Penicillium chrysogenum Takes up the Penicillin G Precursor Phenylacetic Acid by Passive Diffusion

Penicillium chrysogenum utilizes phenylacetic acid as a side chain precursor in penicillin G biosynthesis. During industrial production of penicillin G, phenylacetic acid is fed in small amounts to the medium to avoid toxic side effects. Phenylacetic acid is taken up from the medium and intracellularly coupled to 6-aminopenicillanic acid. To enter the fungal cell, phenylacetic acid has to pass the plasma membrane. The process via which phenylacetic acid crosses the plasma membrane was studied in mycelia and liposomes. Uptake of phenylacetic acid by mycelium was nonsaturable, and the initial velocity increased logarithmically with decreasing external pH. Studies with liposomes demonstrated a rapid passive flux of the protonated species through liposomal membranes. These results indicate that phenylacetic acid passes the plasma membrane via passive diffusion of the protonated species. The rate of phenylacetic acid uptake at an external concentration of 3 mM is at least 200-fold higher than the penicillin production rate in the Panlabs P2 strain. In this strain, uptake of phenylacetic acid is not the rate-limiting step in penicillin G biosynthesis.     

Plasma concentrations of p- and m-hydroxyphenylacetic acid and phenylacetic acid in humans : Gas chromatographic—high-resolution mass spectrometric analysis

Conjugated and unconjugated phenylacetic acid and m- and p-hydroxyphenylacetic acid have been determined in the plasma of normal, healthy subjects after fasting, consumption of a meal and ingestion of deuterium-labelled amine precursors, by high-resolution gas chromatography—high-resolution mass spectrometry with selected ion monitoring of their trifluoroethyl-pentafluoropropionyl derivatives.We observed that all three conjugated acids are higher in fasting than in non-fasting subjects, and unconjugated phenylacetic acid was lower. Ingestion of deuterium-labelled amine precursors resulted in the appearance in the blood of the correspondingly labelled acids, a peak in the concentrations being reached about 1 h after consumption. Conjugated and unconjugated acids as expected increased following the consumption of a meal.Unconjugated phenylacetic acid was significantly higher in females than in males. Most values tended to increase with age, with male unconjugated and conjugated m-hydroxyphenylacetic acid and female conjugated phenylacetic and m-hydroxyphenylacetic acids increasing significantly.     

Uptake of phenylacetic acid by two strains of Penicillium chrysogenum

Uptake of phenylacetic acid, the side-chain precursor of benzylpenicillin, was studied in Penicillium chrysogenum Wisconsin 54-1255 and in a strain yielding high levels of penicillin. In penicillin fermentations with the high-yielding strain, 100% recovery of phenylacetic acid in benzylpenicillin was found, whereas in the Wisconsin strain only 17% of the supplied phenylacetic acid was incorporated into benzylpenicillin while the rest was metabolized. Accumulation of total phenylacetic acid-derived carbon in the cells was nonsaturable in both strains at high external concentrations of phenylacetic acid (250-3500 microM), and in the high-yielding strain at low phenylacetic acid concentrations (2. 8-100 microM), indicating that phenylacetic acid enters the cells by simple diffusion, as concluded earlier for P. chrysogenum by other authors. However, at low external concentrations of phenylacetic acid saturable accumulation appeared in the Wisconsin strain. HPLC-analyses of cell extracts from the Wisconsin strain showed that phenylacetic acid was metabolized immediately after entry into the cells and different [14C]-labeled metabolites were detected in the cells. Up to approximately 50% of the accumulated phenylacetic acid was metabolized during the transport-assay period, the conversion having an impact on the uptake experiments. Nevertheless, accumulation of free unchanged phenylacetic acid in the cells showed saturation kinetics, suggesting the possible involvement of a high-affinity carrier in uptake of phenylacetic acid in P. chrysogenum Wisconsin 54-1255. At high concentrations of phenylacetic acid, contribution to uptake by this carrier is minor in comparison to simple diffusion and therefore, of no importance in the industrial production of penicillin.     

