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Y Hatada

Publications and source records attributed to Y Hatada.

At least 37 records · Page 2Linked to original sources
PubMed1999

Enzymatic properties and deduced amino acid sequence of a high-alkaline pectate lyase from an alkaliphilic Bacillus isolate.

A high-alkaline pectate lyase (pectate trans-eliminase, EC 4.2.2.2.) from alkaliphilic Bacillus sp. strain KSM-P7, designated Pel-7, was purified to homogeneity. The purified Pel-7 had a molecular mass of approximately 33 kDa as determined by SDS-polyacrylamide gel electrophoresis. The isoelectric point was close to or higher than pH 10.5. In the presence of Ca2+ ions, Pel-7 trans-eliminated polygalacturonate in random manner to generate oligogalacturonides; it exhibited optimal activity at pH 10.5 and around at 60 to 65 degrees C in glycine-NaOH buffer. Mn2+ and Sr2+ ions can serve as cofactors at almost the same level of Ca2+ ions. It also exhibited a protopectinase-like activity, liberating soluble pectin and/or oligogalacturonides from cotton fibers. The pel gene was cloned and sequenced, and the deduced amino acid sequence of mature Pel-7 (302 amino acids, 33, 355 Da) showed some conserved regions in Pel superfamily, although homology to amino acid sequences of known Pels with 27 to 32% identity. Furthermore, Pel-7 appears to have similar core structure of parallel beta-helix and active site topology with other Pels as revealed by secondary structure prediction in the Pel proteins. These results suggest that Pel-7 is basically grouped into Pel superfamily although the enzymatic and molecular properties are different.

Amino Acid Sequence↗
PubMed1999

Purification and properties of a low-molecular-weight, high-alkaline pectate lyase from an alkaliphilic strain of Bacillus.

A low-molecular-weight, high-alkaline pectate lyase (pectate transeliminase, EC 4.2.2.2) was found in an alkaline culture of Bacillus sp. strain KSM-P15, purified to homogeneity, and crystallized. The enzyme had a relative molecular weight of approximately 20,300 as measured by sedimentation equilibrium, with a sedimentation coefficient (s20,w0) of 1.73 S. It was a basic protein with an isoelectric point of pH 10.3, and the alpha-helical content was only 6.6%. In the presence of Ca2+ ions, the enzyme degraded polygalacturonic acid in a random manner to yield 4,5-unsaturated oligo-galacturonides and had its optimal activity around pH 10.5 and 50-55 degrees C. It also had a protopectinase-like activity on cotton fibers. The N-terminal amino acid sequences of the intact protein (28 amino acids) and its two lysyl endopeptidase-cleaved peptide fragments (8 and 12 amino acids) had very low sequence similarity with pectate lyases reported to date. These results strongly suggest that the pectate lyase of Bacillus sp. strain KSM-P15 may be a novel enzyme and belongs in a new family.

Amino Acid Sequence↗
PubMed1999

Cloning and sequencing of a high-alkaline pectate lyase gene from an alkaliphilic Bacillus isolate.

Alkaliphilic Bacillus sp. strain KSM-P103 was found to exoproduce a high-alkaline pectate lyase (pectate transeliminase, EC 4.2.2.2). The gene for this enzyme from the alkaliphile was cloned and sequenced for the first time. The structural gene contained a 1,038-bp open reading frame encoding 345 amino acids. The deduced amino acid sequence of the mature enzyme (302 amino acids, 33,312 Da), designated Pel-103, showed very low similarity to those of known pectate lyases with 28-36% identity: the loop regions were very short and the amino acid usage in the parallel beta-helix core structure was considerably different. Moreover, physicochemical and catalytic properties of Pel-103 were different from those of other enzymes reported so far. Pel-103 was a very basic protein with an isoelectric point close to pH 10.5 and had optimal activity at 60-65 degrees C and at pH as high as 10.5. However, Pel-103 appeared to have a similar core and active site topology to the enzymes of known structure from Erwinia chrysanthemi and Bacillus subtilis. Expression of the gene for Pel-103 in B. subtilis resulted in high pectate lyase activity in the culture broth, concomitant with the appearance of a main protein band on an SDS gel at 33 kDa.

Alkalies↗
PubMed1998

Improved thermostability of a Bacillus alpha-amylase by deletion of an arginine-glycine residue is caused by enhanced calcium binding.

alpha-Amylase from alkaliphilic Bacillus KSM-1378 (LAMY) is a novel semi-alkaline enzyme which has a high specific activity, a value 5-fold higher than that of a Bacillus licheniformis enzyme at alkaline pH. Thermostability of this enzyme could be improved by deletion of the Arg181-Gly182 residue by means of site-directed mutagenesis. The wild-type and engineered LAMYs were very similar with respect to specific activity, pH-activity curve, temperature-activity curve, susceptibility to inhibitors, and pattern of hydrolysis products from soluble starch and maltooligosaccharides. However, the engineered enzyme also acquired increased pH stability and resistance to sodium dodecyl sulfate and especially chelating reagents, such as ethylenediaminetetraacetate and ethyleneglycol-bis (beta-aminoethylether)tetraacetate. This is the first report that thermostability of alpha-amylase is improved by enhanced calcium binding to the enzyme molecule.

