Enzymatic conversion in aqueous two-phase systems: deacylation of benzylpenicillin to 6-aminopenicillanic acid with penicillin acylase

The conversion of benzylpenicillin (BP) to 6-aminopenicillanic acid (6-APA) using penicillin acylase (penicillin amidohydrolase, EC 3.5.1.11) has been studied in aqueous two-phase systems. In a system composed of 8.9% (w/w) PEG 20000/7.6% (w/w) potassium phosphate the enzyme was almost completely partitioned to the bottom phase (K < 0.01), which allowed repeated batch conversions, recirculating the enzyme several times. The initial specific productivities were 0.31–1.47 μmol 6-APA mg protein^?1 min^?1 in repeated conversions over five steps. The yield obtained from the top phase was 0.47–0.71 mol 6-APA mol BP^?1. The results are discussed in relation to recirculating the enzyme by immobilizing it to a solid matrix. Despite the high phosphate concentration in the bottom phase the system needs to be titrated in order for the reaction to proceed. Titration of the top phase alone protected the enzyme from denaturation by strong alkali used for the titration.     

Concentration of ultrafiltered benzylpenicillin broths by reverse osmosis

Concentration of benzylpenicillin filtered broths purified by ultrafiltration and fermented broths clarified by ultrafiltration was carried out by reverse osmosis. This study was done using a reverse osmosis laboratory/pilot installation from Paterson Candy International Ltd. whose module has a tubular configuration and a membrane with a molecular weight cut-off of 100 Dalton (ZF99) made of a polyamide film with a rejection of 99% of NaCl. It was concluded that reverse osmosis is an adequate technique to concentrate benzylpenicillin ultrafiltered broths, leading to low losses of benzylpenicillin in the permeate and high benzylpenicillin recovery for high volumetric concentration factors.     

Notes on preparation of 6-aminopenicillanic acid by bacterial hydrolysis of benzylpenicillin

Using a cell suspension of a bacterial strain isolated from natural material, benzylpenicillin solution was hydrolysed in water to which toluene was added. The resultant 6-aminopenicillanic acid was isolated in crystalline form. The yield from 0.5% penicillin solution was 69% of the theoretical yield, from 1% solution 64% and from 2% solution up to 45%. The cell suspension could be used several times, the highest yield being obtained on the second or third occasion.     

Enzymatic hydrolysis of penicillin V to 6-aminopenicillanic acid byFusarium oxysporum

Summary A strain ofFusarium oxysporum was identified as having an intracellular penicillin V acylase activity (penicillin V amidohydrolase EC 3.5.1.11). Activity was induced by phenoxyacetic acid and had a good tolerance for high substrate and product concentrations. Washed cells could be used repeatedly for the complete hydrolysis of 5% penicillin V solutions. The enzyme was partially purified and concentrated from disrupted cells by fractional precipitation with water miscible solvents.     

Thermodynamics of the conversion of penicillin G to phenylacetic acid and 6-aminopenicillanic acid

The thermodynamics of the enzymatic conversion (penicillin acylase) of aqueous penicillin G to phenylacetic acid and 6-aminopenicillanic acid have been studied using both high-pressure liquid-chromatography and microcalorimetry. The reaction was carried out in aqueous phosphate buffer over the pH range 6.0-7.6, at ionic strengths from 0.10 to 0.40 mol kg-1, and at temperatures from 292 to 322 K. The data have been analyzed using a chemical equilibrium model with an extended Debye-Hückel expression for the activity coefficients. For the reference reaction, penicillin G- (aq) + H2O(l) = phenylacetic acid-(aq) + 6-aminopenicillanic acid-(aq) + H+ (aq), the following parameters have been obtained: K = (7.35 +/- 1.5) X 10(-8) mol kg-1, delta G0 = 40.7 +/- 0.5 kJ mol-1, delta H0 = 29.7 +/- 0.6 kJ mol-1, and delta C0p = -240 +/- 50 J mol-1 K-1 at 298.15 K and at the thermochemical standard state. The extent of reaction for the overall conversion is highly dependent upon the pH.     

Conversion of benzylpenicillin to 6-aminopenicillanic acid in a batch reactor and continuous feed stirred tank reactor using immobilized penicillin amidase

A rate equation has been derived to describe the hydrolysis of benzylpenicillin to 6-aminopenicillanic acid by penicillin amidase. The integrated from of the rate equation has been shown to predict satisfactorily the progress of the reaction in a batch reactor using either soluble or immobilized penicillin amidase. The rate equation was also used to predict the performance of a continuous feed stirred tank reactor containing immobilized enzyme. There was good agreement with experimental measurements.     

The conversion of benzyl penicillin to 6-aminopenicillanic acid using an insoluble derivative of penicillin amidase

Penicillin amidase was extracted from Escherichia coli ATCC 9637, grown on phenylacetic, acid and glutamate, and purified by fractional ion with streptomycin sulphate, ammonium sulphate and polyethylene glycol, followed by chromatography on DEAE–cellulose. The purification factor was 100–200 × and the overall yield was about 115%. The enzyme was chemically attached to derivatives of cellulose and the kinetics of these insolubilized penicillin amidase preparations was investigated.     

