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Biomedical subjects

K Ohmiya

Publications and source records attributed to K Ohmiya.

At least 73 records · Page 4Linked to original sources
PubMed1996

Cloning, DNA sequencing, and expression of the gene encoding Clostridium thermocellum cellulase CelJ, the largest catalytic component of the cellulosome.

The Clostridium thermocellum F1 celJ gene, encoding endoglucanase J (CelJ), consists of an open reading frame (ORF) of 4,803 nucleotides and encodes a protein of 1,601 amino acids with a molecular weight of 178,055. The ORF was confirmed as celJ by comparison with the N-terminal sequence of a truncated CelJ derivative. CelJ is a modular enzyme composed of N-terminal signal peptide and six domains in the following order: an S-layer homology domain, a domain of unknown function (UD-1), a subfamily E1 endoglucanase domain, a family J endoglucanase domain, a docking domain, and another domain of unknown function (UD-2). UD-1 has no significant similarity to UD-2. CelJ hydrolyzed carboxymethylcellulose and xylan, and xylanase activity was ascribed to the family J domain. Antiserum raised against the truncated CelJ cross-reacted with proteins contained in the cellulosome of C. thermocellum F1. These results strongly suggest that CelJ is equivalent to S2, which was identified as the largest catalytic component in the cellulosome of C. thermocellum YS. A second but incomplete ORF encoding an enzyme classified in subfamily E2 endoglucanase, was located downstream of celJ.

Amino Acid Sequence↗
PubMed1995

Analysis of the sequence of a new cryptic plasmid, pRJF2, from a rumen bacterium of the genus Butyrivibrio: comparison with other Butyrivibrio plasmids and application in the development of cloning vector.

A small cryptic plasmid, pRJF2, from Butyrivibrio fibrisolvens strain OB157 was isolated and sequenced. The plasmid is similar in organisation to the previously sequenced Butyrivibrio plasmid, pRJF1, with two open reading frames, ORF1 and ORF2, flanking a region tentatively identified as the replication origin, and a region of unknown function defined by terminal 79 bp invert repeats. The sequences of ORF1, ORF2, and the presumptive replication origin are highly conserved. The sequence between the 79, bp invert repeats is not, and is therefore presumed to be of lesser functional significance, although the 5' and 3' termini are still highly conserved. The functional importance for plasmid replication of these regions was tested by constructing potential shuttle vectors, each lacking one or more of the regions of interest. When the region between the invert repeats was deleted and replaced by the erythromycin resistance gene from pAM beta 1 together with pUC18, to produce the 7.9 kb chimaeric plasmid pYK4, the construct was successfully transformed into E. coli and B. fibrisolvens by electroporation, and was stably maintained in both hosts. Both ORF1 and ORF2 were required for successful transformation of B. fibrisolvens.

Amino Acid Sequence↗
PubMed1995

Cloning and sequencing of some genes responsible for porphyrin biosynthesis from the anaerobic bacterium Clostridium josui.

The 6.2-kbp DNA fragment encoding the enzymes in the porphyrin synthesis pathway of a cellulolytic anaerobe, Clostridium josui, was cloned into Escherichia coli and sequenced. This fragment contained four hem genes, hemA, hemC, hemD, and hemB, in order, which were homologous to the corresponding genes from E. coli and Bacillus subtilis. A typical promoter sequence was found only upstream of hemA, suggesting that these four genes were under the control of this promoter as an operon. The hemA and hemD genes cloned from C. josui were able to complement the hemA and hemD mutations, respectively, of E. coli. The COOH-terminal region of C. josui HemA and the NH2-terminal region of C. josui HemD were homologous to E. coli CysG (Met-1 to Leu-151) and to E. coli CysG (Asp-213 to Phe-454) and Pseudomonas denitrificans CobA, respectively. Furthermore, the cloned 6.2-kbp DNA fragment complemented E. coli cysG mutants. These results suggested that both C. josui hemA and hemD encode bifunctional enzymes.

