A potentiometric rapid method of determining the level of phenylacetic acid during the production of beta-lactam antibiotics

A potentiometric rapid method for control of phenylacetic acid (PAA) concentration in production of ++beta-lactam antibiotics is described. The method is based on ion selective electrodes with a film membrane. The results of the theoretical and experimental studies on estimation of the electrode selectivity specific of PAA in the presence of various interfering ions are presented. It was shown possible to use the electrodes for PAA control in the media containing nitrates, bicarbonates and chlorides. Recommendations how to use the ion selective electrodes at various stages in production of ++beta-lactam antibiotics are given. Prospects for improving the method and designing an instrument for rapid assay of phenyl acetate ion activity are discussed.     

A simple method for hyaluronic acid quantification in culture broth

The carbazole assay has been used for determination of the percentage of hyaluronic acid in biological fluids. However, it is difficult to measure the concentration of hyaluronic acid in culture broth because glucose and polysaccharides remaining after cultures can react with sulfuric acid and carbazole. The glucose and polysaccharide remnants must be completely removed in order to get the correct value for hyaluronic acid. The turbidity assay, another method for estimating the concentration of hyaluronic acid, is based on the formation of insoluble complexes between hyaluronic acid and cetyltrimethylammonium bromide. This method is very easy and fast compared with the carbazole assay. Because concentrations of hyaluronic acid measured by the turbidity assay were ranged around 100% of those measured by the carbazole assay, the content of hyaluronic acid in culture broth can be determined by the turbidity assay. The turbidity method also has the advantage of being safer than the carbazole assay.     

Auxin activity of phenylacetic acid in tissue culture

The ability of phenylacetic acid (PAA), a naturally occurring auxin, to initiate and support growth of callus and suspension cultures of several species is reported. Callus tissue of tobacco (Nicotiana tabacum L. var. WI-38), initiated and maintained on a medium with 2,4-dichlorophenoxyacetic acid (2,4-D), was transferred to and maintained on media supplemented with 25–500 μM PAA as the only plant growth regulator (PGR). Optimal concentrations of PAA were determined for tobacco callus proliferation in the dark (250 μM PAA) and with a 16-h light/8-h dark photoperiod (500 μM PAA). Tobacco suspension cultures were maintained for over 28 transfers in media containing 20–40 μM PAA as the sole PGR. When tobacco callus tissue maintained on PAA-supplemented media for over 18 months was transferred to liquid media containing kinetin, plantlets were regenerated. Callus of sunflower (Helianthus annuus L. var. Russian Mammoth) proliferated on media containing PAA at 5–250 μM as the sole PGR. Similar PAA concentrations inhibited normal development and promoted callus formation in tobacco and pea (Pisum sativum L. vars. common, Frogel, and Frimas) epicotyl tissue. PAA as the sole PGR did not support the growth of soybean (Glycine max (L.) Merrill var. Fiskeby) callus or suspension cultures. Chickpea (Cicer arietinum L. var. UC-5) and lentil (Lens culinaris Medic. var. Laird) callus cultures proliferated on media containing 25–500 μM PAA, but habituation of the cultures was common. PAA was not toxic to tobacco, chickpea, and lentil tissues at levels as high as 500 μM.     

The role of phenylacetic acid in biosynthesis of penicillin amidase in E. coli

For improved biosynthesis of penicillin amidase by E. coli, NCIM 2400, the participation of different carboxylic acids and polyols has been studied in association with the effect of phenylacetic acid. A modified defined medium has been devised for this purpose.     

Production of phenylacetic acid derivatives and 4-epiradicinol in culture by Curvularia lunata

Phenylacetic acid derivatives, methyl 2-acetyl-3,5-dihydroxyphenylacetate (1) and methyl 2-acetyl-5-hydroxy-3-methoxyphenylacetate (3); curvulin or ethyl 2-acetyl-3,5-dihydroxyphenylacetate (4), a known metabolite of Curvularia siddiqui, and 4-epiradicinol (5) have been isolated from the culture mycelia of Curvularia lunata grown on YMG, a medium consisting of yeast, malt extract and glucose. Compounds 1, 3 and 4 lack antimicrobial and antioxidant activity, but 4-epiradicinol (5) inhibited the growth of Escherichia coli, Staphylococcus aureus, Salmonella choleraesuis and Bacillus subtilis. The structures of compounds 1, 3-5 were determined by analyses of IR, MS, 1D and 2D NMR data, assisted by chemical shift comparison to related and model compounds. The relative stereochemistry of the vicinal diol in 5 was determined from the 1H NMR signals for the methyl protons of the resulting cyclic acetonide prepared from 5.     

Differential spectrophotometric method of analysing levorin in the culture broth and in the mycelium]

It was shown on model experiments that the microbiological method was not applicable for determination of levorin content in industrial intermediate products containing in addition levoristatin, since the presence of the latter made higher the results of the microbiological assay. Because of this till to the present date the quantitative content of levorin in the industrial intermediate products was determined photometrically using alcohol (pure solvent) as the reference solution. Still, this method also made higher the results of the assay, especially when the content of levorin was determined in the fermentation broth. In the solid phase levorin is contained in the mycelium which occupies only 1 to 2 per cent of the fermentation broth, while the liquid phase or the fermentation broth filtrate occupies 98 to 99 per cent. It was found that the fermentation broth filtrate contained protein admixtures which coagulated on addition of alcohol to the fermentation broth and formed fine colloid solutions. As a result the absorption values became higher. In the present study not the pure solvent but an extract of the fermentation broth filtrate containing neither levorin, nor levoristatin was used as the reference solution. Such a differential method provided elimination of all errors due to the presence of various metabolites in the fermentation broth filtrate which varied both qualitatively and quantitatively.     

Stability of phenylacetic acid in liquid media

The stability of phenylacetic acid (PAA) in H₂O and B5 and MS culture media was determined by HPLC. There was no loss of PAA when a nonsterile 10 mM stock solution was held at 5°C for 2 months. PAA was stable to autoclaving in full-strength MS and B5 media. After storage at 5°C or after agitation at 125 rpm at room temperature for 28 days, 100% of the PAA remained in B5 medium. Under comparable conditions, up to 15% of the PAA was lost in MS medium.