[Antibiotic production by variants of Penicillium chrysogenum isolated after protoplast formation and exposure to mutagens at the protoplast stage]. / Antibiotikoobrazovanie u variantov Penicillium chrysogenum, poluchennykh posle protoplastirovaniia i obrabotki mutagenami na stadii protoplastov.

A procedure for protoplast formation in the penicillin-producing organism Penicillium chrysogenum was developed. The yield of the protoplasts was high, the protoplasts were stable and capable of regeneration. Two types of the protoplast regeneration were revealed. The spores and protoplasts were treated with UV light and N-nitroso-N'-methyl biuret and their effect on production of the antibiotic by the isolated variants was studied. It was shown that the protoplasts of P. chrysogenum were more liable to the mutagenic effect of UV light and nitroso methyl biuret than the fungus conidia. It is possible to use this specific feature in intensification of selection aimed at isolation of highly productive strains of P. chrysogenum.

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.

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.

Synthesis of benzylpenicillin by cell-free extracts from Streptomyces clavuligerus.

Cell-free enzyme concentrates from Streptomyces clavuligerus were found to convert phenylacetyl-L-cysteinyl-D-valine (PCV) directly into benzylpenicillin when incubated under reaction conditions which support the activity of isopenicillin N synthetase. The formation of benzylpenicillin was detected both by biological assay and by high performance liquid chromatography. Supplementation of PCV-containing reaction mixtures with cofactors required for ring expansion activity did not result in the production of cephalosporins. Incubation of phenoxyacetyl-L-cysteinyl-D-valine (PoCV) under the same reaction conditions did not result in the formation of penicillin V or any cephalosporin product.

Kinetic studies on the mechanism of the penicillin amidase-catalysed synthesis of ampicillin and benzylpenicillin.

Hydrophobic protein chromatography was used to prepare homogeneous fractions of penicillin amidase (EC 3.5.1.11) from E. coli. The apparent ratios of the rate constants for the deacylation of the acyl-penicillin amidase formed in the hydrolysis of phenylacetylglycine or D-phenylglycine methyl ester, by H2O and 6-aminopenicillanic acid (6-APA), were determined at different concentrations of the latter compound. The ratios were obtained from direct measurements of the initial rates of formation of phenylacetic acid and benzylpenicillin or D-phenylglycine and ampicillin. For the semisynthesis of ampicillin as well as of benzylpenicillin the ratio was found to depend on the concentration of 6-APA. This was observed for heterogeneous and homogeneous enzyme preparations. These results show that 6-APA must be bound to the acyl-enzyme before the deacylation, yielding ampicillin and benzylpenicillin, occurs. The dissociation constant KN for the formation of the complex was estimated to be approximately 10mM. This mechanism in which acyl-enzyme with and without bound nucleophile is involved, is in agreement with the principle of microscopic reversibility. Both acyl-enzymes can be deacylated by H2O. The finding that there is a specific binding site for 6-APA adjacent to the binding site for the phenylacetyl-(D-phenylglycyl-) group in the active site of the enzyme is supported by the observation that 6-APA acts as a mixed inhibitor in the hydrolysis of D-phenylglycine methyl ester. The ionic strength dependence indicates that the binding site for 6-APA of the acyl-enzyme is positively charged.

[Mass exchange conditions in penicillin biosynthesis in an industrial fermenter with aerodynamic foam suppressor]. / Izuchenie uslovii massobmena pri biosinteze penitsillina v promyshlennom fermentatore s aérodinamicheskim penogasitelem.

The mass exchange characteristics of 50 m3 industrial fermentors with aerodynamic foam suppression and the effect of the specific power input on biosynthesis of penicillin were studied. A change in the specific power input from 1.3 to 1.9 kW/m3 had no effect on the level of the antibiotic accumulation when the medium with 8 per cent of lactose was used. An increase in the aeration rate from 1 to 1.2 m3/m3 X min provided a 1.1-fold increase in the penicillin activity of the fermentation broth. The use of the device for aerodynamic foam suppression with a system of automatic control of the partial pressure of dissolved carbon dioxide allowed decreasing 1.22-fold the oil use and increasing the process productivity by 10 per cent.

Penicillin production: biotechnology at its best.

A brief description is given of the history of penicillin production in the Netherlands. The development of today's penicillin production technology is analysed in terms of changes in the quality and intensity of the production process. Technological as well as genetical developments are shown to be of influence on the quality and the intensity of the production process. The analysis is illustrated by a brief description of the productivity improvement of the penicillin fermentation as it occurred at Gist-brocades during the past 20 years.

Simple assay and extraction of periplasmic penicillinase in Escherichia coli.