Amino Acid Sequence↗
PubMed1998

Alkaline detergent enzymes from alkaliphiles: enzymatic properties, genetics, and structures.

The cleaning power of detergents seems to have peaked; all detergents contain similar ingredients and are based on similar detergency mechanisms. To improve detergency, modern types of heavy-duty powder detergents and automatic dishwasher detergents usually contain one or more enzymes, such as protease, amylase, cellulase, and lipase. Alkaliphilic Bacillus strains are often good sources of alkaline extracellular enzymes, the properties of which fulfil the essential requirements for enzymes to be used in detergents. We have isolated numbers of alkaliphilic Bacillus that produce such alkaline detergent enzymes, including cellulase (CMCase), protease, alpha-amylase, and debranching enzymes, and have succeeded in large-scale industrial production of some of these enzymes. Here, we describe the enzymatic properties, genetics, and structures of the detergent enzymes that we have developed.

Amino Acid Sequence↗
PubMed1998

Enzymatic properties of a novel liquefying alpha-amylase from an alkaliphilic Bacillus isolate and entire nucleotide and amino acid sequences.

A novel liquefying alpha-amylase (LAMY) was found in cultures of an alkaliphilic Bacillus isolate, KSM-1378. The specific activity of purified LAMY was approximately 5,000 U mg of protein-1, a value two- to fivefold greater between pH 5 and 10 than that of an industrial, thermostable Bacillus licheniformis enzyme. The enzyme had a pH optimum of 8.0 to 8.5 and displayed maximum activity at 55 degreesC. The molecular mass deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 53 kDa, and the apparent isoelectric point was around pH 9. This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltopentaose, maltohexaose, and maltose as major end products after completion of the reaction. Maltooligosaccharides in the maltose-to-maltopentaose range were unhydrolyzable by the enzyme. The structural gene for LAMY contained a single open reading frame 1, 548 bp in length, corresponding to 516 amino acids that included a signal peptide of 31 amino acids. The calculated molecular mass of the extracellular mature enzyme was 55,391 Da. LAMY exhibited relatively low amino acid identity to other liquefying amylases, such as the enzymes from B. licheniformis (68.9%), Bacillus amyloliquefaciens (66.7%), and Bacillus stearothermophilus (68.6%). The four conserved regions, designated I, II, III, and IV, and the putative catalytic triad were found in the deduced amino acid sequence of LAMY. Essentially, the sequence of LAMY was consistent with the tertiary structures of reported amylolytic enzymes, which are composed of domains A, B, and C and which include the well-known (alpha/beta)8 barrel motif in domain A.

Amino Acid Sequence↗
PubMed1998

Hyperexpression of the gene for a Bacillus alpha-amylase in Bacillus subtilis cells: enzymatic properties and crystallization of the recombinant enzyme.

We have constructed a new excretion vector, pHSP64, to develop a hyperexcretion system for Bacillus subtilis [Sumitomo et al., Biosci. Biotech. Biochem., 59, 2172-2175 (1995)]. The structural gene for a novel liquefying semi-alkaline alpha-amylase from the alkaliphilic Bacillus sp. KSM-1378 was amplified by PCR. It was cloned into a SalI-SmaI site of pHSP64 and the recombinant plasmid obtained was introduced into B. subtilis. The transformed B. subtilis hyperproduced the alpha-amylase activity extracellularly, corresponding to approximately 1.0 g (5 x 10(6) units) per liter of an optimized liquid culture. The recombinant enzyme was purified to homogeneity by a simple purification procedure with very high yield. No significant differences in physiochemical and catalytic properties were observed between the recombinant enzyme and the native enzyme produced by Bacillus sp. KSM-1378. The enzymatic properties of the recombinant enzyme were further examined with respect to the responses to various metal ions. The recombinant enzyme could easily be crystallized at room temperature within one day in a buffered solution of 10% (w/v) ammonium sulfate (pH 6.5).

Amino Acid Sequence↗
PubMed1997

Amino acid sequence and stereoselective hydrolytic reaction of an endo-1,4-beta-glucanase from a Bacillus strain.

