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S Udaka

Publications and source records attributed to S Udaka.

At least 55 records · Page 3Linked to original sources
PubMed1993

Very efficient extracellular production of cholera toxin B subunit using Bacillus brevis.

We have constructed a very efficient synthesis and secretion system for cholera toxin B subunit (CTB) of Vibrio cholerae 569B using Bacillus-brevis. The constructed expression-secretion vector has 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, directly followed by the gene encoding the mature CTB. A large amount of mature CTB (1.4 g per liter of culture) was secreted into the medium. It had the same amino terminal amino acid sequence as that of authentic CTB and was fully active in GM1 ganglioside binding assay.

Amino Acid Sequence↗
PubMed1993

Production of a fungal protein, Taka-amylase A, by protein-producing Bacillus brevis HPD31.

An expression-secretion vector, pMK300, was constructed to express the Aspergillus oryzae Taka-amylase A (Taa) cDNA. The promoter and signal peptide regions of the HWP (a major cell wall protein of Bacillus brevis HPD31) gene on pMK300 were efficiently utilized in B. brevis HPD31 and a large amount of Taa (22 mg/l) was secreted into the medium. The HWP signal peptide utilized for secretion of Taa was correctly processed during the protein transport across the membrane. The enzymatic properties of Taa produced by B. brevis HPD31 were the same as those of the Aspergillus oryzae Taa in several respects; specific activity, thermal and pH stabilities, and temperature and pH optima. These results, in combination with previous results, indicate that B. brevis HPD31 could be used to produce extracellularly foreign proteins of diverse origins as functional proteins.

Amino Acid Sequence↗
PubMed1993

Antimutagenic effects of N-methyl-valyl-amiclenomycin (BA-2) isolated from the metabolites of Streptomyces sp.

A novel antimutagenic factor, BA-2, active against UV-induced mutagenesis in Escherichia coli WP2 was isolated from the metabolites of Streptomyces sp. strain AJ9455. BA-2 also suppressed mutations induced by 4-nitroquinoline N-oxide (4-NQO) and furylfuramide (AF-2) in E. coli WP2s (uvrA) without any decrease of cellular viability. BA-2 strongly inhibited the UV induction of SOS repair functions when it was monitored by beta-galactosidase activity expressed from the sulA::lacZ fusion gene of strain PQ37. It is assumed that the antimutagenic effect of BA-2 on mutagenesis induced by UV, 4-NQO or AF-2 was the result of inhibition of induction of the inducible error-prone SOS repair. The structure of BA-2 was considered to be N-methyl-valyl-amiclenomycin, and the structural unit of 4-amino-2,5-cyclohexadiene must be essential for the antimutagenic activity, since deamination by heating results in the loss of antimutagenic activity of BA-2.

Aminobutyrates↗
PubMed1993

Characterization of Bacillus brevis with descriptions of Bacillus migulanus sp. nov., Bacillus choshinensis sp. nov., Bacillus parabrevis sp. nov., and Bacillus galactophilus sp. nov.

Thirty-five Bacillus brevis strains obtained from culture collections, including protein-producing isolates, were taxonomically studied by using numerical analysis, DNA base composition, and DNA-DNA hybridization. Six DNA relatedness groups were represented, and these groups correlated well with clusters based on the numerical analysis. The B. brevis strains were separated into B. brevis sensu stricto, four new species, and an unidentified species of the genus Bacillus. Bacillus migulanus sp. nov., Bacillus choshinensis sp. nov., Bacillus parabrevis sp. nov., and Bacillus galactophilus sp. nov. are proposed.

Bacillus↗
PubMed1993

Characterization of the gene encoding an intracellular proteinase inhibitor of Bacillus subtilis and its role in regulation of the major intracellular proteinase.

The gene (ipi) for an intracellular proteinase inhibitor (BsuPI) from Bacillus subtilis was cloned and found to encode a polypeptide consisting of 119 amino acids with no cysteine residues. The deduced amino acid sequence contained the N-terminal amino acid sequence of the inhibitor, which was chemically determined previously, and showed no significant homology to any other proteinase inhibitors. Analysis of the transcription initiation site and mRNA showed that the ipi gene formed an operon with an upstream open reading frame with an unknown function. The transcriptional control of ipi gene expression was demonstrated by Northern (RNA) blot analysis, and the time course of transcriptional enhancement roughly corresponded to the results observed at the protein level. Strains in which the ipi gene was disrupted or in which BsuPI was overexpressed constitutively sporulated normally. Analysis of the time course of production of the intracellular proteinase and proteinase inhibitor in these strains suggested that BsuPI directly regulated the major intracellular proteinase (ISP-1) activity in vivo.

Amino Acid Sequence↗
PubMed1993

Efficient production of Bacillus stearothermophilus alpha-amylase in Bacillus brevis by altering its signal peptide.

