Showing papers on "Penicillin amidase published in 1976"

Continuous production of 6-aminopenicillanic acid from penicillin by immobilized microbial cells

Abstract: For continuous production of 6-aminopenicillanic acid (6-APA) the microbial cells ofEscherichia coli ATCC 9637 having high penicillin amidase (penicillin amidohydrolase, E.C. 3. 5. 1. 11) activity were immobilized by entrapment in a polyacrylamide gel lattice. Enzymatic properties of penicillin amidase of the immobilizedE. coli cells were investigated and compared with those of the intact cells. With regard to optimal pH and temperature, no marked difference was observed. The heat stability was somewhat increased by immobilization of the cells. The enzyme activity of the immobilized cell column was stable, and its half-life was 17 days at 40°C and 42 days at 30°C. From the effluent of the column, 6-APA was easily obtained in a good yield.

Study of E. coli penicillin amidase. The pH dependence of the equilibrium constant of the enzymatic hydrolysis of benzylpenicillin

Abstract: The equilibrium constant for penicillin amidase-catalyzed hydrolysis of benzylpenicillin(Keg =3.00 +/- 0.24 x 10(-3) M at pH 5.0) and the ionization constants for phenylacetic acid (PAA) and the amino groups of 6-aminopenicillanic acid (6-APA) were determined (4.20 and 4.60 under conditions of the kinetic experiments respectively). The experimental data at pH 6.0 satisfactorily correlated with the theoretical pH-dependence for Keg constructed according to the hypothesis that benzylpenicillin synthesis has a thermodynamic optimum at pH 4.4 equal to a half-sum of the pK values for the carboxylic and amino groups of the PAA and 6-APA respectively.