Amino Acid Sequence↗
PubMed1995

The nitrate reductase gene from a shoyu koji mold, Aspergillus oryzae KBN616.

A niaD gene encoding nitrate reductase was isolated from Aspergillus oryzae KBN616 and sequenced. The structural gene comprises 2973 bp and 868 amino acids, which showed a high degree of similarity to nitrate reductases from other filamentous fungi. The coding sequence is interrupted by six introns varying in size from 48 to 98 bp. The intron positions are all conserved among the niaD genes from A. oryzae, Aspergillus nidulans, and Aspergillus niger. A homologous transformation system was developed for an industrial shoyu koji mold, A. oryzae KBN616, based on the nitrate reductase (niaD) of the nitrate assimilation pathway.

Amino Acid Sequence↗
PubMed1995

Nucleotide sequence of the Clostridium stercorarium xynB gene encoding an extremely thermostable xylanase, and characterization of the translated product.

The nucleotides of the xynB gene of Clostridium stercorarium F-9 were sequenced. The structural gene consists of an open reading frame of 1161 bp encoding a xylanase (XynB) in family F of 387 amino acids with a molecular weight of 44,377. The molecular weight of the enzyme purified from a recombinant Escherichia coli was around 41,000, smaller than the predicted value, on SDS-polyacrylamide gel electrophoresis due to the lack of 32 amino acids at the N-terminus. Intact XynB with a molecular weight of around 43,000 was immunologically detected in the total cell proteins of a recombinant E. coli and C. stercorarium F-9. The purified XynB was active toward xylan, carboxymethylcellulose, p-nitrophenyl-beta-D-xylopyranoside and p-nitrophenyl-beta-D-cellobioside. The pH optimum was 7.0 and it was quite stable over the pH range of 5 to 12 at 4 degrees C. This enzyme was optimally active at 80 degrees C and retained about 50% of the original activity even after incubation at 100 degrees C for 10 min.

Amino Acid Sequence↗
PubMed1995

Process of thermal denaturation of xylanase (XynB) from Clostridium stercorarium F-9.

The thermal denaturation process of Clostridium stercorarium F-9 xylanase (XynB) was studied by monitoring remaining activity and recovered activity of the enzyme. At pH 5.5, aggregation occurred rapidly after the thermal denaturation initiated. The aggregated protein could be dissolved in 8 M urea solution, and the enzyme activity was recovered by diluting the urea. The extent of the recovered activity was gradually decreased with two phases as the reaction time of the thermal denaturation became longer. These results suggested the thermal denaturation process to be as follows: [formula: see text] where N is the native state of the enzyme; D1 is the denatured state of the enzyme that is formed rapidly after the reaction started and can be renatured by the urea treatment, and D2 and D3 are the denatured states of the enzyme that cannot be renatured even by the urea treatment. The rate constants were k1 > 9.2, k2 = 0.33, and k2 = 0.57, and k3 = 0.13 (in min-1 unit).

Calorimetry, Differential Scanning↗
PubMed1994

Purification and characterization of xylanase A from Clostridium stercorarium F-9 and a recombinant Escherichia coli.

Xylanase A encoded by the Clostridium stercorarium F-9 xynA gene was purified to homogeneity from a recombinant clone of Escherichia coli. The N-terminal amino acid sequence and molecular weight (54,000) estimated by SDS-PAGE of the purified enzyme were consistent with those deduced from the nucleotide sequence [Biosci. Biotech. Biochem., 57, 273-277 (1993)]. A xylanase was also purified to homogeneity from a culture supernatant of C. stercorarium F-9. Its N-terminal amino acid sequence, molecular weight, and enzymatic properties were quite in agreement with those of the recombinant enzyme, indicating that the xynA gene was predominantly expressed as a xylanase gene in C. stercorarium F-9. The purified enzyme hydrolyzed xylotriose to yield xylobiose and xylose while it was less active toward xylobiose. It was optimally active at 75 degrees C and pH 7.0 Km and Vmax were estimated to be 1.9 mg/ml and 2.8 mumol of xylose equivalent/min/micrograms for oat spelt xylan, respectively.