Comparison of efficiencies of hollow tubes and hollow zones biocatalytic reactors for the enzymatic hydrolysis of benzylpenicillin to 6-aminopenicillanic acid

The comparative efficiencies of two special biocatalytic reactors designed to handle gel-like immobilized biocatalysts, the hollow tubes and the hollow zones reactors, have been assessed for the performance of the hydrolysis of benzylpenicillin to 6-aminopenicillanic acid and phenyl acetic acid by an immobilized penicillin amidase preparation. On the basis of the efficiency factor η15/35, representing the ratio of activity expressed in the reactor at the end of 15 minutes in relation to the activity loaded in the reactor, and on the conversion achieved at the end of a given time close to that required to attain conversions near those required in industrial operation the hollow tubes reactor revealed to be slightly superior to the hollow zones reactor. The application to the conversion data obtained in both reactors of the α, β kinetic model derived by Cardoso and Costa [1] also gave evidence of the superior performance of the hollow tubes reactor. The influence of the linear velocity on the final conversion achieved for the hollow tubes reactor was investigated allowing the conclusion that from a velocity across the reactor of at least about 17.6 cm min⁻¹ the final conversion time is independent of this parameter. The application of the α, β kinetic model to the conversion data obtained in the hollow tubes reactor for different linear velocities also supported this conclusion. Although the kinetic behaviour of the conversion reaction for linear velocities higher than 17.6 cm min⁻¹ varied with the linear velocity the final conversion was not affected by this parameter.     

Enzymic acylation of 6-aminopenicillanic acid

The enzymic synthesis of benzylpenicillin from 6-aminopenicillanic acid in the presence of poly (ethylene glycol) has been studied. With equimolar initial concentrations (20 mM) of 6-aminopenicillanic acid and phenylacetic acid a 60% conversion to benzylpenicillin can be achieved at 10°C and pH 5.2 in the presence of 45% (w/v) poly(ethylene glycol). Under these conditions the lactam ring of the benzylpenicillin and 6-aminopenicillanic acid and the enzyme, penicillin acylase (penicillin amidase, penicillin amidohydrolase, EC 3.5.1.11), were more stable than in the absence of the polyol.     

Advances in enzymatic transformation of penicillins to 6-aminopenicillanic acid (6-APA)

This article elaborates on the important recent developments in the enzymatic transformation of penicillins to 6-aminopenicillanic acid (6-APA), which is the basic raw material for the industrial production of semisynthetic penicillins such as amoxycillin and ampicillin. Particular emphasis is placed on the improvements in purification, stability, and immobilization of the enzymes, (i.e. penicillin acylases) used for these transformations.     

A quantitative microbiological determination of 6-aminopenicillanic acid

Several complicated chemical and microbiological methods have been described for the quantitative assay of 6-aminopenicillanic acid in the presence of benzyl- and phenoxymethylpenicillin. In all these methods the penicillins must be removed since they interfere with the assay. A specific microbiological plate assay withSerratia marcescens D 217 (ATCC 27117) is described for estimating even small amounts of 6-APA in benzyl- and phenoxymethylpenicillin samples. The contents of this paper were presented at the Xth International Congress for Microbiology at Mexico City 1970. The author wishes to express his gratitude to Mrs. I. Struyk-Hoette and Messrs. A. P. Struyk and R. G. W. Spickett for their valuable help and discussions.     

Enzymatic conversion of norsolorinic acid to averufin in aflatoxin biosynthesis

5'-Hydroxyaverantin (HAVN) was isolated from a mold, Emericella heterothallica IFO 30842. Aspergillus parasiticus NIAH-26, a UV-irradiated mutant of A. parasiticus SYS-4, produced neither aflatoxins nor precursors in yeast extract-sucrose (YES) medium. When the postmicrosome (cytosol) fraction of NIAH-26, which had been prepared from the culture in YES medium, was incubated with norsolorinic acid (NA) in the presence of NADH or NADPH, averantin (AVN) was produced. The reverse reaction from AVN to NA was promoted by the addition of NAD or NADP (dehydrogenase reaction). When the microsome fraction of NIAH-26 was incubated with AVN, HAVN was produced in the presence of NADPH (monooxygenase reaction). HAVN was, furthermore, oxidized to averufin (AVR) by the cytosol fraction of NIAH-26 in the presence of NAD or NADP (dehydrogenase reaction). In the feeding experiments with A. parasiticus NIAH-26, aflatoxins were produced from AVN, HAVN, NA, and AVR but not from averufanin or averythrin. These results indicate that the reaction sequence NA in equilibrium AVN----HAVN----AVR is involved in the biosynthetic pathway of aflatoxins. The enzyme activities described here were dependent on the culture medium, and no enzyme activities were observed in the nonaflatoxigenic strain A. oryzae SYS-2 (IFO 4251).