoter and signal peptide sequence of the cellobiohydrolase 1 gene (cbh1) of T. viride, and the hygromycin B resistance gene was used to transform protoplasts of T. viride. Using hygromycin B resistance, a frequency of 3 transformants per microgram DNA on average was obtained. One transformant showed highly elevated alpha-amylase production, 1.0 g/l, which was shown to be under the control of the cbh1 gene promoter. Analysis of the chromosomal DNA of the transformant showed the integration of more than one copy of the vector.

Base Sequence↗
PubMed1990

Nucleotide sequence of the raw-starch-digesting amylase gene from Bacillus sp. B1018 and its strong homology to the cyclodextrin glucanotransferase genes.

The gene coding for the raw-starch-digesting amylase from Bacillus sp. B1018 was cloned into Escherichia coli, and its nucleotide sequence was determined. Starting at an ATG codon, there was an open reading frame composed of 2139 bp (713 amino acids). The NH2-terminal portion encoded a 27 amino acid-long signal peptide. S1 mapping revealed the presence of a typical promoter region upstream from the transcription initiation site. The deduced amino acid sequence of the extracellular mature enzyme was very similar to those of cyclodextrin glucanotransferases (CGTases), especially that of alkalophilic Bacillus sp. 1011 (87% homology). In fact, the B1018 amylase showed CGTase activity. The COOH-terminal portion of the B1018 amylase shows significant homology with other raw-starch-digesting enzymes.

Amino Acid Sequence↗
PubMed1990

Efficient production of human alpha-amylase by a Bacillus brevis mutant.

A cDNA for mature human salivary alpha-amylase was directly joined to a sequence encoding the signal peptide of the middle wall protein (MWP) gene of Bacillus brevis 47. This hybrid gene was placed downstream from the multiple promoter region of the MWP gene on a low copy-number plasmid vector, pHW1. B. brevis 47 carrying the plasmid produced 0.9 mg/l of active human alpha-amylase in the medium. A B. brevis 47 mutant obtained on mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine produced an increased amount of the alpha-amylase (6 mg/l). When the fused gene was inserted into a high copy-number expression vector, pNU200, and then introduced into the mutant, a large amount (60 mg/l) of the alpha-amylase was produced in the medium. The alpha-amylase showed approximately the same specific activity and molecular weight as those of the natural enzyme. The mutant showed higher sensitivity to various antibiotics than the original strain, and altered cell wall and cytoplasmic membrane protein compositions. The results of reversion analysis suggested that a single mutation is responsible for the above phenotypes and hyper-productivity of human alpha-amylase.

Amino Acid Sequence↗
PubMed1990

Use of both translation initiation sites of the middle wall protein gene in Bacillus brevis 47.

The middle wall protein gene of Bacillus brevis 47 has two potential translation initiation sites located tandemly in the same reading frame. We demonstrate here that both sites are utilized to start translation in B. brevis 47. Translation from the first site (located upstream) gives rise to a precursor of the middle wall protein with an extension peptide of 31 amino acids preceding the signal peptide. The precursor was cleaved at the same position as that of the precursor translated from the second site. The TTG codon seems to play an appreciable role in the initiation of translation in B. brevis 47.

Bacillus↗
PubMed1990

Conserved structures of cell wall protein genes among protein-producing Bacillus brevis strains.

Bacillus brevis HPD31 contains a surface (S)-layer protein, termed the HWP, which forms a hexagonal array in the cell wall. The 5' region of the HWP gene was isolated from a DNA library constructed in bacteriophage vector EMBL3 from a partial BamHI digest of the chromosomal DNA. The 3' region contained in a 2.7-kilobase BglII fragment of the DNA was cloned into Escherichia coli, using pUC118 as a vector. On the basis of the chemically determined N-terminal amino acid sequence, the HWP gene was found to encode a polypeptide consisting of 1,087 amino acid residues with a signal peptide of 53 or 23 amino acid residues. The deduced amino acid composition was similar to the chemical amino acid compositions of other S-layer proteins in the predominance of acidic relative to basic amino acids and in the very low content of sulfur-containing amino acids. The deduced amino acid sequence showed high homology (78%) with that of the middle wall protein of B. brevis 47. Furthermore, the multiple 5' ends of the HWP gene transcripts detected on S1 nuclease analysis closely resembled those of the middle wall protein gene transcripts. This complex structure was also conserved (greater than 85%) in the regulatory regions of two other cell wall protein genes isolated from B. brevis HPD52 and HP033, suggesting that the synthesis of the cell wall proteins is intricately regulated through a similar mechanism in protein-producing B. brevis.

