Biomedical subjects

T Himeno

Publications and source records attributed to T Himeno.

21 records · Page 2Linked to original sources

PubMed1985

Effect of in vitro DNA rearrangement in the NH2-terminal region of the penicillinase gene from Bacillus licheniformis on the mode of expression in Bacillus subtilis.

We have constructed secretion vector plasmids that have unique BglII sites within or near the signal sequence of Bacillus licheniformis penicillinase, and have also constructed penicillinase cartridges that lack either one, two or three of the processing sites for the membrane-bound, exo-large and exo-small enzymes. Each of these penicillinase cartridges was cloned on secretion vectors in Bacillus subtilis, and enzyme production was examined. The presence of both the signal sequence and the three host-specific processing sites on the secretion vector was required for an effective expression of the enzyme in B. subtilis. The presence of any of the processing sites on the cartridge reduced the accumulation of penicillinase in the culture medium. When a vector plasmid lacking part of the hydrophobic region of the signal sequence and lacking the three processing sites was used, total penicillinase production decreased and enzyme accumulation in the medium was extremely low, despite the complete or incomplete presence of the processing sites on the cartridge. Molecular mass determination of these extracellular penicillinases suggested the existence of a new cleavage site for the enzyme.

Amino Acid Sequence↗

PubMed1984

Characterization of Saccharomycopsis lipolytica mutants that express temperature-sensitive synthesis of isocitrate lyase.

Four mutants specifically deficient in the activity of isocitrate lyase were independently isolated in the alkane yeast Saccharomycopsis lipolytica. Genetic analysis by means of protoplast fusion and mitotic haploidization revealed that the mutations were recessive and non-complementary at a single genetic locus, icl. icl is a structural gene for isocitrate lyase, because some revertants from icl-1 and icl-3 mutants produced thermolabile isocitrate lyase in comparison with the wild-type enzyme, and also because the gene dosage effect was observed on the specific activity of isocitrate lyase in icl+/icl-1 and icl+/icl-3 heterozygotes. The icl-3 mutation also gave rise to temperature-sensitive revertants that could grow on acetate at 23 degrees C but not at 33 degrees C, exhibiting temperature-sensitive synthesis as well as thermostable activity of isocitrate lyase. Studies on purified isocitrate lyase showed that this enzyme is tetrameric and that the enzyme synthesized at 23 degrees C by a temperature-sensitive synthesis mutant was indistinguishable from the wild-type enzyme with respect to the subunit molecular weight (59,000), the isoelectric pH (5.3), the thermostability, and the Km value for threo-Ds-isocitrate (0.2 mM). When induced by acetate at 33 degrees C, the temperature-sensitive synthesis mutant did not express isocitrate lyase activity but did synthesize polypeptides whose electrophoretic mobilities were equal to that of the purified mutant enzyme. Hence, the temperature-sensitive mutation assumed in the structural gene for isocitrate lyase might have prevented the maturation of the polypeptide chains synthesized at the restrictive temperature.

Acetates↗

PubMed1983

Comparative studies on extracellular penicillinases of the same structural gene, penP, expressed in Bacillus licheniformis and Bacillus subtilis.

Extracellular penicillinases produced by Bacillus licheniformis ATCC 9945A and Bacillus subtilis from the same structural gene, penP, were compared. The two strains secreted the same exo-large penicillinase (mol. wt, 305000; isoelectric point, pI = 5.00-5.04; NH2-terminal amino acid, Ser). In contrast, the exo-small enzyme from Bacillus subtilis (mol. wt, 29500; pI = 5.00-5.04; NH2-terminal amino acid, Glu or Asn) was slightly different from that of Bacillus licheniformis (mol. wt, 29500; pI = 5.13; NH2-terminal amino acid, Lys). The difference in the NH2-terminal residue is most probably due to differences in degradation by host-specific proteolytic enzymes.

Amino Acid Sequence↗