Bacillus sp. KSM-522 produces three different extracellular endo-1,4-beta-glucanases [EGs; Okoshi et al., Agric. Biol. Chem., 54, 83-89 (1990)]. Here, we report the molecular cloning and sequencing of the gene for the fourth EG (EG-IV) of the organism and the mechanism of its hydrolytic reaction. The structural gene contained an open reading frame of 1911 bp, corresponding to 636 amino acids, the amino acid sequence of which was very close to that of an EG of Clostridium cellulovorans, belonging to the cellulase family E2. The molecular mass of the extracellular mature enzyme (Ser26 through Lys636) was calculated to be 69,076 Da, a value close to the 69.2 kDa measured for the recombinant EG-IV expressed in Bacillus subtilis. The optimum pH and temperature for activity of the recombinant enzyme were pH 8.0 and 50 degrees C, respectively. By 1H-NMR spectroscopy, we demonstrated that the hydrolysis of p-nitrophenyl beta-D-cellotrioside by EG-IV proceeded with inversion of the anomeric configuration.

Amino Acid Sequence↗
PubMed1996

Amino acid sequence and molecular structure of an alkaline amylopullulanase from Bacillus that hydrolyzes alpha-1,4 and alpha-1,6 linkages in polysaccharides at different active sites.

An amylopullulanase from alkalophilic Bacillus sp. KSM-1378 hydrolyzes both alpha-1,6 linkages in pullulan and alpha-1,4 linkages in other polysaccharides, with maximum activity in each case at an alkaline pH, to generate oligosaccharides (Ara, K., Saeki, K., Igarashi, K., Takaiwa, M., Uemura, T., Hagihara, H., Kawai, S., and Ito, S. (1995) Biochim. Biophys. Acta 1243, 315-324). Here, we report the molecular cloning and sequencing of the gene for and the structure of this enzyme and show that its dual hydrolytic activities are associated with two independent active sites. The structural gene contained a single, long open reading frame of 5,814 base pairs, corresponding to 1,938 amino acids that included a signal peptide of 32 amino acids. The molecular mass of the extracellular mature enzyme (Glu33 through Leu1938) was calculated to be 211,450 Da, a value close to the 210 kDa determined for the amylopullulanase produced by Bacillus sp. KSM-1378. The amylase and the pullulanase domains were located in the amino-terminal half and in the carboxyl-terminal half of the enzyme, respectively, being separated by a tandem repeat of a sequence of 35 amino acids. Four regions, designated I, II, III, and IV, were highly conserved in each catalytic domain, and they included a putative catalytic triad Asp550-Glu579-Asp645 for the amylase activity and Asp1464-Glu1493-Asp1581 for the pullulanase activity. The purified enzyme was rotary shadowed at a low angle and observed by transmission electron microscopy; it appeared to be a "castanet-like" or "bent dumbbell-like" molecule with a diameter of approximately 25 nm.

Amino Acid Sequence↗
PubMed1995

Nucleotide sequence of a principal sigma factor gene (hrdB) of Streptomyces griseus.

The hrdB homologue was isolated from a streptomycin-producing Streptomyces griseus 2247 strain, which is independent of A-factor. The nucleotide sequence of the cloned DNA fragment revealed the presence of an open reading frame (ORF) of 1,542bp, which predicted a primary product of 514 amino acids and Mr 56,100. The N-terminal sequence of the purified HrdB protein of S. griseus was identical to the amino acid sequence deduced from the nucleotide sequence. The deduced amino acid sequence contains an "rpoD box" conserved in the principal sigma factors of eubacteria, and shows high similarity to the hrdB products of S. coelicolor A3(2)(89.9%) and S. aureofaciens (88.1%). The cloned gene encodes a principal sigma factor of S. griseus. The promoter region was identified by using a promoter-probe vector and by means of primer extensions experiments. The transcription start point is located 158-bp upstream of the initiation codon.

Amino Acid Sequence↗
PubMed1994

A fate map of the epiblast of the early chick embryo.

We have used carbocyanine dyes (DiI and DiO) to generate fate maps for the epiblast layer of the chick embryo between stage X and the early primitive streak stage (stages 2-3). The overall distribution of presumptive cell types in these maps is similar to that described for other laboratory species (zebrafish, frog, mouse). Our maps also reveal certain patterns of movement for these presumptive areas. Most areas converge towards the midline and then move anteriorly along it. Interestingly, however, some presumptive tissue types do not take part in these predominant movements, but behave in a different way, even if enclosed within an area that does undergo medial convergence and anterior movement. The apparently independent behaviour of certain cell populations suggests that at least some presumptive cell types within the epiblast are already specified at preprimitive streak stages.

Animals↗
PubMed1993

The pedunculated type of primary peritoneal pregnancy implanted on the infundibulopelvic ligament.

