Kinetics of the enzymatic synthesis of benzylpenicillin

The kinetics of the enzymatic synthesis of benzylpenicillin catalysed by penicillin amidase (EC 3.5.1.11) from Escherichia coli have been studied. Both free phenylacetic acid (PAA) and its activated derivative, phenylacetylglycine (PAG), were used in the synthesis as acylating agents for 6-aminopenicillanic acid (6-APA). The catalytic rate constants for synthesis carried out at pH 6.0 were 11.2 and 25.2 s⁻¹, respectively, i.e. they are close and have high absolute values. The main feature of the enzymatic synthesis of benzylpenicillin from phenylacetylglycine, compared with the synthesis from phenylacetic acid, is the shape of the progress curve of antibiotic accumulation. In the former case, benzylpenicillin gradually accumulates until equilibrium is reached. Thus, if the reaction is carried out at the thermodynamically optimum pH of synthesis (low pH), penicillin can be obtained in high yield. In the case of phenylacetylglycine, the kinetic curves are more complex and are characterized by a clear-cut maximum. The presence of the maximum, its value and position on the time axis depend on reagent concentration and on the pH used. A kinetic scheme is proposed which describes well the experimental dependencies. The possibility of using activated acid derivatives in synthesis and the advantages of using computer calculations for process optimization are discussed.     

Penicillin acylase catalyzed synthesis of penicillin-G from substrates anchored in cyclodextrins

Penicillin acylase (EC 3.5.1.11) catalyses the condensation of phenylacetic acid (PAA) and 6-aminopenicillanic acid (6-APA) to form benzylpenicillin (BP). Both PAA and 6-APA were found to form host-guest complexes with beta-methylcyclodextrin (beta m-CD) and gamma-cyclodextrin (gamma-CD) respectively. The rate of the reaction catalyzed by the enzyme remained unaffected if one of the substrates used was in the cyclodextrin complexed form. However, in this case, the reaction lasted longer and yielded about 20 per cent more products compared to the condensation reaction involving only uncomplexed substrates. There was distinct increase in the rate of formation of the antibiotic, if both substrates used are in CD-complexed form.     

Immobilization of permeabilized Escherichia coli cells with penicillin acylase activity.

Escherichia coli cells with penicillin acylase activity were sequentially treated at pH 7.8 with aqueous solutions of N-cetyl-N,N,N-trimethylammonium bromide and glutaraldehyde and then immobilized within porous polyacrylamide beads. The immobilized whole cells showed enhanced hydrolysis rates in the conversion of benzylpenicillin to 6-aminopenicillanic acid (6-APA) compared to untreated cells immobilized and used under identical conditions. The immobilized system showed no apparent loss in enzyme activity when used repeatedly over 90 cycles for 6-APA production from 4% benzylpenicillin.     

Effect of penicillin precursors on antibiotic biosynthesis in various strains]

The regularities of biosynthesis of 6-aminopenicillanic acid (6-APA), benzylpenicillin (BP) and phenoxymethylpenicillin (PMP) by the strains under the investigation did not significantly differ. In the absence of the precursor both the strains mainly synthesized 6-APA. Phenylacetic acid (PAA) and phenoxyacetic acid (POAA) provided directed biosynthesis: the fungus synthesized BP or PMP depending on the precursor nature. When the amount of the precursors was not sufficient, 6-APA was synthesized along with the penicillins. PAA proved to be a more active precursor than POAA. When both precursors were present in the fermentation broth, only BR was synthesized. An important distinction of strain 316A was its increased sensitivity to PAA especially in the initial period. After an increase in the PAA concentration the growth rate of strain 316A lowered to a greater extent than that of strain 284A. This was likely to determine the higher levels of penicillin production by strain 316A in the presence of POAA, a nontoxic precursor. A procedure for supplying the precursors was developed. Under the laboratory conditions it provided high levels of the penicillin production.     

Enhancement of penicillin G hydrolysis using penicillin acylase to 6-aminopenicillanic acid by simultaneous chromatographic separation

Penicillin G (Pen-G) was hydrolyzed to 6-aminopenicillanic acid (6-APA) and phenylacetic acid (PAA) in a chromatographic reactor-separator using the mixture of immobilized Escherichia coli cells and a macroporous adsorbent as stationary phase and a phosphate buffer of pH 7.8 as eluant. Pen-G conversion of 98% was observed without adjustment of the eluant pH due to the effective separation of 6-APA from Pen-G and PAA. At a sample load of 600 mg Pen-G, the volume overload gave higher Pen-G conversion (86%) than the mass overload (68%), while their difference in product resolution (0.9 and 1.0, respectively) was insignificant.     