High-performance liquid chromatographic determination of phenylacetic acid in human plasma extracted with ethyl acetate

This paper describes a high-performance liquid chromatographic method with ultraviolet detection for measuring plasma phenylacetic acid. This simple and reliable method consists of an acid hydrolysis of conjugated phenylacetic acid before extraction with an organic solvent: washed ethyl acetate saturated in sodium chloride. The recovery of extraction was estimated by internal standardization with phenylpropionic acid, and validated by addition of phenylacetic acid standards. A preliminary application to plasma phenylacetic acid in patients suffering from depression is described.     

Mechanism of specific inhibition of phototropism by phenylacetic Acid in corn seedling

Using geotropism as a control for phototropism, compounds similar to phenylacetic acid that photoreact with flavins and/or have auxin-like activity were examined for their ability to specifically inhibit phototropism in corn seedlings using geotropism as a control. Results using indole-3-acetic acid, napthalene-1-acetic acid, naphthalene-2-acetic acid, phenylacetic acid, and β-phenylpyruvic acid suggest that such compounds will specifically inhibit phototropism primarily because of their photoreactivity with flavins and not their auxin activity. For example, strong auxins, indole-3-acetic acid and naphthalene-1-acetic acid, affected both tropic responses at all concentrations tested whereas weak auxins, phenylacetic acid and naphthalene-2-acetic acid, exhibited specific inhibition. In addition, the in vivo concentration of phenylacetic acid required to induce specificity was well below that required to stimulate coleoptile growth. Estimates of the percentage of photoreceptor pigment inactivated by phenylacetic acid (>10%) suggest that phenylacetic acid could be used to photoaffinity label the flavoprotein involved in corn seedling phototropism.     

Uremic toxins inhibit transport by breast cancer resistance protein and multidrug resistance protein 4 at clinically relevant concentrations

During chronic kidney disease (CKD), there is a progressive accumulation of toxic solutes due to inadequate renal clearance. Here, the interaction between uremic toxins and two important efflux pumps, viz. multidrug resistance protein 4 (MRP4) and breast cancer resistance protein (BCRP) was investigated. Membrane vesicles isolated from MRP4- or BCRP-overexpressing human embryonic kidney cells were used to study the impact of uremic toxins on substrate specific uptake. Furthermore, the concentrations of various uremic toxins were determined in plasma of CKD patients using high performance liquid chromatography and liquid chromatography/tandem mass spectrometry. Our results show that hippuric acid, indoxyl sulfate and kynurenic acid inhibit MRP4-mediated [(3)H]-methotrexate ([(3)H]-MTX) uptake (calculated Ki values: 2.5 mM, 1 mM, 25 μM, respectively) and BCRP-mediated [(3)H]-estrone sulfate ([(3)H]-E1S) uptake (Ki values: 4 mM, 500 μM and 50 μM, respectively), whereas indole-3-acetic acid and phenylacetic acid reduce [(3)H]-MTX uptake by MRP4 only (Ki value: 2 mM and IC(50) value: 7 mM, respectively). In contrast, p-cresol, p-toluenesulfonic acid, putrescine, oxalate and quinolinic acid did not alter transport mediated by MRP4 or BCRP. In addition, our results show that hippuric acid, indole-3-acetic acid, indoxyl sulfate, kynurenic acid and phenylacetic acid accumulate in plasma of end-stage CKD patients with mean concentrations of 160 μM, 4 μM, 129 μM, 1 μM and 18 μM, respectively. Moreover, calculated Ki values are below the maximal plasma concentrations of the tested toxins. In conclusion, this study shows that several uremic toxins inhibit active transport by MRP4 and BCRP at clinically relevant concentrations.     

Capsaicinoid Formation in the Protoplast from the Placenta of Capsicum Fruits

Sixty-two phenylacetic acid derivatives having phenylpropane skeleton were synthesized. Their physiological effects on the growth of lettuce seedlings were distinguished from those of plant hormones and an antiauxin. The derivatives retarded the growth in a similar manner to the auxin transport inhibitors, NPA and CPD. A few derivatives including 2-[3′-(m-chlorophenyl)isoxazol-5′-yl]phenylacetic acid were up to 10 times as potent as CPD in inhibition of the hypocotyl growth and IAA transport. The phenylacetic acids did not cause, even at high concentrations, any serious damage such as a herbicidal would. Their remarkable growth-retarding effects may be due, at least in part, to their ability to prevent the polar movement of IAA within tissues.     