BACKGROUND: Primary peritoneal pregnancy is extremely rare in the category of extrauterine pregnancy and usually has wide attachment to the peritoneal surface at the implantation site. CASE: A 37-year-old woman had severe lower abdominal pain 13 days before laparotomy for a preoperative diagnosis of extrauterine pregnancy. The conceptus was implanted on the pedicle originating from the left infundibulopelvic ligament. A fresh embryo of approximately 6 weeks' gestation was found in the conceptus. CONCLUSION: Primary peritoneal pregnancy may become pedunculated, and implantation on the infundibulopelvic ligament may be involved in the formation of the pedicle. A pedunculated pregnancy may increase the risk of early termination. The reflux theory may be one of the mechanisms in this case.

Adult↗
PubMed1992

Relationships between mesoderm induction and the embryonic axes in chick and frog embryos.

The hypoblast is generally thought to be responsible for inducing the mesoderm in the chick embryo because the primitive streak, and subsequently the embryonic axis, form according to the orientation of the hypoblast. However, some cells become specified as embryonic mesoderm very late in development, towards the end of the gastrulation period and long after the hypoblast has left the embryonic region. We argue that induction of embryonic mesoderm and of the embryonic axis are different and separable events, both in amniotes and in amphibians. We also consider the relationships between the dorsoventral and anteroposterior axes in both groups of vertebrates.

Animals↗
PubMed1991

Synthesis and platelet aggregation inhibitory activities of 3-(2-oxopropylidene)azetidin-2-one derivatives. II.

A series of 3-acylidene-4-methylazetidin-2-one derivatives bearing various substituents at the 1-position of the azetidin-2-one ring was synthesized. These compounds were evaluated for platelet aggregation inhibitory activities. Most of the compounds synthesized showed potent inhibitory activities against rabbit platelet aggregation induced by adenosine diphosphate or collagen in vitro. Structure-activity relationships are also discussed.

Animals↗
PubMed1991

N-(p-isothiocyanatophenethyl)spiperone, a selective and irreversible antagonist of D2 dopamine receptors in brain.

N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), an irreversible and nonselective protein-modifying reagent, has been used extensively in studies involving inactivation of receptors. Here, we present N-(p-isothiocyanatophenethyl)spiperone (NIPS), a novel and highly selective irreversible inactivator of D2 but not D1 receptors. In in vitro studies, NIPS exhibited an apparent Ki of 10 nM for [3H]methylspiperone binding to D2 receptors in rat striatum. Preincubation of the striatal membranes with NIPS followed by extensive washing resulted in up to an 80% reduction of the D2 receptor maximum binding (Bmax). Coincubation with the D2 receptor antagonist domperidone could protect against this reduction. NIPS was additionally shown to irreversibly inactivate D2 receptor binding activity in cultured cells expressing the D2 receptor protein. In in vivo administration studies, using [3H]SCH 23390 and [3H]spiperone to assay D1 and D2 receptors in vitro, 24 hr after injection (s.c.) with 5 to 40 mg/kg of NIPS D2 receptor, Bmax was decreased by 58 to 76%, without a change in D2 receptor affinity. In contrast, there was no effect on D1 receptor Bmax or affinity. There was also a small (24%) reduction in frontal cortex 5-hydroxytryptamine2 receptors by 20 mg/kg of NIPS. However, there was no effect on alpha-1 or alpha-2 adrenergic receptors in the frontal cortex, or on muscarinic cholinergic or 5-hydroxytryptamine1A receptors in the hippocampus. After single doses of either 20 mg/kg of NIPS or 10 mg/kg of EEDQ, the D2 receptor recovery rate was much slower after NIPS (half-time of receptor recovery = 170 hr) than after EEDQ (half-time of receptor recovery = 76.7 hr).(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Antagonists↗
PubMed1990

Synthesis and platelet aggregation-inhibitory activities of novel 3-(2-oxopropylidene)azetidin-2-one derivatives. I.

Treatment of (E)-3-(2-hydroxypropylidene)-4-methyl-1-phenylazetidin-2-one (11) with 10% Pd/C gave (E)-(12), (Z)-3-(2-oxopropylidene)-4-methyl-1-phenylazetidin-2-one (13), 3,4-cis-(14a) and 3,4-trans-3-(2-oxopropyl)-4-methyl-1-phenylazetidin-2-one (14b). Among them, 12 and 13 were found to show potent inhibitory activities against rabbit platelet-rich plasma aggregation induced by adenosine diphosphate or collagen. Ring-expanded homologous derivatives and an acyclic analogue of 12 were also synthesized and tested for the biological activities. The azetidin-2-one skeleton bearing a 2-oxoalkylidene moiety at the 3 position was found to be essential for the platelet aggregation inhibitory activities of these compounds.

Animals↗