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Biomedical subjects

A L Demain

Publications and source records attributed to A L Demain.

At least 91 records · Page 5Linked to original sources
PubMed1990

Achievements in microbial technology.

In 1973, recombinant DNA technology was born and the age of the "new biotechnology" came upon us. Today we are seeing the amazing results of recombinant DNA technology, hybridoma technology, enzyme engineering and protein engineering. These techniques are exerting major effects on basic research and on health care, diagnostics and agriculture and soon will bring about changes in other industries such as petroleum, mining, foods and chemicals. Entire pathways of primary and secondary metabolism have been cloned and expressed in foreign microorganisms. The development of recombinant DNA technology is having its major impact on the production of rare polypeptides such as mammalian enzymes, hormones, antibodies and biological response modifiers. In addition to natural polypeptides, analogs are being produced by recombinant DNA technology and this has added an extra dimension of excitement to the field. The future is thus insured for the expanded use of microorganisms in the biotechnological world and the continued improvement in microbial processes to reduce the cost of drugs, enzymes and specialty chemicals.

Journal Article↗
PubMed1990

Subcellulosome preparation with high cellulase activity from Clostridium thermocellum.

We have prepared a much simpler cellulase preparation than that of cellulosomes from the extracellular broth of Clostridium thermocellum. This "subcellulosome" preparation from C. thermocellum was obtained by column chromatography on CM-Bio-Gel A and then on a lectin-affinity material (Jacalin). The subcellulosome preparation is a macromolecular complex, composed of six main protein subunits (molecular weight, 210,000 to 58,000) revealed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activities of carboxymethylcellulase (CMCase) and Avicelase are 15- and 8-fold-higher, respectively, than those of crude extracellular cellulase. We could not further fractionate this preparation without denaturing it. The optimum pH and temperature of the subcellulosome preparation are 5.5 to 7.0 and 70 degrees C for CMCase and 5.5 to 7.0 and 65 degrees C for Avicelase. The subcellulosome preparation acted on various types of carboxymethyl cellulose, cellulose, and p-nitrophenyl-beta-D-cellobioside but not on p-nitrophenyl-beta-D-glucoside. Sulfhydryl reagents and N-bromosuccinimide inhibited both CMCase and Avicelase activities, whereas EDTA and o-phenanthroline inhibited Avicelase activity only.

Bacterial Proteins↗
PubMed1990

Rapid microbial detection and enumeration using gel microdroplets and colorimetric or fluorescence indicator systems.

A new micromethod employing gel microdroplets (GMDs) and optical measurements can be used for rapid detection and enumeration of viable microorganisms (J. C. Weaver, G. B. Williams, A. M. Klibanov, and A. L. Demain, Bio/Technology 6:1084-1089, 1988) and has several potential applications in clinical microbiology. This method involves entrapping microorganisms in GMDs (10 to 100 microns in diameter) which are surrounded by a hydrophobic (low dielectric) fluid, subsequently distinguishing occupied and unoccupied GMDs with colorimetric or fluorescence indicators, counting both occupied and unoccupied GMDs, and applying Poisson statistical analysis. Acid-producing microorganisms were used to compare colorimetric and fluorescence pH indicator systems. Fluorescence systems were generally superior, particularly for detection before microbial growth occurred. Although colorimetric detection was reasonably fast for fast-growing microorganisms, significantly longer times were needed for slow-growing microorganisms. We investigated the dependence of the detection time on microbial division time, GMD size, and buffering capacity of the medium within GMDs. It was found possible to use a single preparation of GMDs, containing a range of GMD sizes, to simultaneously provide a viable enumeration of growing and nongrowing (e.g., stressed) cells. This was possible because small GMDs responded rapidly to both growing and nongrowing cells, while large GMDs, although slower, responded much more rapidly to growing cells than to nongrowing cells. Separate analysis of small and large GMDs in the same preparation yielded two enumerations, one of nongrowing cells and the other of growing cells. GMDs can also be used with conventional light microscopy to detect and enumerate fast-growing acid-producing bacteria much more quickly than conventional plating methods.

Bacteria↗
PubMed1990

Regulation of ACV synthetase: biosynthesis and action.

The biosynthesis of cephalosporins has been studied for almost 30 years. Development of cell-free systems began with the later enzymes of the pathway and, in recent years, moved to the early part of the pathway. The first enzyme, delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase was one of most difficult to demonstrate in cell-free extracts. We recently developed a reproducible system from Cephalosporium acremonium (Banko et al., 1987) and similar activity has been found in Streptomyces clavuligerus (Jensen et al., 1988). With this assay, we have been able to study the regulation of ACV synthetase formation and action. Our results are summarized in this communication. Our comparison of the intracellular specific activities of the beta-lactam synthetases in both C. acremonium c-10 and S. clavuligerus NRRL 3585 indicates that ACV synthetase possesses the lowest activity and appears to be the rate-limiting step in the cephalosporin biosynthetic process. Since it is the initial enzyme in the biosynthetic pathway common to both penicillins and cephalosporins, this makes sense from a cellular economy viewpoint. It is therefore understandable that ACV synthetase would be subject to greater regulatory control than other steps.