Structure-Activity Relationship in the Auxin Activity of Mono-Substituted Phenylacetic Acids

The analysis of substituent constants for the lipophilic and electronic factors in the auxin activity of substituted phenylacetic acids in elongation of coleoptile segments shows that these factors parallel those for the phenoxyacetic acids but assign reactivity in growth promotion to the meta position of phenylacetic acid. The inhibitory effects with supra-optimal concentrations are highly dependent on the lipophilic character of the molecules.     

Effect of media components on the gynogenic regeneration of onion (Allium cepa L.) cultivars and analysis of regenerants

Summary Two separate experiments were performed to analyze the effects of different media on gynogenic regeneration in four onion cultivars. In a two step flower/ovary culture procedure, 2,4-dichlorophenoxyacetic acid added to the induction medium was superior to phenylacetic acid in the highly regenerating cultivar, while the effect of thidiazuron in the regeneration medium was generally optimal in higher (2 mg/l) rather than in lower (0.2 mg/l) concentrations. Gellan-gum was compared to agar solidified media. A higher number of regenerants was achieved on the former, but an undesirable hyperhydricity of regenerants formed on gellan-gum solidified media greatly reduced the final survival of formed embryos. Analysis of the time interval needed for regeneration showed high variability (from 46 to 152 days after inoculation), which was particularly pronounced in genotypes with lower regeneration capacity. Simpler isozyme patterns of regenerants showed that all analysed regenerants of the cultivar with a high regeneration capacity were homozygous, while in the other three cultivars, a considerable percentage (11.1 to 36.4%) of heterozygous regenerants were also detected. Ploidy analysis of the regenerants with simpler isozyme patterns revealed that the majority of lines remain haploid. Identification of 2 homozygous triploid regenerants demonstrated that as in androgenesis, nuclear fusions can occur during gynogenic haploid regeneration.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - PAA phenylacetic acid - TDZ thidiazuron - B5 Gamborg et al. (1968) medium - BDS Dunstan and Short (1977) medium     

Accumulation of polyhydroxyalkanoate from styrene and phenylacetic acid by Pseudomonas putida CA-3

Pseudomonas putida CA-3 is capable of converting the aromatic hydrocarbon styrene, its metabolite phenylacetic acid, and glucose into polyhydroxyalkanoate (PHA) when a limiting concentration of nitrogen (as sodium ammonium phosphate) is supplied to the growth medium. PHA accumulation occurs to a low level when the nitrogen concentration drops below 26.8 mg/liter and increases rapidly once the nitrogen is no longer detectable in the growth medium. The depletion of nitrogen and the onset of PHA accumulation coincided with a decrease in the rate of substrate utilization and biochemical activity of whole cells grown on styrene, phenylacetic acid, and glucose. However, the efficiency of carbon conversion to PHA dramatically increased once the nitrogen concentration dropped below 26.8 mg/liter in the growth medium. When supplied with 67 mg of nitrogen/liter, the carbon-to-nitrogen (C:N) ratios that result in a maximum yield of PHA (grams of PHA per gram of carbon) for styrene, phenylacetic acid, and glucose are 28:1, 21:1, and 18:1, respectively. In cells grown on styrene and phenylacetic acid, decreasing the carbon-to-nitrogen ratio below 28:1 and 21:1, respectively, by increasing the nitrogen concentration and using a fixed carbon concentration leads to lower levels of PHA per cell and lower levels of PHA per batch of cells. Increasing the carbon-to-nitrogen ratio above 28:1 and 21:1 for cells grown on styrene and phenylacetic acid, respectively, by decreasing the nitrogen concentration and using a fixed carbon concentration increases the level of PHA per cell but results in a lower level of PHA per batch of cells. Increasing the carbon and nitrogen concentrations but maintaining the carbon-to-nitrogen ratio of 28:1 and 21:1 for cells grown on styrene and phenylacetic acid, respectively, results in an increase in the total PHA per batch of cells. The maximum yields for PHA from styrene, phenylacetic acid, and glucose are 0.11, 0.17, and 0.22 g of PHA per g of carbon, respectively.     