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.     

Enzymatic hydrolysis of penicillin in mixed ionic liquids/water two-phase system

In this paper, an integrated process involving the mixed ionic liquids/water two-phase system (MILWS) is proposed to improve the efficiency for enzymatic hydrolysis of penicillin G. First, hydrophilic [C4mim]BF4 (1-butyl-3-methylimidazolium tetrafluoraborate) and NaH2PO4 salt form an ionic liquids aqueous two-phase system (ILATPS), which could extract penicillin from its fermentation broth efficiently. Second, a hydrophobic [C4mim]PF6 (1-butyl-3-methylimidazolium hexafluoraphosphate) is introduced into the ionic liquids-rich phase of ILATPS containing penicillin and converses it into MILWS. Penicillin is hydrolyzed by penicillin acylase in the water phase of MILWS at pH 5. The byproduct phenylacetic acid (PAA) is partitioned into the ionic liquids mixture phase, while the intended product 6-aminopenicillanic acid (6-APA) is precipitated at this pH. In comparison with a similar butyl acetate/water system (BAWS) at pH 4, MILWS exhibits two advantages. (1) The selectivity between PAA and penicillin is greatly optimized at pH 5 by varying the mole ratio of [C4mim]PF6/[C4mim]BF4 in MILWS, whereas in BAWS the unalterable nature of the organic solvent restricts the optimized pH for maximum selectivity between PAA and penicillin at pH 4. (2) The pH for 6-APA precipitation in BAWS is 4, whereas it shifts to pH 5 in MILWS due to the complexation between negatively charged 6-APA and the cationic surface of the ionic liquids micelle. As a result, the removal of the two products from the enzyme sphere at relatively high pH is permitted in MILWS, which is beneficial for enzymatic activity and stability in comparison with the acidic pH 4 environment in BAWS.     

Factors affecting the synthesis of ampicillin and hydroxypenicillins by the cell-bound penicillin acylase of Escherichia coli

1. The effect of pH, temperature, reactant concentration and reaction time has been investigated for the synthesis of benzylpenicillin, dl-alpha-hydroxybenzylpenicillin and d-alpha-aminobenzylpenicillin from 6-aminopenicillanic acid by the penicillin acylase of Escherichia coli. 2. Synthesis of penicillins from carboxylic acids proceeds most rapidly at pH5; with amides the optimum pH is higher (6-7) but the reverse reaction rapidly sets in. This can be counteracted by lowering the pH or adding more amide. Optimum temperatures are 35-40 degrees . 3. Most rapid synthesis of penicillin was obtained with the N-acylglycine and methyl ester derivatives of carboxylic acids. Increasing the amide/6-APA ratio above 1:1 raised the rate of synthesis of penicillins. 4. Preferential synthesis of d-alpha-hydroxybenzylpenicillin takes place in a reaction mixture containing dl-mandelic acid. 5. From d- and l-mandelamide, d- and l-alpha-hydroxybenzylpenicillins were prepared, the former being more bioactive than the latter. p-Hydroxy- and 3,4-dihydroxybenzylpenicillins were also prepared, the latter being more active against some Gram-negative bacteria than benzylpenicillin.     

Formation of 6-Aminopenicillanic Acid, Penicillins, and Penicillin Acylase by Various Fungi

Several penicillin-producing fungi were examined for ability to produce 6-aminopenicillanic acid (6-APA) and penicillin acylase. 6-APA was found in corn steep liquor fermentations of Trichophyton mentagrophytes, Aspergillus ochraceous, and three strains of Penicillium sp. 6-APA was not detected in fermentations of Epidermophyton floccosum although penicillins were produced. 6-APA formed a large part of the total antibiotic production of T. mentagrophytes. The types of penicillins produced by various fungi were identified by paper chromatography, and it was found that all cultures produced benzylpenicillin. T. mentagrophytes and A. ochraceous showed increased yields of benzylpenicillin and the formation of phenoxymethylpenicillin in response to the addition to the fermentation medium of phenylacetic acid and phenoxyacetic acid, respectively. Washed mycelia of the three Penicillium spp. and two high penicillin-yielding strains of P. chrysogenum possessed penicillin acylase activity against phenoxymethylpenicillin. A. ochraceous, T. mentagrophytes, E. floccosum, and Cephalosporium sp. also had penicillin acylase activity against phenoxymethylpenicillin. Only two of the above fungi, T. mentagrophytes and E. floccosum, showed significant penicillin acylase activity against benzylpenicillin; in both cases it was very low. The acylase activity of A. ochraceous was considerably increased by culturing in the presence of phenoxyacetic acid. It is concluded that 6-APA frequently but not invariably accompanies the formation of penicillin, and that penicillin acylase activity against phenoxymethylpenicillin is present in all penicillin-producing fungi.     