Amino Acid Sequence↗
PubMed1993

Structural features of a polygalacturonase gene cloned from Aspergillus oryzae KBN616.

A genomic gene encoding a polygalacturonase from Aspergillus oryzae, used in soy sauce production, was cloned and sequenced. The structural gene comprises 1227 bp coding for 363 amino acids with a putative prepropeptide of 28 amino acids and the open reading frame is disrupted by two short introns of 57 bp and 81 bp. The deduced amino acid sequence of the mature protein showed 63, 63, 63 and 64% homology with those of Aspergillus niger polygalacturonase I, Aspergillus niger polygalacturonase II, Aspergillus tubingensis polygalacturonase II and Cochliobolus carbonum polygalacturonase, respectively. There is, however, little homology among fungal, plant and bacterial polygalacturonases.

Amino Acid Sequence↗
PubMed1993

Nucleotide sequence of the Clostridium stercorarium xynA gene encoding xylanase A: identification of catalytic and cellulose binding domains.

The nucleotides of the xynA gene of Clostridium stercorarium were sequenced. The structural gene consists of an open reading frame of 1533 bp encoding 511 amino acids with an M(r) of 56,519. The signal peptide cleavage site was identified by comparison with the N-terminal amino acid sequence of the enzyme produced by a recombinant Escherichia coli. Xylanase A consists of a catalytic domain belonging to family G at the N-terminus and two direct repeats of about 90 amino acids with a short spacing at the C-terminus. Deletion analysis showed that the repeated sequences were responsible for binding the enzyme to Avicel and were not essential for catalytic activity. The catalytic domain of this enzyme is highly homologous to xylanase A of Clostridium acetobutylicum (identity: 69%) and xylanase B of Bacillus pumilus (identity: 64%).

Amino Acid Sequence↗
PubMed1993

Nucleotide sequence of the Clostridium stercorarium xylA gene encoding a bifunctional protein with beta-D-xylosidase and alpha-L-arabinofuranosidase activities, and properties of the translated product.

The nucleotides of the beta-xylosidase (xylA) gene from Clostridium stercorarium were sequenced. A single open reading frame of 473 codons specifying the subunit (MW 53,340) of xylosidase was identified. The N-terminal amino acid sequence and molecular weight estimated by SDS-polyacrylamide gel electrophoresis of the purified enzyme were quite in agreement with those deduced from the nucleotide sequence. Analysis of the enzyme by gel filtration on an HPLC column gave a molecular weight of 220,000, suggesting that the native enzyme is a tetramer composed of 4 identical subunits. The pH optimum was 7.0 and quite stable over the pH range of 5 to 10 at 4 degrees C. The optimum temperature was 65 degrees C. Vm was estimated to be 5.9 nmol/min/micrograms for p-nitrophenyl-beta-D-xylopyranoside and 16.7 nmol/min/micrograms for p-nitrophenyl-alpha-L-arabinofuranoside, while Km was estimated to be 2.5 mM for p-nitrophenyl-beta-D-xylopyranoside and 17.6 mM for p-nitrophenyl-alpha-L-arabinofuranoside.

Amino Acid Sequence↗
PubMed1992

[Development of computer software in ramp slope controller for treadmill ergometer].

We developed computer software to produce a treadmill ramp protocol through which oxygen uptake (VO2) is increased in a linear fashion, thus enabling subjects to walk until the end of exercise. The developed software simulates the increases in speed and grade of the treadmill displayed on a personal computer screen, and produces the ramp protocol by arbitrarily determining the variables of the following formula: 1) Increments of VO2 = a1t+a2 (ml/min/kg) [t = exercise time (min)] 2) Predicted VO2 by speed (S) and grade (G) = a3S2 + a4G2 + a5SG + a6S + a7G + a8 (ml/min/kg) [S = speed (km/hr); G = grade (%)] 3) Speed suitable for desired exercise time (S or S2) = a9t2 + a10t + a11 (km/hr) 4) Grade suitable for desired exercise time (G or G2) = a12t2 + a13t + a14 (%) In this study, the increment of VO2 was determined by considering the subject's exercise capacity (VO2 = 4t + 7 ml/min/kg), using the Ito's formula (VO2 = 0.067S2 + 0.289SG + 7.73 ml/min/kg). The formula of grade was determined following the formula arbitrarily (G2 = 25t + 5%). The formula of speed for exercise time was calculated automatically. The new ramp protocol, which was applied to 10 healthy subjects (mean age: 24.8 +/- 4.8 years old), disclosed a similar linear relationship between the predicted VO2 and the measured VO2.