Acremonium↗
PubMed1989

Solid state fermentation for cephalosporin production by Streptomyces clavuligerus and Cephalosporium acremonium.

Solid state fermentation systems were developed for the production of cephalosporins with Streptomyces clavuligerus and Cephalosporium acremonium. S. clavuligerus NRRL 3585 was grown on moistened barley under optimum solid state fermentation conditions for 7 days; approximately 300 micrograms cephalosporins per g substrate were extracted from the kernels. C. acremonium C-10 produced approximately 950 micrograms cephalosporin C per g substrate after 10 days of solid state fermentation.

Acremonium↗
PubMed1989

Addition of cloned beta-glucosidase enhances the degradation of crystalline cellulose by the Clostridium thermocellum cellulose complex.

A thermostable beta-glucosidase from Clostridium thermocellum which is expressed in Escherichia coli was used to determine the substrate specificity of the enzyme. A restriction map of the beta-glucosidase gene cloned in plasmid pALD7 was determined. Addition of the E. coli cell extract (containing the beta-glucosidase) to the cellulase complex from C. thermocellum increased the conversion of crystalline cellulose (Avicel) to glucose. The increase was specifically due to hydrolysis of the accumulated cellobiose. A cellulose degradation process using beta-glucosidase to assist the potent cellulase complex of C. thermocellum, as shown here can open the way for industrial saccharification of cellulose to glucose.

Cellulase↗
PubMed1989

Phosphate regulation of ACV synthetase and cephalosporin biosynthesis in Streptomyces clavuligerus.

Cephalosporin production by Streptomyces clavuligerus was reduced sharply by 60 mM phosphate added to a chemically-defined medium. All the four synthetases in the pathway examined, i.e., ACV synthetase, cyclase, epimerase and expandase, were repressed by phosphate, with ACV synthetase being the main repression target and expandase the next. ACV synthetase activity was inhibited by phosphate to a lesser extent than expandase and cyclase, and this inhibition could be reversed by adding Fe2+. Fe2+ itself was inhibitory to ACV synthetase action.

Cephalosporins↗
PubMed1988

Synthesis of bacilysin by Bacillus subtilis branches from prephenate of the aromatic amino acid pathway.

Previously characterized mutants of Bacillus subtilis 168 were analyzed for the accumulation of the antibiotic bacilysin in culture broths and for the presence of bacilysin synthetase in cell extracts. All aro mutants tested were deficient in bacilysin biosynthesis but had synthetase activity. Mutants with lesions in tyrA and pheA produced normal levels of bacilysin, suggesting that prephenate is the primary metabolic precursor of bacilysin.

Anti-Bacterial Agents↗
PubMed1988

Bacillus subtilis mutant deficient in the ability to produce the dipeptide antibiotic bacilysin: isolation and mapping of the mutation.

A Bacillus subtilis mutant which carries a lesion in a gene specific to the synthesis of the dipeptide antibiotic bacilysin was isolated. A derivative strain in which transposon Tn917 had inserted near the bacilysin lesion was isolated and used as the donor in PBS-1 transduction mapping experiments. The bac-1 locus was mapped between the ctrA and sacA loci, near 90% on the standard B. subtilis 168 chromosome map.

Anti-Bacterial Agents↗
PubMed1987

Efficient plasmid transformation of the beta-lactam producer Streptomyces clavuligerus.

The conditions for optimal formation and regeneration of protoplasts of Streptomyces clavuligerus were established. The optimal temperature for regeneration of protoplasts and for transformation was 26 degrees C in three different regeneration media. The best efficiency of transformation was obtained with 40% polyethylene glycol 1000. The efficiencies of regeneration and transformation increased greatly when protoplasts were obtained from cultures in the early stationary phase of growth. The number of transformants per assay increased linearly with rising concentrations of protoplasts. However, the number of transformants per protoplast decreased at concentrations of protoplasts above 1.5 X 10(9). The total number of transformants rose linearly at increasing plasmid DNA concentrations, but the number of the transformants per microgram of DNA became constant at concentrations above 1 microgram of DNA. Transformation frequencies as high as 5 X 10(5) transformants per microgram of DNA were obtained when plasmid pIJ702 was isolated from S. clavuligerus but not when isolated from Streptomyces lividans.

Anti-Bacterial Agents↗
PubMed1987

Effect of ammonium as nitrogen source on production of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase by Cephalosporium acremonium C-10.