Release of periplasmic penicillin amidase from Escherichia coli by chloroform shock

Penicillin amidase is a periplasmic enzyme in Escherichia coli. Conventionally, the periplasmic enzymes are released into the medium by osmotic shock which is tedious involving a number of centrifugation steps. The present communication deals with a simple technique for the release of penicillin amidase by chloroform shock. Experimental findings show that the periplasmic penicillin amidase does not show any variation by the chloroform treatment. This analysis was also extended to the E. coli cells grown at various concentrations of phenylacetic acid, optimal concentration of phenylacetic acid plus glucose and lactic acid.     

Anaerobic degradation of phenylacetic acid by mixed and pure cultures

Summary A phenylacetic acid-degrading mixed culture was enriched from effluent of an anaerobic reactor for the treatment of waste water from cellulose bleaching. From this consortium a phenylacetic acid-degrading pure culture, strain DSU3, was isolated and, due to its typical morphology and substrate spectrum, tentatively classified as a Desulfosarcina sp. It could grow on and degrade phenylacetic acid, cyclohexane carboxylate, cyclohexylacetate, benzoate, fumaric acid and several volatile fatty acids, while phenol, o-hydroxybenzoate, p-hydroxybenzoate and glucose were not utilized. Production of mandelic acid from phenylacetic acid by the enrichment culture and utilization of benzoate, an intermediate of the mandelic acid pathway, by strain DSU3 may presumably indicate degradation of phenylacetic acid via the mandelic acid pathway.     

Accumulation of Polyhydroxyalkanoate from Styrene and Phenylacetic Acid by Pseudomonas putida CA-3

Pseudomonas putida CA-3 is capable of converting the aromatic hydrocarbon styrene, its metabolite phenylacetic acid, and glucose into polyhydroxyalkanoate (PHA) when a limiting concentration of nitrogen (as sodium ammonium phosphate) is supplied to the growth medium. PHA accumulation occurs to a low level when the nitrogen concentration drops below 26.8 mg/liter and increases rapidly once the nitrogen is no longer detectable in the growth medium. The depletion of nitrogen and the onset of PHA accumulation coincided with a decrease in the rate of substrate utilization and biochemical activity of whole cells grown on styrene, phenylacetic acid, and glucose. However, the efficiency of carbon conversion to PHA dramatically increased once the nitrogen concentration dropped below 26.8 mg/liter in the growth medium. When supplied with 67 mg of nitrogen/liter, the carbon-to-nitrogen (C:N) ratios that result in a maximum yield of PHA (grams of PHA per gram of carbon) for styrene, phenylacetic acid, and glucose are 28:1, 21:1, and 18:1, respectively. In cells grown on styrene and phenylacetic acid, decreasing the carbon-to-nitrogen ratio below 28:1 and 21:1, respectively, by increasing the nitrogen concentration and using a fixed carbon concentration leads to lower levels of PHA per cell and lower levels of PHA per batch of cells. Increasing the carbon-to-nitrogen ratio above 28:1 and 21:1 for cells grown on styrene and phenylacetic acid, respectively, by decreasing the nitrogen concentration and using a fixed carbon concentration increases the level of PHA per cell but results in a lower level of PHA per batch of cells. Increasing the carbon and nitrogen concentrations but maintaining the carbon-to-nitrogen ratio of 28:1 and 21:1 for cells grown on styrene and phenylacetic acid, respectively, results in an increase in the total PHA per batch of cells. The maximum yields for PHA from styrene, phenylacetic acid, and glucose are 0.11, 0.17, and 0.22 g of PHA per g of carbon, respectively.     

