[The immobilization of enzyme-membrane complexes--a new approach to creating highly efficient biocatalysts].
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Biomedical subjects
Publications and source records attributed to S M Navashin.
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Sensitivity of different species of Lactobacillus i.e. L. casei, L. plantarum, L. acidophillus, L. buchneri, L. jugurti and others to penicillins and cephalosporins of various generations was studied. Penicillin binding proteins (PBPs) of the Lactobacillus species were specified. It was shown that the number of PBPs depended on the Lactobacillus species. L. casei had the least number of PBPs (4) and L. brevis had the highest number of PBPs (11). Competition of 14C-benzylpenicillin with ampicillin, cefotaxime, ceftizoxime and cefoperazone for binding to separate PBPs in three strains of different Lactobacillus species was investigated.
A system providing a high frequency genetic transfer of various markers making the reference strain Lactobacillus buchneri 1837 resistant to Lm, Em and Fus, able to ferment some carbohydrates and antagonistic against Pseudomonas diminuta CCM 2657 was developed. The frequency of the marker transfer during the lactobacilli joint cultivation was 1.5 X 10(-5) = 5.5 X 10(-5) to 1.5 X 10(-4) = 5.5 X 10(-4) which was 3-4 orders of magnitude higher than the marker transfer frequency during transformation of L. buchneri NRRLB 1837 with plasmid DNA of different lactobacilli. The recombinants and transformants resulting from the joint cultivation of lactobacilli contained a plasmid about 60 kb in length which provided cells of L. buchneri NRRLB 1837 with resistance to fusidin, antagonistic activity against Pseudomonas diminuta and capacity for utilizing sucrose and sorbite. It was shown possible to integrate the plasmid DNA about 26.5 kb in length contained in the cells of L. casei MT 205 to the chromosomes of erythromycin resistant transformants. The results of the investigation of the biological properties of the recombinants and transformants and their plasmid profiles were confirmed with the Southern DNA-DNA hybridization.
The erythromycin resistance gene (ermE) and part of erythromycin biosynthesis genes located in the same cluster with the ermE gene were cloned from S. erythraeus 3 subjected to improvement with respect to erythromycin production. For isolating the erythromycin biosynthesis genes, the plasmid vector pUC18 and the phage vector lambda EMBL3 were used. The ermE gene DNA was used as a labeled probe for analysis of the recombinant plasmids and phages. The recombinant phages lambda ermE1 and ermE4 containing fragments of the chromosomal DNA collinear to the genome DNA of S. erythraeus 3 were analyzed. The size of the cloned fragment of the chromosomal DNA of S. erythraeus 3 was about 20 kb. Subcloning with the vector pUS18 resulted in isolation of plasmids pSU235-pSU244 containing BamHI fragments of chromosomal DNA from S. erythraeus 3. The restriction map of the chromosomal region of S. erythraeus 3 containing the ermE gene was constructed. The cloned genes of erythromycin biosynthesis are useful in the study of their structure and functions, construction of integrative vectors, improvement of cultures producing macrolide antibiotics and isolation of genes responsible for biosynthesis of other polyketide antibiotics.
The contents of high-energy phosphorous compounds, i.e. three fractions of polyphosphates, pyrophosphate, and ATP were determined in isogenic strains of Tolypocladium sp. differing in cyclosporine production levels. The content of polyphosphates was 1 to 2 orders of magnitude greater than that of pyrophosphate or ATP and did not depend on the strain productivity. During the period of the mycelial intensive growth and at the beginning of antibiotic synthesis, the level of polyphosphates lowered 2-3-fold and the content of pyrophosphate markedly decreased as well. The activities of polyphosphatase and pyrophosphatase during the culture growth and cyclosporine biosynthesis was higher in the highly productive strain.
An attempt was made to show a correlation between definite bioenergetic parameters of the cells of the cyclosporine-producing culture and biosynthesis of cyclosporine. It was found that the three strains producing cyclosporine used in the study had an alternative cyanide-resistant pathway along with the classical cytochrome chain. In the strain forming only traces of the cyclopeptide during fermentation of the cyanide-resistant respiration constituted 60 to 80%. In the isogenic highly productive strains the cyanide-resistant respiration appeared to be markedly decreased beginning from the 1st day of fermentation and during the maximum biosynthesis of cyclosporine (on day 4 or 5 of fermentation) it reached zero. The ATP content in the cells of the highly productive strain, despite its decrease by the antibiotic biosynthesis peak, remained at a much higher level than that in the strain producing only traces of cyclosporine. A procedure for isolating functionally active mitochondria from the protoplasts was developed and a bioenergetic characterization of the mitochondria isolated from the strains with different antibiotic productions is presented.
A bireplicone plasmid pSU901,4.6 kb in length, was constructed on the basis of plasmid pUC19 and the pstIB fragment, 1.9 kb in length, from mitochondrial DNA of A. chrysogenum. Based on the hybrid plasmid pSU901 and kanamycin resistance determinant, an autonomically replicating vector for A. chrysogenum, a culture producing cephalosporin C, is being constructed.
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Circular mDNAs 26.85 and 26.94 kb in length were isolated from two isogenic strains of A. chrysogenum producing cephalosporin C. The strains differed in antibiotic production capacity. Restriction analysis of the mDNAs was performed with using 6 endonucleases. Comparison of the restriction data revealed identity of mDNAs. A restriction map of the mDNAs was constructed. It is useful as a basis for further studies with molecular cloning.
The general principles of in-vitro simulation of drug pharmacokinetic profiles for linear one-, two- and multi-compartment models are described. An in-vitro dynamic model constructed on the basis of these, incorporating a novel filtration unit to provide efficient filtration of drugs at constant inoculum size, was used to study the antimicrobial action of sisomicin on Escherichia coli A 20363, in conditions simulating the pharmacokinetic profile observed in humans after a single intramuscular dose of 1 mg/kg.
It was shown morphologically and microbiologically that cephalexin administered intragastrically in a dose of 2 g/kg induced deep microecological changes in the cecum of Wistar albino rats. Scanning electron microscopy revealed that a single use of cephalexin in the above dose resulted in almost complete elimination of the biofilm from the surface of the cecum mucosa and it inflammation. This was accompanied by an increase in the number of potentially pathogenic bacteria and broadening of the spectrum of the phage variants of coliform bacilli present in the animal feces, as well as by change the periods of coliform bacilli persisting in the large intestine.
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To investigate the evolutionary relationships between the aph(3') genes from different plasmids, the nucleotide sequence of the aph(3') gene from the E. coli R plasmid was determined and compared with the known aph(3') genes of Tn903 and Tn4352. Three point mutations in the structural part of the cloned aph(3') gene caused amino acid changes in the enzyme molecule at positions 19, 27 and 48 beginning from the start codon. The structural part of the gene was followed by two stop codons and a long DNA region containing no nucleotide sequences homologous to the sequences of Tn903 or Tn4352. Both the cloned aph(3') gene and Tn4352 were limited on the left by the spacer sequence and the insertion sequence IS176. Twenty one base pairs deletion abolished the -35 sequence of the promoter suggested for the aph(3') gene of Tn4352 and resulted in formation of a fusion promoter utilizing the -35 box of IS176 and the -10 box of the aph(3') gene. The distance between the -35 and -10 sequences changed from 18 to 17 bp. Changes in the cloned aph(3') gene and the flanking DNA regions resulted in formation of a new promoter and loss of the right IS176 element.
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