Ca2+ and calmodulin antagonists inhibit the proton gradients associated with non-cyclic and cyclic photophosphorylation in spinach chloroplasts

The synthesis of benzylpenicillin (BP) after mixing phenyl-acetyl-glycine(PAG), 6-aminopenicillanic acid (6-APA) and free or immobilized penicillin amidase (E.C.3.5.1.11.) was studied as a function of pH and ionic strength. Before the final equilibrium was reached a kinetically controlled synthesis of BP was observed. Then a transient maximum concentration in BP much larger than the final equilibrium content was synthesized in the acyl-transfer process. The factors influencing this maximum have been analyzed. Increasing ionic strength markedly decreased the maximum in BP and the rate of deacylation of phenyl-acetyl-penicillin amidase by 6-APA. The change was largest when the enzyme was immobilized in a positively charged support, where at low ionic strength the concentration of 6-APA around the enzyme is larger than the bulk concentration due to the partitioning of charged solutes.     

Production of lovastatin examined by an integrated approach based on chemometrics and DOSY-NMR

The penicillin acylase-catalyzed synthesis of ampicillin by acyl transfer from D-(-)-phenylglycine amide (D-PGA) to 6-aminopenicillanic acid (6-APA) becomes more effective when a judiciously chosen pH gradient is applied in the course of the process. This reaction concept is based on two experimental observations: 1) The ratio of the initial synthesis and hydrolysis rates (V(S)/V(H)) is pH-dependent and exhibits a maximum at pH 6.5-7.0 for a saturated solution of 6-APA; 2) at a fixed 6-APA concentration below saturation, V(S)/V(H) increases with decreasing pH. Optimum synthetic efficiency could, therefore, be achieved by starting with a concentrated 6-APA solution at pH 7 and gradually decreasing the pH to 6.3 in the course of 6-APA consumption. A conversion of 96% of 6-APA and 71% of D-PGA into ampicillin was accomplished in an optimized procedure, which significantly exceeds the efficiency of enzymatic synthesis performed at a constant pH of either 7.0 or 6.3.     

An integrated process for the production and biotransformation of penicillin.

The extraction of Penicillin G (PG) from the filtered cultivation medium of Penicillium chrysogenum and its conversion into 6-amino penicillanic acid (6-APA) and phenyl acetic acid (PhA) at pH 8 was performed in a 10 l kühni extractor during the production by means of penicillin-G-amidase immobilized in a liquid membrane carrier system (LM). 6-APA was enriched in LM, and the PhA returned to the cultivation medium. After electrocoalescence of LM, the 6-APA was converted into ampicillin with the same enzyme at pH 6, while the liquid membrane phase and enzyme were recycled and reused.     

Inhibition of penicillin biosynthetic enzymes by halogen derivatives of phenylacetic acid

Summary The effect of phenylacetic acid (PAA) and several analogs on the activity of isopenicillin N synthase (IPNS) and acyl-CoA: 6-APA acyltransferase (AT) fromPenicillium chrysogenum Wis 54-1255 has been tested. Whereas the substitution on the ring of a hydrogen atom by hydroxy-, methyl- or methoxy- groups did not cause any effect, the presence of halogens (Cl or Br) at positions 3 and/or 4 of PAA strongly inhibited these two enzymes. The replacement of hydrogen atoms by fluorine in certain positions also caused inhibition, but to a lesser extent.     

In vivo transport of the intermediates of the penicillin biosynthetic pathway in tailored strains of <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis>

Penicillium chrysogenum npe10 (Δpen; lacking the 56.8-kbp amplified region containing the penicillin gene cluster), complemented with one, two, or three penicillin biosynthetic genes, was used for in vivo studies on transport of benzylpenicillin intermediates. 6-Aminopenicillanic acid (6-APA) was taken up efficiently by P. chrysogenum npe10 unlike exogenous δ(l-α-aminoadipyl)-l-cysteinyl-d-valine or isopenicillin N (IPN), which were not taken up or were taken up very poorly. Internalization of exogenous IPN and 6-APA inside peroxisomes was tested by quantifying their peroximal conversion into benzylpenicillin in strains containing only the penDE gene. Exogenous 6-APA was transformed efficiently into benzylpenicillin, whereas IPN was converted very poorly into benzylpenicillin due to its weak uptake. IPN was secreted to the culture medium. IPN secretion decreased when increasing levels of phenylacetic acid were added to the culture medium. The P. chrysogenum membrane permeability to exogenous benzylpenicillin was tested in the npe10 strain. Penicillin is absorbed by the cells by an unknown mechanism, but its intracellular concentration is kept low. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