Identification of metabolites of phenylacetic acid in rat brain by plasma desorption mass spectrometry

Mass spectra of acyl hydroxamates may be obtained directly, without prior derivatization or gas chromatography, by the new technique of plasma desorption mass spectrometry. Authentic alkyl and aryl hydroxamates showed the expected protonated molecular ions when isobutane was used as the carrier gas. Advantage was taken of this novel technique to identify phenylacetyl-CoA and phenylbutyric acid in brain extracts obtained from young rats after a loading dose of phenylacetic acid. The formation of these metabolic products lends strong support to our suggestion that brain dysfunction induced by phenylacetic acid in experimental phenylketonuria may be due to decreased availability of CoA and acetyl-CoA in the rapidly developing brain.     

Phenylacetic acid production by Bacteroides gingivalis from phenylalanine and phenylalanine-containing peptides

Phenylacetic acid production and growth of Bacteroides gingivalis were directly proportional to the trypticase content of the medium. L-Phenylalanine enhanced phenylacetic acid production; 5 mg L-phenylalanine per millilitre stimulated maximum production of phenylacetic acid. Peptides (2-4 amino acids) containing L-phenylalanine also stimulated phenylacetic acid production as did phenylpyruvic acid. Resting cell suspensions of B. gingivalis also produced phenylacetic acid when incubated aerobically in the presence of L-phenylalanine and phenylpyruvic acid. Hydrocinnamic acid (3-phenylpropionic acid) and phenyllactic acid were also produced by resting cell suspensions. Our results suggest that L-phenylalanine and phenylpyruvic acid are both precursors to phenylacetic acid.     

Effect of sex and gonadal hormones on rat plasma lipids during the development of an essential fatty acid deficiency

1. Male, female and castrated rats treated with oestradiol (30mug./week) or testosterone (2mg./week) were given an essential fatty acid-deficient diet containing 10% of hydrogenated coconut oil for 9 weeks. The concentrations and fatty acid composition of plasma phospholipids, cholesteryl esters and triglycerides were determined. 2. Between the second and third weeks of the deficiency, concentrations of plasma cholesteryl esters, phospholipids and triglycerides decreased, then remained relatively constant. There were no significant differences between males and females, but oestradiol caused a significant rise in plasma phospholipids and triglycerides as compared with testosterone-treated animals. 3. During the first 2 weeks of the deficiency, linoleic acid in the plasma lipids of all groups decreased to low concentrations and changed very little thereafter. 4. Female rats maintained higher percentages and concentrations of arachidonic acid and stearic acid in plasma phospholipids and arachidonic acid in cholesteryl esters than did males. Males had higher proportions of eicosatrienoic acid and oleic acid. There was no sex difference in the fatty acid composition of plasma triglycerides. 5. Oestradiol-treated rats had concentrations of cholesteryl and phospholipid arachidonate comparable with those of female rats and higher than the testosterone-treated group. Eicosatrienoic acid in the oestradiol-treated rats was high and resembled that of the male rats, apparently because of the higher concentration of plasma phospho lipids in this group. 6. Supplementation of the essential fatty acid-deficient rats with linoleate restored plasma cholesteryl and phospholipid linoleate and arachidonate nearly to normal concentrations in a single day. The increase in arachidonic acid in these fractions was accompanied by a similar quantitative decrease in eicosatrienoic acid. 7. These sex differences appear to be related to the smaller size of the female rat and to a more direct influence of oestradiol on the formation or maintenance of phospholipids rich in arachidonic acid.     

De novo formation of organochlorines in a sewage treatment plant

The de novo formation of organochlorines was observed in a municipalsewage treatment plant. Due to this formation, the amount of organically boundhalogens (AOX) increased 15-fold inside the sewage treatment plant. Per day,more than 6 kg of organically-bound chlorine were produced. Thisformation is not based on a metabolism of present organochlorines, it is a denovo formation out of inorganic chloride and organic substrates. The AOXtriggerconcentration in sewage sludge in Germany is 500 mgkg^–1 and was sometimes exceeded by a factor of 10. Noknown anthropogenic organohalogens were found which could explain the elevatedAOX concentrations. Instead many chlorinated compounds could be identifiedwhichwere not known to be of anthropogenic origin. The compound with the highestconcentration was the 3,4-dichlorophenylacetic acid (3,4-CPAc). In one case,more than 1 g kg^–1 of this compound was detected.A slaughterhouse that emits phenylacetic acid is probably the origin of thatformation. In model experiments phenylacetic acid was chlorinated with HOCl butchlorinated phenylacetic acids other than 3,4-CPAc were found. Therefore it canbe excluded that the chlorination in the sewage treatment plant takes place byan abiotic reaction with hypochlorite that might have been introduced there. Weassume that the occurring microorganisms are responsible for the de novoformation in the sewage treatment plant. The obtained knowledge could also beuseful to understand natural chlorination processes.     