Process for preparing 6-aminopenicillanic acid

Abstract: A simple and efficient process for the deacylation of penicillins to 6-aminopenicillanic acid in which an aqueous penicillin solution is rapidly recirculated through a shallow bed comprising particulate immobilized penicillin acylase at 15°-45° C and pH 6.5-9.0 until substantial conversion results. BACKGROUND OF THE INVENTION This invention relates to penicillins. More specifically, it relates to the enzymatic deacylation of penicillins to 6-aminopenicillanic acid. 6-Aminopenicillanic acid, commonly referred to as 6-APA, is an intermediate in the manufacture of synthetic penicillins and is prepared among other means by the deacylation of penicillins. This conversion has been effected by both chemical and biochemical techniques. The chemical conversion, as exemplified by U.S. Pat. No. 3,499,909, suffers from being a multi-step process requiring energy-intensive low-temperature conditions and specialized equipment. The biochemical conversion utilizes the enzyme penicillin acylase, or penicillin amidase. In U.S. Pat. No. 3,260,653, the enzyme activity is supplied by certain bacteria or bacterial extracts. This approach is not entirely satisfactory for the industrial production of 6-APA since the product stream is contaminated with the enzyme and/or microbial cells, which must then be removed during product recovery, and the enzyme is used only once. The problems of product contamination and poor enzyme utilization are purportedly overcome in U.S. Pat. No. 3,953,291 by the use of immobilized penicillin amidase-producing microbial cells. Such a process using immobilized cells is still characterized by low productivity, however, since in batch operation the process suffers from its non-continuous nature and excessive handling of the immobilized cell material, while in column operation it suffers from poor pH control and less than optimum enzyme utilization. The use of a shallow bed of microbial cell catalyst for the continuous isomerization of glucose to fructose is disclosed in U.S. Pat. Nos. 3,694,314 and 3,817,832. The shallow bed is reportedly employed to minimize the pressure drop through the catalyst, the desired conversion being achieved by passing the aqueous process stream through several beds in series. SUMMARY OF THE INVENTION It has now been found that penicillins can be converted to 6-APA in a simple and efficient manner by rapidly recirculating an aqueous penicillin solution through a shallow bed comprising particulate penicillin acylase catalyst under controlled temperature and pH conditions. Accordingly, the present invention entails a process for the enzymatic conversion of a penicillin to 6-APA which comprises recirculating an aqueous solution of the penicillin through a bed up to about 6 cm deep comprising particulate immobilized penicillin acylase catalyst at a flow rate of at least 0.4 bed volume per minute while maintaining the solution at a temperature of from about 15° to 45° C. and a pH from about 6.5 to 9.0 and continuing the recirculation until the penicillin is substantially converted to 6-APA. Preferably the penicillin is potassium penicillin G, the bed has a depth of from about 2 to 3 cm, the particulate catalyst comprises immobilized Proteus rettgeri cells containing the enzyme, the temperature is about 35° to 40° C. and the pH is from about 7.5 to 8.2. DETAILED DESCRIPTION OF THE INVENTION The process of the present invention, in rapidly recirculating the process stream through a shallow bed of particulate catalyst, is thus able to optimize the conversion of penicillins to 6-APA since it overcomes the heretofore unsolved problems of pH and flow control associated with continuous column deacylation and of excessive catalyst handling with batch deacylations. The capability of the process to control pH is especially significant since the deacylation generates a carboxylic acid which must be neutralized, the enzyme is optimally active over a narrow pH range and both the reactant and product are sensitive to pH extremes. Since this control is accomplished with a minimum of pressure drop through the bed, the process has the further advantage of utilizing standard process equipment. The process is suitable for the deacylation of any water-soluble penicillin. Representative penicillins include but are not limited to penicillin G (benzylpenicillin), penicillin X (p-hydroxybenzylpenicillin) and penicillin V (phenoxymethylpenicillin). Preferred is penicillin G in the form of the potassium or sodium salt. The concentration of the penicillin substrate in the aqueous solution is not critical and normally varies from about 1 to 20 g/100 ml solution. By particulate immobilized penicillin acylase catalyst is meant the enzyme penicillin acylase, or any penicillin acylase-producing microorganism, entrapped within or attached to or on a water-insoluble particulate matrix of organic or inorganic origin in such a manner as to retain the enzyme's activity. Suitable penicillin acylase-producing microorganisms include those belonging to the general of Proteus, Escherichia, Streptomyces, Nocardia, Micrococcus, Pseudomonas, Alkaligenes and Aerobacter such as disclosed in U.S. Pats. Nos. 3,260,653 and 3,953,291. The common methods employed for such immobilization of enzymes and microbial cells include covalent bonding to the matrix, entrapment within the matrix, physical adsorption on the matrix and cross-linking with a bifunctional reagent to form the matrix. Illustrative of these immobilization techniques are those of U.S. Pats. Nos. 3,645,852, 3,708,397, 3,736,231, 3,779,869, 3,925,157, 3,953,291 and 3,957,580. As indicated by these references, the matrix is typically a polymer or copolymer of such monomers as glycidyl methacrylate methacrylic acid anhydride, acryloylamide, acrylamide, styrene, divinylbenzene or glucose, or the matrix may be of such substances as bentonite, powdered carbon, titania, alumina or glass. Preferred catalyst is one in which Proteus rettgeri cells containing penicillin acylase are immobilized by the process of U.S. Pat. No. 3,957,580. Such particulate catalysts will normally have an activity of from about 200 to 5,000 units (micromoles penicillin G deacylated per hour) per gram of dry catalyst. The particulate catalyst is utilized in the form of a shallow bed through which the process stream is rapidly recirculated. By shallow bed is meant a bed having a depth of up to about 6 cm. The actual depth of the bed is determined by the desired productivity of the conversion unit, the activity of the particulate catalyst and, in the case of immobilized microbial cell catalyst, the concentration of cells in the particulate catalyst. The bed should be deep enough to supply sufficient enzyme activity for the desired productivity of the unit and not so deep as to prevent the desired flow discussed hereinafter. At times it may be advisable to admix the catalyst with a particulate material such as diatomaceous earth, perlite or powdered cellulose added in the amount of up to about 80 volume percent of the bed to give the bed a more porous structure. Beds about 1 to 6 cm deep normally meet the desired productivity and flow requirements. Particularly suitable units for preparing such beds include standard filtration equipment such as a horizontal pressure leaf filter or a plate-and-frame filter press. The conversion is run under controlled temperature as well as pH conditions to maximize productivity while minimizing substrate, product and catalyst degradation. The temperature is limited to between about 15° to 45° C and preferably between about 35° and 40° C. The activity of the catalyst drops off considerably at temperatures much below 15° C while temperatures much above 45° C result in considerable decomposition of the penicillin and 6-APA and appreciable denaturization of the catalyst. As indicated hereinbefore, pH control is critical to high conversion of penicillin to 6-APA, and the pH of the process is therefore limited to from about 6.5 to 9.0. A pH much below 6.5 results in considerably reduced enzyme activity and in enhanced penicillin degradation, while a pH much greater than 9.0 accelerates not only penicillin degradation but also enzyme denaturization. The pH is preferably maintained between about 7.5 and 8.2 when the catalyst is derived from Proteus rettgeri. In practice, the bulk of the process stream is maintained at the desired temperature and pH in a stirred tank or reservoir. A small portion of the stream is continuously passed through the catalyst bed and quickly returned to the reservoir where the acid formed during the passage through the bed is neutralized by a suitable base such as sodium hydroxide to maintain the pH within the desired range. Such a system can be adapted to batch, semi-continuous or continuous operation. The flow rate of the process stream through the catalyst bed is also of critical importance in assuring optimum conversion of penicillins to 6-APA, and should be at least 0.4 bed volume per minute. Flow rates much below this value not only cause a considerable reduction of catalyst utilization resulting from poorer diffusion of the substrate and product within the catalyst bed but also enhance the degradation of substrate, product and catalyst from the increase localized acidity present in the bed. The recycling of the process stream through the catalyst bed is continued until the penicillin in the stream has been substantially (at least 80 percent) converted. The 6-APA in the stream may then be either isolated or reacted to a desired penicillin by conventional means.