颗粒状固定化青霉素酰化酶的研究

将巨大芽孢杆菌 (Bacillusmegaterium)胞外青霉素酰化酶通过共价键结合到聚合物载体EupergitC颗粒环氧基团上 ,制成的颗粒状固定化青霉素酰化酶表现活力达 1 40 0 μ/g左右。固定化酶水解青霉素的最适 pH8 0 ,最适温度为 55℃。在pH6 0～ 8 5、温度低于 40℃时固定化酶活力稳定。在 pH8 0、温度 37℃时 ,固定化酶对青霉素的表现米氏常数Ka为 2×1 0 - 2 mol/L ;苯乙酸为竞争性抑制剂 ,抑制常数Kip为 2 8× 1 0 - 2 mol/L ;6 APA为非竞争性抑制剂 ,抑制常数Kia为 0 1 2 5mol/L。固定化酶水解青霉素 ,投料浓度为 8% ,在使用 2 0 0批后 ,保留活力 80 %左右 ,6 APA收率平均达 89 48%。     

Acyl coenzyme A: 6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum and Aspergillus nidulans

A study of the final stages of the biosynthesis of the penicillins in Penicillium chrysogenum has revealed two types of enzyme. One hydrolyses phenoxymethyl penicillin to 6-aminopenicillanic acid (6-APA). The other, also obtained from Aspergillus nidulans, transfers a phenylacetyl group from phenylacetyl CoA to 6-APA. The acyltransferase, purified to apparent homogeneity, had a molecular mass of 40 kDa. It also catalyses the conversion of isopenicillin N (IPN) to benzylpenicillin (Pen G) and hydrolyses IPN to 6-APA. In the presence of SDS it dissociates, with loss of activity, into fragments of ca 30 and 10.5 kDa, but activity is regained when these fragments recombine in the absence of SDS.     

Cell aggregates ofEscherichia coli with benzylpenicillin amidase activity

Intact cellsEscherichia coli CCM 2843, exhibiting substantial benzylpenicillin amidase activity, were bound mutually with supporting waste microbial cells, native or treated, to obtain an inexpensive biocatalyst for the production of 6-aminopenicillanic acid (6-APA). The bond was effected by glutaraldehyde (GA) and Sedipur CL-930 (PEI), without any carrier. The optimal concentration of GA was 2%, that of PEI 1%. The optimal biocatalyst was obtained by immobilization of productive cells with their fragments at a mass ratio of 4∶1. The cell aggregates were used for hydrolysis of potassium benzyl-penicillin at a concentration of 5 % to 6-APA. After 25 repeated batch conversions the degree of conversion did not decrease; its average value was 96.4%.     

Enzyme immobilization on ultrafine cellulose fibers via poly(acrylic acid) electrolyte grafts

Ultrafine cellulose fiber (diameter 200-400 nm) surfaces were grafted with polyacrylic acid (PAA) via either ceric ion initiated polymerization or methacrylation of cellulose with methacrylate chloride (MACl) and subsequent free-radical polymerization of acrylic acid. PAA grafts by ceric ion initiated polymerization increased with increasing reaction time (2-24 h), monomer (0.3-2.4 M), and initiator (1-10 mM) concentrations, and spanned a broad range from 5.5-850%. PAA grafts on the methacrylated cellulose fibers also increased with increasing molar ratios of MACl to cellulosic hydroxyl groups (MACl/OH, 2-6.4) and monomer acrylic acid (AA) to initiator potassium persulfate (KPS) ratios ([AA]/[KPS], 1.5-6), and were in a much narrower range between 12.8% and 29.4%. The adsorption of lipase (at 1 mg/ml lipase and pH 7) and the activity of adsorbed lipase (pH 8.5, 30 degrees C), in both cases decreased with increasing PAA grafts. The highest adsorption and activity of the lipase on the ceric ion initiated grafted fibers were 1.28 g/g PAA and 4.3 U/mg lipase, respectively, at the lowest grafting level of 5.5% PAA, whereas they were 0.33 g/g PAA and 7.1 U/mg lipase, respectively, at 12.8% PAA grafts on the methacrylated and grafted fibers. The properties of the grafted fibers and the absorption behavior and activity of lipase suggest that the PAA grafts are gel-like by ceric-initiated reaction and brush-like by methacrylation and polymerization. The adsorbed lipase on the ceric ion-initiated grafted surface possessed greatly improved organic solvent stability over the crude lipase. The adsorbed lipases exhibited 0.5 and 0.3 of the initial activity in the second and third assay cycles, respectively.     

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.