产黄青霉谷胱甘肽S-转移酶基因PcgstA的克隆与鉴定

从青霉素工业生产菌产黄青霉(Penicilliumchrysogenum)中首次克隆了一个谷胱甘肽S-转移酶(GST)基因,定名为PcgstA.该基因的开放阅读框长840bp,含有两个内含子,编码一个238氨基酸残基的蛋白质.其推断的氨基酸序列与一些已经鉴定的丝状真菌GST具有50%左右的序列一致性.PcgstA的完整编码区经RT-PCR扩增、验证,插入原核表达载体pET11a,转化大肠杆菌BL21(DE3)-RP菌株,表达得到重组PcGSTA蛋白.酶活测定证实,重组PcGSTA具有GST活性,其对底物CDNB(1-chloro-2,4-dinitrobenzene)的比活为(0.159±0.031)μmol/(min·mg).利用TaqMan探针法,对PcgstA的表达情况进行了比较.结果表明,在添加了侧链前体苯乙酸的青霉素生产培养基中,PcgstA的表达水平和在不含苯乙酸培养基中的表达相比明显下调,显示了该基因与苯乙酸代谢的关系.     

Catabolism of dl-α-phenylhydracrylic,phenylacetic and 3- and 4-hydroxyphenylacetic acid via homogentisic acid in a Flavobacterium sp.

A degradation pathway for dl--phenylhydracrylic, phenylacetic, 3- and 4-hydroxyphenylacetic acid by a Flavobacterium is presented. Experiments with washed cells and enzyme studies revealed that dl--phenylhydracrylic acid in an initial reaction was oxidatively decarboxylated to phenylacetaldehyde. Whole cells oxidized both stereoisomers of phenylhydracrylic acid at different rates. The product phenylacetaldehyde in turn was oxidized to phenylacetic acid. No hydroxylation of phenylacetic acid was detected in cell extracts, but on the basis of experiments with washed cells it is assumed that phenylacetic acid is mainly metabolized via 3-hydroxyphenylacetic acid. This latter product was subsequently hydroxylated yielding the ring-cleavage substrate homogentisate. 4-Hydroxyphenylacetic acid was also degraded via homogentisate. Ringcleavage of homogentisate gave maleylacetoacetate which was further degraded through a glutathione-dependent pathway. Homoprotocatechuate was not an intermediate in the metabolism of dl-phenylhydracrylic acid, phenylacetic, 3- and 4-hydroxyphenylacetic acid metabolism, but it could be hydroxylated aspecifically to 2,4,5-trihydroxyphenylacetic acid by the action of the 3-hydroxyphenylacetic acid-6-hydroxylase.Abbreviations HPLC high-performance liquid chromatography - PHA phenylhydracrylic acid - PA phenylacetic acid - HPA hyxdroxyphenylacetic acid - PMS phenazine methosulphate - PMA phenylmalonic acid - GSH glutathione     

Comparisons of Acids in Smoke of Lamina and Midrib of Flue-cured Tobacco Leaves

Acids of lamina and midrib cigarette smoke were converted into trimethylsilyl derivatives and they were analyzed with glass capillary column gas chromatography. Then compositional differences of acids between lamina and midrib cigarette smoke were discussed. The concentrations of organic acids were higher for lamina cigarette smoke than for midrib cigarette smoke. Large concentration difference were found in formic, acetic, propionic, lactic, glycolic, furoic, benzoic, phenylacetic, fumalic and m-hydroxybenzoic acid. Succinic and methylsuccinic acid were similar in lamina smoke and in midrib smoke.

A large amount of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one(amino-carbonyl reaction product) was identified for the first time in lamina smoke.