Adult↗
PubMed1992

[Hemodynamic effects of alcohol ingestion on anaerobic threshold].

Hemodynamic influence of alcohol ingestion was evaluated using expiratory gas analysis in 10 healthy men whose mean age was 26.1 +/- 2.2 years. Protocol of the control study (C) was as follows: Oxygen uptake (VO2) at rest and during warm-up, anaerobic threshold (AT) and peak VO2 were measured with ramp protocol using a bicycle ergometer. AT was determined by the V-slope method. Protocol of the alcohol study (A) was as follows: On a different day, the same parameters as C were measured after the ingestion of 0.7 g ethanol per kg body weight. The following results were obtained: 1) The serum ethanol concentration was 79.1 +/- 13.5 mg/dl. 2) VO2 in the resting state was higher in A than in C (4.51 +/- 0.74 vs 3.61 +/- 0.62 ml/min/kg), (p < 0.05). 3) AT and peak VO2 in C and A were 17.15 +/- 2.76, 32.09 +/- 6.77 ml/min/kg and 15.43 +/- 2.86, 29.60 +/- 5.35 ml/min/kg, respectively. 4) Exercise time to the AT in C and A was 222.90 +/- 37.32 and 172.0 +/- 33.1 sec, respectively. 5) The heart rate was higher in A than in C. It was concluded that peak VO2 and AT decreased by alcohol ingestion.

Adult↗
PubMed1991

Cloning of a beta-glucosidase gene from Ruminococcus albus and its expression in Escherichia coli.

A HindIII fragment of R. albus DNA encoding beta-glucosidase was cloned into E. coli. The DNA sequence (3158 bp) was determined, and the longest potential encoding sequence consisted of 2,841 bp (947 amino acids with the calculated molecular weight of 104,276. The deduced NH2-terminal amino acid sequence from the first (methionine) to the twentieth (glycine) was identical to that of the purified enzyme, suggesting that the gene for beta-glucosidase does not encode a signal peptide. The enzyme purified from the culture supernatant of the transformant had a molecular weight of 120,000 and its maximum activity was revealed at pH 6.5 and 30 degrees C. Reducing reagents activated the enzyme, whereas the sulfhydryl group-blocking reagents and reaction products (glucose) inhibited the activity. Hydrolyzates of celloorigomers contained glucose as a major product, indicating that the enzyme acts as beta-glucosidase. The enzyme from the transformant revealed similar properties to that from R. albus, and both enzyme proteins were immunologically the same to each other, indicating that the cloned gene encodes beta-glucosidase from R. albus.

Amino Acid Sequence↗
PubMed1991

Modification of the properties of a Ruminococcus albus endo-1,4-beta-glucanase by gene truncation.

An endo-1,4-beta-glucanase (EgI) gene isolated from Ruminococcus albus was deleted at the 5'-flanking region by gene truncation or at the 3'-flanking region by insertion of an omega (omega) fragment with a universal stop codon at the EcoRI or BamHI site. These modified genes were integrated into pUC vectors to construct chimera plasmids for Escherichia coli. The truncated EgIs were produced from transformants (E. coli) harboring the chimera plasmids. An EgI with a 15-amino-acid N-terminal deletion exibited higher activity at lower pH and temperature compared with the activity of the original EgI. The EgIs with 59- and 75-amino-acid deletions from the N and C terminals, respectively, had no activity, indicating that both terminal moieties are essential for enzyme activity.

Amino Acid Sequence↗