[Pediatric and social aspects of the child with a chronic disease. Round table].
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Age Factors↗
Biomedical subjects
J M Luengo
Publications and source records attributed to J M Luengo.
Cyclization of phenylacetyl-L-cysteinyl-D-valine to benzylpenicillin using cell-free extracts of Streptomyces clavuligerus.
Benzylpenicillin, a typical antibiotic produced by some species of fungi, was obtained by direct cyclization of the heteropeptide phenylacetyl-L-cysteinyl-D-valine using cell-free extracts of Streptomyces clavuligerus. This is the first description of evidence of the synthesis of benzylpenicillin from a non natural molecule using a bacterial enzyme.
Cell-Free System↗
Direct evaluation of phenylacetyl-CoA: 6-aminopenicillanic acid acyltransferase of Penicillium chrysogenum by bioassay.
The enzyme phenylacetyl-CoA: 6-Aminopenicillanic acid acyltransferase of Penicillium chrysogenum was evaluated by direct bioassay against Micrococcus luteus ATCC 9341. The enzyme required dithiothreitol, was inactivated by 0.2 mM Hg2+ (100%), Zn2+ (80%), Cu2+ (60%), 1 mM N-ethylmaleimide (80%), and showed maximal catalytic activity at pH 8.4 and 20 degrees C. The V50 values for phenylacetyl-CoA and 6-aminopenicillanic acid were 0.55 mM and 1 microM, respectively. When octanoyl-CoA was employed as substrate similar results were obtained. In both cases the product generated showed strong antibacterial activity which was quickly lost when incubation was carried out with beta-lactamase. Reactions performed in the presence of Escherichia coli penicillin acylase did not generated active products when phenylacetyl-CoA was the substrate; they did with octanoyl-CoA. Time-course experiments revealed that the highest enzyme levels are found in 36 hours mycelium and remained almost constant from 48 to 96 hours; thereafter the level of the enzyme slowly decreased.
Acetyl Coenzyme A↗
Direct enzymatic synthesis of natural penicillins using phenylacetyl-CoA: 6-APA phenylacetyl transferase of Penicillium chrysogenum: minimal and maximal side chain length requirements.
In vitro synthesis of different natural penicillins (hexanoyl, heptanoyl and octanoyl-penicillin) have been carried out by direct acylation of 6-aminopenicillanic acid (6-APA) with several fatty acid-CoA derivatives (hexanoyl-CoA, heptanoyl-CoA and octanoyl-CoA). The reactions were catalyzed by the enzyme Acyl-CoA: 6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum AS-P-78. This enzyme only accepts as substrate, aliphatic side chain precursors whose carbon length is between 6 and 8 atoms. Although the enzymatic synthesis of octanoylpenicillin has been previously reported the in vitro synthesis of hexanoyl and heptanoyl penicillins is described here for the first time.
Acyltransferases↗
Precipitation of phenyl and phenoxypenicillin from solutions using ammonium sulfate.
An easy, rapid, and available method for separating 6-aminopenicillanic acid (6-APA), benzylpenicillin (penicillin G), and other related molecules from aqueous solutions or complex industrial broths is described. A high concentration of ammonium sulphate induces partially or totally the precipitation of the penicillin present in the solutions, while 6-APA, phenylacetic, and phenoxyacetic acid always remain in the supernatant. The filtration through No. 4 Pyrex glass-fiber filter or Whatman 3MM paper permits the separation of the compounds present in the supernatant from the other ones precipitated. The precipitated product was identified, in all cases, as ammonium penicillin. This method is described here for the first time.
Ammonium Sulfate↗
Carbon catabolite repression of penicillin biosynthesis by Penicillium chrysogenum.
The addition of glucose to batch cultures of Penicillium chrysogenum AS-P-78 reduced the biosynthesis of penicillin. This regulatory effect was also observed in penicillin biosynthesis by nitrogen-limited resting cells when cultures were previously grown in high concentrations of glucose. The effect of glucose was concentration-dependent in the range of 28-140 mM. Incorporation of L-[U-14C]valine into penicillin in nitrogen-limited resting cultures was reduced by 70% when cells were grown on 140 mM glucose, as compared with that grown on lactose. It was not affected when the sugar was added to the resting cell system, in which penicillin biosynthesis took place without growth. Fructose, galactose and sucrose exerted the regulatory effect to the same extent as glucose (64 to 70%). Lactose did not exert suppression of penicillin biosynthesis. Penicillin-synthesizing activity in control cultures with lactose reached a peak at 24 hours of incubation and decreased slowly thereafter, as studied with resting cell cultures in which further protein synthesis was blocked with cycloheximide. Glucose repressed the formation of penicillin-synthesizing enzymes, but had no effect on the activity of these enzymes. These results suggest that glucose represses but does not inhibit penicillin biosynthesis.
Glucose↗
Inhibition and repression of homocitrate synthase by lysine in Penicillium chrysogenum.
Homocitrate synthase in the first enzyme of the lysine biosynthetic pathway. It is feedback regulated by L-lysine. Lysine decreases the biosynthesis of penicillin (determined by the incorporation of [14C]valine into penicillin) by inhibiting and repressing homocitrate synthase, thereby depriving the cell of alpha-aminoadipic acid, a precursor of penicillin. Lysine feedback inhibited in vivo the biosynthesis and excretion of homocitrate by a lysine auxotroph, L2, blocked in the lysine pathway after homocitrate. Neither penicillin nor 6-aminopenicillanic acid exerted any effect at the homocitrate synthase level. The molecular mechanism of lysine feedback regulation in Penicillium chrysogenum involved both inhibition of homocitrate synthase activity and repression of its synthesis. In vitro studies indicated that L-lysine feedback inhibits and represses homocitrate synthase both in low- and high-penicillin-producing strains. Inhibition of homocitrate synthase activity by lysine was observed in cells in which protein synthesis was arrested with cycloheximide. Maximum homocitrate synthase activity in cultures of P. chrysogenum AS-P-78 was found at 48 h, coinciding with the phase of high rate of penicillin biosynthesis.
Cycloheximide↗
Lysine regulation of penicillin biosynthesis in low-producing and industrial strains of Penicillium chrysogenum.
The inhibitory effect of L-lysine on penicillin biosynthesis by Penicillium chrysogenum has been compared in a low-producing strain (Wis. 54-1255) and a high-producing strain (ASP-78). Lysine inhibited total penicillin synthesis to a similar extent in both strains. However, in the high-producing strain the onset of penicillin synthesis occurred even at a high lysine concentration, whereas in the low-producing strain lysine had to be depleted before penicillin production commenced.
Culture Media↗