To investigate the role of signal peptides in extracellular production in Bacillus brevis, the Bacillus stearothermophilus alpha-amylase signal peptide was systematically altered and the effects were analyzed in B. brevis. Efficient extracellular production of the amylase in B. brevis was accomplished either by introducing point mutations at positions between -6 and -4 or by replacing the whole signal peptide with that of B. brevis middle wall protein.

Amino Acid Sequence↗
PubMed1993

Purification of protein disulfide isomerase from a thermophilic fungus.

A protein disulfide isomerase (PDI) was purified to homogeneity from the thermophilic fungus Humicola insolens by a rapid three-step procedure, anion-exchange chromatography, concanavalin A-affinity chromatography, and reverse phase high performance liquid chromatography. Forty-one micrograms of PDI was obtained from 100 g of wet mycelium. Concanavalin A-Sepharose chromatography is available for purification of the fungal PDI, indicating that the enzyme is also glycosylated like the yeast PDI. The fungal PDI exists as a dimer (2 x 60 kDa), has a pI of 3.5, and is fairly heat-stable. The amino acid composition of the PDI is similar to those of yeast and bovine liver PDI, and the high content of acidic amino acid residues agrees with the lower acidic pI.

Amino Acid Sequence↗
PubMed1992

Efficient production of thermostable Thermus thermophilus xylose isomerase in Escherichia coli and Bacillus brevis.

The xylose (glucose) isomerase from the thermophile Thermus thermophilus seems to have potential for the development of new isomerization processes using high temperatures and slightly acidic pH. The isomerase has an optimum temperature at 95 degrees C, and is also very stable at high temperatures. The optimum pH is around 7.0, close to where by-product formation is minimal. Since Thermus produces only a little of this useful isomerase, the production of the cloned gene in Escherichia coli and Bacillus brevis were compared. Especially B. brevis was able to produce the isomerase efficiently, more than 1 g/l, in spite of the high G + C content (67%) of the Thermus gene, and the presence of codons not frequently used in E. coli or B. brevis.

Aldose-Ketose Isomerases↗
PubMed1992

Mass production of sphingomyelinase of Bacillus cereus by a protein-hyperproducing strain, Bacillus brevis 47, and its purification.

Sphingomyelinase (sphingomyelin cholinephosphohydrolase) [EC 3.1.4.12] of Bacillus cereus was overproduced in a protein-hyperproducing strain, B. brevis 47, by cloning the gene into an expression vector pNU211, which has been developed to express a foreign gene utilizing a promoter and a signal sequence of an outer cell wall protein gene. From 1 liter of culture, about 10 mg of protein was purified to near-homogeneity by two steps of column chromatography; this is almost 500 times higher production compared to the conventional preparation from the original strain, B. cereus IAM 1208. The N-terminal amino acid sequence of the secreted enzyme was identical to that of the authentic enzyme, indicating that the signal sequence for secretion of B. cereus was processed properly in B. brevis 47.

Amino Acid Sequence↗
PubMed1992

Characterization of an extracellular protease inhibitor of Bacillus brevis HPD31 and nucleotide sequence of the corresponding gene.

A novel proteinaceous protease inhibitor was isolated from the culture supernatant of Bacillus brevis HPD31. The protease inhibitor of B. brevis (designated BbrPI) was produced extracellularly in multiple forms having at least three different molecular weights. One of them, BbrPI-a, was purified to near homogeneity and only showed inhibitory activity toward serine proteases, such as trypsin, chymotrypsin, and subtilisin. BbrPI was presumed to form a trypsin-inhibitor complex in a molar ratio of 1:1. The inhibitor was found to be heat resistant at neutral and acidic pHs. The gene coding for BbrPI was cloned into Escherichia coli, and its nucleotide sequence was determined. The sequence suggested that BbrPI is produced with a signal peptide of 24 amino acid residues. The amino acid sequence of the protein deduced from the DNA sequence contained the amino acid sequences of amino termini of the inhibitors, a, b, and c, and their putative precursor determined chemically. The molecular weight of the precursor was about 33,000, and the molecular weights of inhibitors a, b, and c were about 22,000, 23,500, and 24,000, respectively. It is presumed that the secreted precursor protein, which is probably inactive, is cleaved by protease into several active protease inhibitor molecules. BbrPI shows no significant homology to the protease inhibitors described previously and is unique in not having any cysteine residues in its molecule.

Amino Acid Sequence↗
PubMed1992

Cloning, characterization, and inactivation of the Bacillus brevis lon gene.

