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K Sonomoto

Publications and source records attributed to K Sonomoto.

28 records · Page 2Linked to original sources
PubMed1994

Purification and characterization of the poly(hydroxyalkanoic acid) synthase from Chromatium vinosum and localization of the enzyme at the surface of poly(hydroxyalkanoic acid) granules.

A recombinant strain of Escherichia coli, which overexpressed phaC and phaE from Chromatium vinosum, was used to isolate poly(3-hydroxyalkanoic acid) synthase. The isolation was performed by a two-step procedure including chromatography on DEAE-Sephacel and Procion Blue H-ERD. The poly(3-hydroxyalkanoic acid) synthase consisted of two different kinds of subunit (PhaC, M(r) 39,500 and PhaE, M(r) 40.500). PhaC was separated from the poly(3-hydroxyalkanoic acid) synthase complex by chromatography on phenyl-Sepharose: PhaE was enriched by solubilization of protein inclusion bodies. The stoichiometry of PhaC and PhaE in the enzyme complex was not determined. The poly(3-hydroxyalkanoic acid) synthase (PhaEC) exhibited a native relative molecular mass of M(r) 400,000 and most probably consists of ten subunits. The Km value of the enzyme for D(-)-3-hydroxybutyryl-CoA was 0.063 mM. The enzyme synthesized poly(3-hydroxybutyric acid) in vitro from D(-)-3-hydroxybutyryl-CoA or, together with propionyl-CoA transferase in a coupled enzyme reaction, synthesized the same product from acetyl-CoA plus D(-)-3-hydroxybutyric acid. Antibodies were raised against both subunits of the poly(3-hydroxyalkanoic acid) synthase. By immunoelectron microscopy, the poly(3-hydroxyalkanoic acid) synthase was localized within the cytoplasm in cells of C. vinosum grown under non-storage conditions. In cells grown under poly(3-hydroxybutyric acid) storage conditions, the enzyme was observed to be located at the surface of the poly(3-hydroxybutyric acid) granules. Immunoblots with anti-PhaC, anti-PhaE IgG and crude extract proteins indicated that poly(3-hydroxyalkanoic acid) synthases with partial sequence similarities are widespread among purple sulphur bacteria.

Acyltransferases↗
PubMed1994

Amperometric flow-injection analysis of creatinine based on immobilized creatinine deiminase, leucine dehydrogenase and L-amino acid oxidase.

Leucine dehydrogenase/L-amino acid oxidase was proposed as an enzymatic conversion system for ammonia and its application to amperometric assay of creatinine was investigated. Ammonia formed by creatinine deiminase catalyzed hydrolysis of creatinine was converted to L-leucine by leucine dehydrogenase, and the oxidation of L-leucine by L-amino acid oxidase was detected with an oxygen electrode. Two approaches were proposed to overcome the problem of endogenous ammonia and L-amino acids. The first was using glutamate dehydrogenase prereactor to remove endogenous ammonia; endogenous L-amino acids were corrected by a separate run. In the second approach, endogenous ammonia and L-amino acids were simultaneously compensated with a two-channel system. It resulted in double peak recording that the flow was split and rejoined between the two ends of creatinine deiminase reactor and a delay coil and a reference column were properly set at one of the two-channels. One gave the sum response of all responsible compounds, the other that of endogenous interferences except creatinine. Both approaches were applied to creatinine assay in urine and the results showed a good agreement with those obtained from the Jaffe method.

Amino Acid Oxidoreductases↗
PubMed1993

A flow-injection biosensor system for the amperometric determination of creatinine: simultaneous compensation of endogenous interferents.

A flow-injection biosensor system was developed for the amperometric determination of creatinine based on coupled reactions of three sequentially aligned enzyme reactors, creatinine deiminase, glutamate dehydrogenase, and glutamate oxidase, using an oxygen electrode as the detector. To overcome the problem of endogenous ammonia and glutamate, the flow was split into two channels after the injector and rejoined before the glutamate dehydrogenase reactor. Double peak recording was obtained by setting a delay coil and a reference column in one of the two channels. The first peak gave the sum response of creatinine, endogenous ammonia, and glutamate, and the second that of endogenous ammonia and glutamate. By this method compensation for endogenous ammonia and glutamate, as well as for interfering ascorbic acid, was achieved simultaneously. The system gave linear calibrations up to 2 mM for the first peak and 3 mM for the second one. The lower detection limits were 0.1 and 0.02 mM for 35- and 100-microliters injection of sample, respectively. One run was completed within 2 min. The system showed good reproducibility (< 3%) and long operational stability (> 1300 runs). The assay results of creatinine in urine showed good correlation with those obtained from the chemical method of Jaffe.

Amino Acid Oxidoreductases↗
PubMed1992

Amperometric assay of creatinine in urine by flow injection analysis based on conjugated reactions of immobilized enzymes. Simultaneous compensation of endogenous ammonia.