Benzylpenicillin was clearly separated from benzylpenicilloic acid by ascending chromatography on a diethylaminoethyl cellulose paper using 0.1 M ammonium acetate as a solvent. Using this chromatographic system, penicillinase was assayed by measuring the formation of [14C]benzylpenicilloic acid from [14C]benzylpenicillin. This assay remedies the lack of specificity of the commonly used iodometric assays. Periplasmic penicillinase was released from Escherichia coli by suspension in a mixture of 1% phenethyl alcohol and 5 mM ethylenediaminetetraacetate (pH 7.0). This simple extraction method not only facilitates the assay of penicillinase in an E. coli culture, but will also be useful for large-scale purification of periplasmic penicillinase.

[Express method of determining phenylacetic acid in the culture broth during the biosynthesis of benzylpenicillin]. / Ekspress-metod opredeleniia feniluksusnoi kisloty v kul'tural'noi zhidkosti pri biosinteze benzilpenitsillina.

Phenylacetate acid (PAA) is transferred by extraction from the fermentation broth filtrate into toluol. The extract is applied to a Silufol plate with an aluminium foil lining (silica gel sorbent, Czechoslovakia). Reference solutions of PAA are also applied to the same plate. The reference and test solutions are applied dropwise (spots of 5--6 x 10(-3)m in diameter). For PAA development the spots are sprayed with a freshly prepared saturated solution of potassium manganate in 6N H2SO4. PAA of the test samples is developed as a dull ring against grey background and that of the reference solution is developed as a circle. The amount of PAA in the spot is determined by using correlation between the spot area and the amount of PAA applied. One plate of 225 X 10(-4) m2 can be used for about 300 analyses. One analysis takes 300--600 seconds.

The ultrastructure of Penicillium chrysogenum in the course of benzyl-penicillin biosynthesis.

The find structures of high- and low-yield mutants of Penicillium chrysogenum, producing 100 and 10,000 units/ml of penicillin G, were compared. The cells of both mutants demonstrated a typical eukaryotic ultrastructure. In the cytoplasm nuclei, mitochondira, lipid bodies, endoplasmic reticulum, and Golgi vesicles were observed. In the cells of high-yield mutant, during the biosynthesis of penicillin, the number of lipid bodies decreased. It is possible that the lipids are metabolized in the process of biosynthesis of penicillin. In the cytoplasm more multivesicular bodies and small vesicles, about 40 nm in diameter, could be seen. These Golgi vesicles, present in largest number in cells of high-yield mutant, fuse with the cell membrane and play an important role in the transport of penicillin from the cytoplasm to the cell environment. The cell walls of the high-yield mutant become three times thicker during the antibiotic biosynthesis. No comparable changes were observed in the ultrastructure of the low-yield mutant. The cell wall thickness did not increase, the cytoplasm contained few Golgi vesicles only, and the lipid bodies can be seen in all cells.

Uptake and metabolism of alpha-aminoadipic acid by Penicillium chrysogenum Wis 54-1255.

The uptake of 1-14C-DL-alpha-aminoadipate in resting mycelium of Penicillium chrysogenum Wis 54-1255 and its metabolism during benzylpenicillin formation were studied. The pH optimum for uptake at 25 degrees C was 6.4. Over a range of concentrations from 0.01--1.0 mM, approximately 45% of 1-14C-DL-alpha-aminoadipate was taken up by carbon-starved mycelium. 14CO2 was formed at a low rate, and the total formed amounted to only 1--3% of the 1-14C-DL-alpha-aminoadipate supplied. The intracellular pool of alpha-aminoadipate appears to be expandable, depending on the concentration of alpha-aminoadipate in the medium. The rate of penicillin synthesis depended on the intracellular concentration of alpha-aminoadipate. Penicillin biosynthesis achieved half of the maximum rate at an intracellular concentration of 0.06 nmol alpha-aminoadipate/mg dry cell weight. This low concentration, the result of adding 0.01 mM DL-alpha-aminoadipate to the medium, was sufficient to reverse the inhibition of penicillin biosynthesis caused by 10 mM extracellular L-lysine. Aminoadipate appears to be recycled during penicillin formation. Labeled alpha-ketoadipate was formed from alpha-aminoadipate to the extent of about 25%.

Theoretical conversion yields for penicillin synthesis.

The efficiency of conversion of the carbon-energy source to product is of primary importance in many fermentation processes. In order to assess the efficiency of a process, one must know how close the actual conversion yield is to the theoretical maximum. Theoretical conversion yields are useful, therefore, as guides in improving a process. This knowledge is particularly important today because the cost of raw materials is rapidly rising. In this study, the biochemical pathway of penicillin synthesis was used to estimate the theoretical yield of penicillin from glucose, ammonia, and sulfate. These values are compared with experimental data from the literature. An analysis of the role of glucose in the synthesis of cell mass and penicillin and in the maintenance of cells makes it possible to assess the efficiency of carbon-source utilization and to direct further advances in penicillin fermentations.

New method of intermittent feeding in penicillin biosynthesis.

Under laboratory conditions, penicillin biosynthesis was improved by feeding sucrose and sodium phenylacetate to the fermentation flasks in the form of coated tablets.