[Study of penicillin amidase from E. coli. pH-dependence of kinetic parameters of enzymatic hydrolysis of benzylpenicillin].

Abstract: The authors studies pH-dependencies of the kinetic parameters (Vm, KM, Vm/KM) and constants of competitive inhibition by phenylacetic acid of penicillinamidase-catalyzed hydrolysis of benzylpenicillin. The experimental data are in agreement with the assumption according to which there are 3 equilibrium ionogenic forms of the enzyme and enzyme-substrate (or enzyme-inhibitor) complexes, i.e. acidic, neutral and alkaline, the neutral form being the only active form of the Michaelis complex. Values of pK in the ionogenic groups controlling interconversions of both the free enzyme (pK1 6.1 and pK2 7.6) and of the enzyme-substrate complex (pKa 6.1 and pK2 10.2 or the enzyzme-inhibitor complex (pK''1 6.1 and pK''2 9.5) were determined. From this and the previously published results it was concluded that the group with pK 6.1 was involved in the catalysis and the group with pK 10.2 in the maintenance of the active conformation of the active centre of penicillinamidase. The ionogenic group with pK 7.6 was apparently involved in the enzyme-substrate binding.

[Study of the penicillin amidase from E. coli. An ultrasonic method of studying the ph- and temperature-conformational transitions in the active center of the enzyme].

Abstract: pH and temperature conformation transitions in the active center of penicillin amidase i.e. penicillinamidohydrolase E.C. 3.5.I.II were investigated by means of the kinetic method and a new ultrasonic method. It was shown that the catalytic activity of the enzyme was controlled by 2 ionogenic groups with pK 6.1 and 10.2. The study of penicillinamidase by means of the ultrasonic method showed that the ionogenic group with pK 10 was responsible for maintaining the catalytically active conformation of the enzyme active center. Investigation of the temperature relation between the kinetic parameters of the enzymatic hydrolysis of benzylpenicillin catalyzed by penicillin amidase and the data on the effect of ultrasound on the enzyme showed that the enzyme was subjected to the temperature conformation transiton. The temperature and thermodynamic parameters of the conformation transition were determinded (T=318 degrees K, delta H=81 kcal/mole and delta S=255 e.u.). The structure of the active center of the enzyme is discussed on the basis of the data obtained.

[Study of penicillin amidase for E. coli. Kinetics of enzymatic hydrolysis of 7-phenylacetamidodeacetoxycephalosporanic acid].

Abstract: Kinetics of hydrolysis of 7-henylacetamidodeacetoxycephalosporanic acid catalyzed by penicillin amidase as a result of which phenylacetic and 7-aminodeacetoxycephalosporanic acids are formed was studied. The kinetic parameters of the reaction were determined on the basis of both the dependence of the initial rate of enzymatic hydrolysis on the substrate concentration and the analysis of progress kinetic curves of the product accumulation. The values of Km determined by the two methods were equal to (10 +/- 1) muM and kcat (50 +/- 5) sec-1 and (50 +/- 10) sec-1 respectively. The study of the inhibition of the enzymatic hydrolysis by the reaction products showed that phenylacetic and 7-aminodeacetoxycephalosporanic acids were competitive and non-competitive inhibitors of the penicillin amidase activity respectively. The inhibition constants were (55 +/- 8) muM and (12 +/- 1) mM respectively. The physiological role of the enzyme and the effect of the structure of the substrate on the specificity of the enzyme are discussed.