A flow-injection analysis biosensor system was developed for the amperometric assay of creatinine based on coupled reactions of three immobilized enzymes, using an oxygen electrode as the detection device. The ammonia produced by creatinine deiminase-catalyzed hydrolysis of creatinine was further converted into L-glutamate with two sequentially aligned enzyme reactors: glutamate dehydrogenase and glutamate oxidase. Endogenous ammonia was simultaneously compensated with a double peak recording system, where the flow was split after sample injection and rejoined before the glutamate dehydrogenase reactor. The system gave linear calibration in a range of 0.1-2.0 mM for creatinine and the first peak of ammonia, and 0.1-3.0 mM for the second peak of ammonia. One run was completed within two minutes. The system can be readily applied to the assay of creatinine in urine and showed good correlation with that from the currently used Jaffe method.

Amino Acid Oxidoreductases↗
PubMed1991

Identification of the DNA region responsible for sulfur-oxidizing ability of Thiosphaera pantotropha.

For the identification of the DNA region responsible for the sulfur-oxidizing ability (Sox) of Thiosphaera pantotropha, we used previously isolated Tn5-mob insertional Sox- mutants. For seven mutants, the Tn5-mob insertion was localized on the chromosome rather than on the megaplasmids pHG41 or pHG42 by using the Tn5-mob-harboring vehicle pSUP5011 as probe. The specific insertion of Tn5-mob into a sox gene was determined for one Sox- mutant, strain TP19. An 18-kb EcoRI fragment was cloned in Escherichia coli by using the mobilizable plasmid pSUP202 as vector and the kanamycin resistance gene of Tn5 as marker. Conjugal transfer of the resulting hybrid plasmid, pKS3-13, to the wild type resulted in two phenotypically different groups of recombinants. Ninety-five percent of the recombinants were Sox+, kanamycin resistant, and tetracycline resistant; 5% were homogenote recombinants exhibiting the Sox-, kanamycin-resistant, tetracycline-sensitive phenotype, and these indicated the specific insertion. To isolate the respective wild-type sox gene, total DNA from a heterogenote recombinant was partially restricted with EcoRI, religated, and transformed in E. coli. Transformants carrying a pSUP202-derived hybrid plasmid with the intact sox gene were identified by screening for a tetracycline-resistant, kanamycin-sensitive, and chloramphenicol-sensitive phenotype and by complementation of the Sox- mutant TP19. A plasmid of this type, pEG12, contained an insert of 13 kb which gave a positive signal in Southern hybridization with the homologous probe of pKS3-13. pEG12 was used to determine the DNA homology of the sulfur-oxidizing enzyme systems of other thiobacteria. Strong hybridization signals were obtained with total DNA of the neutrophilic sulfur-oxidizing bacteria Paracoccus denitrificans, Thiobacillus versutus, and Rhodobacter capsulatus. No hybridization signal was obtained with DNA of other neutrophilic or acidophilic thiobacteria examined.

Blotting, Southern↗
PubMed1987

Novel Leu-Lys-specific peptidase (Leulysin) produced by gel-entrapped yeast cells.

Saccharomyces cerevisiae cells entrapped with a neutral hydrophilic photo-crosslinkable resin prepolymers specifically excreted into a cultured medium a new type of a peptidase, which cleaved Leu-Lys bond of alpha-mating factor. The enzyme was purified by membrane filtration followed by ion exchange chromatography and gel filtration chromatography. The purified enzyme showed a strict substrate specificity on internal Leu-Lys bond. Leu-Lys bond near terminus of a molecule and Leu-X and X-Lys bonds examined so far were not hydrolyzed by the enzyme.

Amino Acid Sequence↗
PubMed1983

11beta-Hydroxylation of Cortexolone (Reichstein Compound S) to Hydrocortisone by Curvularia lunata Entrapped in Photo-Cross-Linked Resin Gels.

Spores of Curvularia lunata were immobilized by entrapment with photo-cross-linkable resin prepolymers and incubated to form mycelium in potato dextrose broth containing cortexolone (Reichstein compound S) as an inducer of steroid 11beta-hydroxylase. In a buffer system containing 2.5% dimethyl sulfoxide, this immobilized mycelium hydroxylated cortexolone to hydrocortisone. The activity of this mycelium was comparable to the activity of free mycelium. Dimethyl sulfoxide did not inhibit hydroxylase activity at the concentration used and was effective in dissolving the product. Of the various photo-cross-linkable resin prepolymers examined, use of ENT-4000, whose main chain was polyethylene glycol 4000 (chain length, approximately 40 nm), resulted in maximum hydroxylation activity of the entrapped mycelium. The chain length of prepolymers affected markedly mycelial growth in the gels and, subsequently, the activity of the entrapped mycelium. The immobilized hydroxylation system was more stable than the system in free mycelium and could be reactivated by incubation of the entrapped mycelium in potato dextrose broth containing cortexolone. The system was tested 50 times during 100 days of operation and was found to carry out the desired transformation with overall yields of 60%.

Journal Article↗