Cephalosporin production by Cephalosporium acremonium strain C-10 was suppressed when the organic nitrogen source (1.2% L-asparagine) was replaced by 1.2% (NH4)2SO4. A higher level of (NH4)2SO4 (3.5%) led to even greater suppression. Ammonium repression was exerted on formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase, together with that of expandase; a lesser effect by ammonium was observed on cyclase production. Inhibition of ACV synthetase activity byammonium was also observed (ca. 50% inhibition at 250 mM NH4+).

Acremonium↗
PubMed1986

Cell-free synthesis of delta-(L-alpha-aminoadipyl)-L-cysteine, the first intermediate of penicillin and cephalosporin biosynthesis.

delta-(L-alpha-aminoadipyl)-L-cysteine synthetase (LL-AC synthetase) activity has been found in extracts of Cephalosporium acremonium C-10. The enzyme extract carries out a linear synthesis of LL-AC from its constituent amino acids for at least 6 hours. The reaction is dependent on active enzyme, time, L-alpha-aminoadipate, L-cysteine, ATP and Mn2+ or Mg2+. The activity is stabilized by glycerol.

Acremonium↗
PubMed1986

Relationship between nitrogen assimilation and cephalosporin synthesis in Streptomyces clavuligerus.

The levels of three enzymes of the beta-lactam antibiotic pathway and overall cephalosporin production were subject to nitrogen source repression in Streptomyces clavuligerus. The specific activities of isopenicillin N synthetase ("cyclase") and deacetoxycephalosporin C synthetase ("expandase") measured during the exponential phase depended on the nitrogen source employed, following a pattern that roughly correlated with the corresponding antibiotic production. The effects on isopenicillin N epimerase ("epimerase") activities were less marked than those on the cyclase and expandase. Production of cephalosporins and enzymatic activities were not related to the growth rate of the cultures. Glutamate, glutamine and alanine inhibited production when added to resting cell systems, while lysine and alpha-aminoadipate were stimulatory. No clear relationship could be drawn between cephalosporin production or beta-lactam synthetase activities and the activities of enzymes of ammonium assimilation (glutamine synthetase, glutamate synthase and alanine dehydrogenase). The intracellular pools of free glutamine, alanine and ammonium were the only ones markedly affected by the nitrogen source in the wild type and mutants, but these amino acids did not seem to play an obvious role as intracellular mediators of nitrogen control.

Alanine Dehydrogenase↗
PubMed1986

A simple and rapid chemical method for the determination of cephalosporins.

A simple and rapid chemical assay for cephalosporins is described. It is a simple modification of the colorimetric determination of penicillins in which the narrow spectrum beta-lactamase (penicillinase) is replaced by a broad spectrum beta-lactamase (cephalosporinase) produced by Enterobacter cloacae. The method can be used for assay of fermentation broths as well as for pure cephalosporins.

Cephalosporinase↗
PubMed1986

Studies on in vivo inactivation of gramicidin S synthetase and its retardation.

The oxygen-dependent in vivo inactivation of gramicidin S synthetase was investigated in Bacillus brevis ATCC 9999. Inhibitors of energy metabolism and of protein synthesis added to aerated cell suspensions did not provide any protection against inactivation, thus indicating that the process does not depend on energy-yielding metabolism nor on de novo protein synthesis. Organic thiols added to anaerobic long-term incubations retarded synthetase inactivation for several hours, whereas in short-term incubations of previously air-exposed cells they resulted in partial restoration of activity. The in vivo inactivation of the enzyme was found to be accompanied by a parallel drop in intracellular thiols. These results implicate enzyme SH oxidation as a mechanism of in vivo inactivation. Retardation of inactivation was achieved upon addition of utilizable carbon sources (glycerol, fructose, inositol) to aerated cell suspensions in buffer, the degree of stabilization being proportional to the ease of uptake and to the concentration of C source. This effect involves actual consumption of the exogenous C source and is accompanied by lower dissolved oxygen levels in the cell suspension. Pulsed additions of C source could retard inactivation but could not restore partly or fully lost activity. The C-source effect was blocked by the uncoupler dinitrophenol, while dissolved oxygen levels in the suspension remained low. C-source-supplemented cell suspensions incubated under air had a decreased intracellular redox state, as revealed by intracellular SH concentration.

2,4-Dinitrophenol↗
PubMed1985

Repression and inhibition of cephalosporin synthetases in Streptomyces clavuligerus by inorganic phosphate.

Cephalosporin production by growing cells of Streptomyces clavuligerus was reduced by 100 mM inorganic phosphate. Resting cell production was repressed by prior growth in high phosphate and inhibited by phosphate. The cell-free activity of desacetoxycephalosporin C synthetase (ring expansion activity) was repressed by prior growth in high phosphate and inhibited by phosphate. Isopenicillin N synthetase (cyclase) was inhibited but not repressed. Penicillin epimerase was neither inhibited nor repressed by phosphate.

Cephalosporins↗