Amino Acid Sequence↗
PubMed1989

Isolation of a cDNA encoding Aspergillus oryzae Taka-amylase A: evidence for multiple related genes.

Complementary and genomic DNAs encoding Aspergillus oryzae Taka-amylase A (Taa) were cloned and sequenced. The coding sequence of the cDNA comprised the signal peptide [21 amino acids (aa)] and mature Taa (478 aa). The deduced aa sequence agrees well with the published aa sequence, except for one insertion, one deletion and ten aa substitutions. These differences might be due to the difference in the strains used. Sequence comparison of the cDNA and genomic DNA indicates the presence of eight introns ranging in size from 55 to 86 bp. Southern-blot analysis showed the presence of at least two Taa genes, and the second gene (Taa-G2) was isolated. All the intron/exon junctions follow the 'GT-AG' rule, except for intron I of the first gene (Taa-G1). The 5'-noncoding region was well conserved among the genomic genes and contained sequences similar to 'CAAT' and 'TATA' boxes at nucleotides -121 and -31, counted from the transcription start point, respectively. The 3'-noncoding regions, however, differed significantly from each other. Taa-G2 contains a sequence identical to that of several independent cDNA clones, suggesting that it may be the major transcribed gene in A. oryzae.

Amino Acid Sequence↗
PubMed1989

Amino acid residues stabilizing a Bacillus alpha-amylase against irreversible thermoinactivation.

The alpha-amylase of Bacillus licheniformis (BLA) is stable and active at high temperature. More than 80% of its activity is retained after heat treatment at 90 degrees C for 30 min, and the optimum temperature for its activity is 80-85 degrees C. In contrast, the alpha-amylase of Bacillus amyloliquefaciens (BAA), the amino acid sequence of which shows 80% homology with that of BLA, is rapidly inactivated at 90 degrees C. Various chimeric genes were constructed from the structural genes for the two enzymes, and their products were analyzed for stability as to irreversible thermoinactivation. Two regions in the amino acid sequence of BLA comprising Gln178 (region I) and the 255th-270th residues (region II), respectively, were shown to determine the thermostability of BLA. Region I plays a major role in determining the thermostability. By means of site-directed mutagenesis of the BAA gene, deletion of Arg176 and Gly177 in region I and substitutions of alanine for Lys269 and aspartic acid for Asn266 in region II were shown to be responsible for the enhancement of the thermostability. Mutant BAAs containing the above deletion and substitutions showed almost the same thermostability as BLA as to irreversible thermoinactivation. Nevertheless, the mutant BAAs showed a temperature optimum as low as that of BAA (65 degrees C), indicating that they are still susceptible to reversible inactivation at temperatures higher than 65 degrees C.

Amino Acid Sequence↗
PubMed1989

Three-dimensional structure of the surface protein layer (MW layer) of Bacillus brevis 47.

The three-dimensional (3D) structure of one surface protein layer from Bacillus brevis 47, the middle wall (MW) layer, has been reconstructed from tilted-view electron micrographs after correlation averaging to a resolution of 2 nm. The MW layer has p6 symmetry with a center-to-center spacing of 18.3 nm and a minimum thickness of 5.5 nm. The reconstruction reveals a distinct domain structure: the heavier domain of six monomers jointly forms a massive core centered at the sixfold symmetry axis, and lighter domains interconnect adjacent unit cells. In addition, the larger domains collectively form a pore by making contact with each other towards the inner surface, while the smaller domains establish a second connectivity towards the outer surface of the S layer. The MW layer of B. brevis resembles the S layer of Acetogenium kivui in various aspects: they have very similar lattice parameters and highly reminiscent 3D structures; the pores penetrate through the whole core and appear to determine the porosity of the S layers.