A gene of Bacillus brevis HPD31 analogous to the Escherichia coli lon gene has been cloned and characterized. The cloned gene (B. brevis lon gene) encodes a polypeptide of 779 amino acids with a molecular weight of 87,400 which resembles E. coli protease La, the lon gene product. Fifty-two percent of the amino acid residues of the two polypeptides were identical. The ATP-binding sequences found in E. coli protease La were highly conserved. The promoter of the B. brevis lon gene resembled that recognized by the major RNA polymerase of Bacillus subtilis and did not contain sequences homologous to the E. coli heat shock promoters. The B. brevis lon gene was inactivated by insertion of the neomycin resistance gene. A mutant B. brevis carrying the inactivated lon gene showed diminished ability for the degradation of abnormal polypeptides synthesized in the presence of puromycin.

ATP-Dependent Proteases↗
PubMed1991

Structural and functional roles of cysteine residues of Bacillus polymyxa beta-amylase.

Bacillus polymyxa beta-amylase contains three cysteine residues at positions 83, 91, and 323, which can react with sulfhydryl reagents. To determine the role of cysteine residues in the catalytic reaction, cysteine residues were mutated to construct four mutant enzymes, C83S, C91V, C323S, and C-free. Wild-type and mutant forms of the enzyme were expressed in, and purified to homogeneity from, Bacillus subtilis. A disulfide bond between Cys83 and Cys91 was identified by isolation of tryptic peptides bearing a fluorescent label, IAEDANS, from wild-type and C91 V enzymes followed by amino acid sequencing. Therefore, only Cys323 contains a free SH group. Replacement of cysteine residues with serine or valine residues resulted in a significant decrease in the kcat/Km value of the enzyme. C323S, containing no free SH group, however, retained a high specific activity, approximately 20% of the wild-type enzyme. None of the cysteine residues participate directly in the catalytic reaction.

Amino Acid Sequence↗
PubMed1991

Structurally different rat liver medium-chain acyl CoA dehydrogenases directed by complementary DNAs differing in their 5'-region.

Different forms of rat liver medium-chain acyl CoA dehydrogenase (MCAD) (EC 1.3.99.3) were produced in Escherichia coli carrying expression plasmids (pRMCADm-1 approximately 9) differing at the 5'-region of the cDNA. The proteins expressed could be readily extracted from the cells. The protein (approximately 44 kDa) directed by pRMCADm-3 showed the highest activity and was readily purified to homogeneity. The purified enzyme contained non-covalently bound FAD and was similar to rat liver mitochondrial enzyme in all respects examined. The purified protein (approximately 45 kDa) directed by pRMCADm-1 did not contain FAD and showed no enzymatic activity. Therefore, the leader peptide disturbs the binding of FAD to the apoprotein. The purified protein (approximately 40 kDa) directed by pRMCADm-6 did not contain FAD. Thus, the deletion of the NH2-terminal portion of the apoprotein to some extent results in its inability to combine with FAD.

Acyl-CoA Dehydrogenase↗
PubMed1991

Xylose (glucose) isomerase gene from the thermophile Thermus thermophilus: cloning, sequencing, and comparison with other thermostable xylose isomerases.

The xylose isomerase gene from the thermophile Thermus thermophilus was cloned by using a fragment of the Streptomyces griseofuscus gene as a probe. The complete nucleotide sequence of the gene was determined. T. thermophilus is the most thermophilic organism from which a xylose isomerase gene has been cloned and characterized. The gene codes for a polypeptide of 387 amino acids with a molecular weight of 44,000. The Thermus xylose isomerase is considerably more thermostable than other described xylose isomerases. Production of the enzyme in Escherichia coli, by using the tac promoter, increases the xylose isomerase yield 45-fold compared with production in T. thermophilus. Moreover, the enzyme from E. coli can be purified 20-fold by simply heating the cell extract at 85 degrees C for 10 min. The characteristics of the enzyme made in E. coli are the same as those of enzyme made in T. thermophilus. Comparison of the Thermus xylose isomerase amino acid sequence with xylose isomerase sequences from other organisms showed that amino acids involved in substrate binding and isomerization are well conserved. Analysis of amino acid substitutions that distinguish the Thermus xylose isomerase from other thermostable xylose isomerases suggests that the further increase in thermostability in T. thermophilus is due to substitution of amino acids which react during irreversible inactivation and results also from increased hydrophobicity.

Aldose-Ketose Isomerases↗
PubMed1991

Repression of the cell wall protein gene operon in Bacillus brevis 47 by magnesium and calcium ions.

Transcription from P2, one of the major promoters of the cell wall protein gene operon of Bacillus brevis 47, was markedly enhanced at the early stationary phase of growth. MgCl2, when added at 1 to 5 mM to the medium, inhibited this enhancement of transcription as well as shedding of the cell wall protein layers from the cell surface. MgSO4 or CaCl2 showed an effect similar to that of MgCl2. The possible coordination of the cell wall structure with regulation of the cell wall protein genes is discussed.

Bacillus↗