Bacillus↗
PubMed1989

Use of Bacillus brevis for efficient synthesis and secretion of human epidermal growth factor.

Using previously isolated Bacillus brevis strains that secrete large amounts of proteins but little protease into the medium, we have developed a host-vector system for very efficient synthesis and secretion of heterologous proteins. The multiple promoters and the signal-peptide-coding region of the MWP gene, a structural gene for one of the major cell wall proteins of B. brevis strain 47, were used to construct expression-secretion vectors. With this system, a synthetic gene for human epidermal growth factor (hEGF) was expressed efficiently and a large amount (0.24 g per liter of culture) of mature hEGF was secreted into the medium. hEGF purified from the culture supernatant had the same NH2-terminal amino acid sequence, COOH-terminal amino acid, and amino acid composition as natural hEGF, and it was fully active in biological assays. These results, in combination with previous results, showed that mammalian proteins can be produced in active form 10-100 times more efficiently in B. brevis than has been reported in other systems.

Amino Acid Sequence↗
PubMed1989

A single gene directs synthesis of a precursor protein with beta- and alpha-amylase activities in Bacillus polymyxa.

The Bacillus polymyxa amylase gene comprises 3,588 nucleotides. The mature amylase comprises 1,161 amino acids with a molecular weight of 127,314. The gene appeared to be divided into two portions by the direct-repeat sequence located at almost the middle of the gene. The 5' region upstream of the direct-repeat sequence was shown to be responsible for the synthesis of beta-amylase. The 3' region downstream of the direct-repeat sequence contained four sequences homologous with those in other alpha-amylases, such as Taka-amylase A. The 48-kilodalton (kDa) amylase isolated from B. polymyxa was proven to have alpha-amylase activity. The amino acid sequences of the peptides generated from the 48-kDa amylase showed complete agreement with the predicted amino acid sequence of the C-terminal portion. The B. polymyxa amylase gene was therefore concluded to contain in-phase beta- and alpha-amylase-coding sequences in the 5' and 3' regions, respectively. A precursor protein, a 130-kDa amylase, directed by a plasmid, pYN520, carrying the entire amylase gene, had both beta- and alpha-amylase activities. This represents the first report of a single protein precursor in procaryotes that gives rise to two enzymes.

Amino Acid Sequence↗
PubMed1989

In vitro reconstitution of a hexagonal array with a surface layer protein synthesized by Bacillus subtilis harboring the surface layer protein gene from Bacillus brevis 47.

Bacillus brevis 47 contains two surface layer proteins, termed the outer wall protein and the middle wall protein (MWP), which form a hexagonal array in the cell wall. Introduction of the MWP structural gene into Bacillus subtilis by using a low-copy-number plasmid led to the synthesis of an immunoreactive polypeptide with a molecular mass almost the same as that of the MWP synthesized by B. brevis 47. Biochemical analysis indicated that most of the MWP synthesized by B. subtilis was localized in the cytoplasmic fraction. This was further confirmed by using immunogold electron microscopy. The amino-terminal amino acid sequence of the MWP purified from the cytoplasm of B. subtilis indicated that the MWP was precursor with a signal peptide of 23 amino acid residues to the amino terminus of the mature protein. The precursor of the MWP possessed the ability to reassemble in vitro on the B. brevis 47 peptidoglycan layer, resulting in the formation of almost the same hexagonal arrays as with the mature MWP purified from B. brevis 47, judging from images averaged at a resolution of about 2.5 nm. Furthermore, a center-to-center distance of the hexagonal lattice on the envelope reconstituted by using the precursor MWP was calibrated as 18.3 nm, which was almost identical to the value of 17.8 nm obtained with the mature protein.

Amino Acid Sequence↗