DE2217745A1 - Penicillin acylase prepn - from e scherichia coli - Google Patents

Abstract

Pure penicillin acylase (I) is obtained from E.coli, by a) Isolating and disintegrating the cells, and acidifying to pH 3.5-5.5 to remove cell debris. b) Adsorbing onto an alumino-silicate, pref. bentonite and eluting with a salt, pref. NaOAc or KOAc c) Purifying the eluate by ion-exchange chromatography an anionic and/or cationic resin. d) Inducing crystallisation by adding (NH4)2SO4. (I) is used in prepn. of 6-aminopenicillanic acid.

Description

Pure crystalline penicillin acylase and process for its preparation The present invention relates to the pure, crystalline enzyme penicillin acylase from Escherichia coli and the process for its preparation.

Penicillin acylase (penicillin amidase, penicillin deacylase) (EC (enzymes Commission) 3.5.1.11) is an enzyme that has been known for a long time (K. Sakaguchi and S. Murao, J. agr. Rohem. Soc., Japan, 23, 411 (1950)), which has the amide bond in the 6-position of benzylpenicillin to form 6-aminopenicillanic acid (6APS) and phenylacetic acid hydrolyzed. This enzyme has been found in large numbers of bacteria and fungi (J.M.T. Hamilton-Miller, Bact. Rev. 30, 761 (1966)). Penicillin acylase from E. coli has a broad specificity; in addition to benzylpenicillin, there will be a large Number of other penicillins, e.g. penicillin X and ampicillin, as well as numerous am Amide nitrogen, aliphatic or aromatic substituted amides of phenylacetic acid and their derivatives substituted on the a-C or on the benzene ring hydrolyzed tM. Oole, Biochem. J. 115, 733 (1969); W. Kaufmann and K. Bauer, Nature 203, 520 (1964)).

The effect of the enzyme is used worldwide in large-scale technical maps for the production of 6APS from benzylpenicillin.

The technical implementation of this process has so far been carried out by Use of the whole cells containing the enzyme (German patent specification 1 111 778) or an enzyme extracted from the cells but not further purified (U.S. Patent 3,297,546), or the crude adsorbed on bentonite Enzyme (U.S. Patent 3,446,7o).

In the latter case, the examples describe the procedure only for the penicillin acylase from Bacillus megaterium and from Streptomyces, so that no evidence for the enzyme from E. coli can be obtained from this.

The adsorption of proteins and enzymes on bentonite is known (Methods in Enzymology, Vol. I, 96), but so far e6 has only been found in the rarest of skins succeeded in producing an adsorptively bound enzyme in good yield and increased purity to elute again.

Use whole cells or extracted, but unpurified However, the enzyme has several disadvantages.

In particular, one such disadvantage is the decomposition of part of the penicillins used by the action of others contained in the raw preparations Enzymes, especially penicillin B-lactamase, and thereby caused a decreased Yield of 6APS. Another important disadvantage is contamination of the 6APS by proteins, which lead to allergic reactions when using the from this 6APS produced synthetic penicillins in humans. These impurities can only be removed from the 6APS through complex and lossy cleaning steps remove.

The disadvantages mentioned when using whole cells or one raw, cell-free enzyme are obtained when using a pure penicillin acylase preparation avoided. So far, however, there has been a simple, technically feasible process not available for the pure preparation of the enzyme.

The partial purification of penicillin acylase in smaller quantities has already been described variously (P.S. Borkar et al., Hindustan Antibiotics Bull. 4, 152, (1961); A. Szentirmai, Acta Microbiologica Acad. Sci. Hung.

12, 395, (1966); THERE. Self et al., Biotechnology and Bioengineering 11, 337, (1969)). These processes run over several precipitation, adsorption or Chromatography stages and only give low yields. A partial cleaning on a larger scale by precipitation with tannin and purification with various ion exchangers is also described in German Offenlegungsschrift 1 907 365. One as electrophoretic Acylase preparation labeled homogeneously was described by Bondareva et al. (Biochemistry (Russ.) 34, 76, (1969)) on a laboratory scale by ammonium sulfate precipitation, chromatography obtained on CM cellulose and chromatography on DEAE cellulose in 27% yield. The crystallization of penicillin acylase has not yet been described. These known processes either only provide a partially purified enzyme or are complicated and lossy.

There has therefore been a need for a simple method of manufacture of pure penicillin acylase.

In addition, the crystallization was desirable because on the one hand it is a purity criterion and, on the other hand, the crystal suspension is stable Represents the storage and transport form of the enzyme.

The present invention describes a method that allows to obtain and crystallize the pure penicillin acylase from E. coli in a simple manner. The adsorption / elution step with betonite greatly reduces the Volume achieved, the implementation of the inventive method in the technical Scale enables.

The activity of the penicillin acylase produced according to the invention u is determined by a novel colorimetric test, which has a broad specificity exploits the enzyme (E. Rauenbusch, ablication in preparation). Serves as a substrate 6-nitro-3 - @ - phenylacetyl) aminobenzoic acid (NIPAB). The measurement is made at 0.002 M solution PH 7.5, 250C at 405 nm. The molar extinction coefficient of the 6-nitro-3-aminobenzoic acid formed in the reaction is 9090 1 mol cm-l 1. One enzymatic unit (E) breaks down 1 µmol of the substrate per minute. The split from Benzylpenicillin under the same conditions is about 1.5 times faster. the specific activities are related to protein determined by the biuret method.

The process according to the invention starts with a crude extract from E. coli cells. This is e.g. made from one according to DP 1 111 778 Culture of the E. coli cells or preferably from the one in the German patent application (p 21 54 213.2) described mutant E. coli ATCC 21 728 by treatment with 3 % Methyl isobutyl ketone at pH 7-8 or obtained by mechanical digestion, with the penicillin acylase is brought into solution to 70-90%.

This crude extract is used according to the inventive method easier precipitation of cell debris and simultaneous purification of penicillin acylase by precipitation of inactive protein and other accompanying substances by adding a Mineral acid, e.g. HC1, HNO3, H2SO4, HSPO4, or another strong one Acid, e.g. acetic acid, brought to a pH of 3.5-5.5, preferably 5.0 and the precipitated material in the usual manner by centrifugation or filtration separated and discarded.

The clear solution obtained as the supernatant becomes pericillin acylase by adding an aluminum silicate material, preferably of the montmorillonite type, preferably se bentonite, bound by adsorption; the mineral is in usual size separated by filtration or centrifugation and the penleillin acylase from the Eluted residue with a suitable salt loess.

The adsorption of penicillin acylase to the mineral can occur in a specific conductivity of the solution of o-5 mS and a pH of 3.5-8.0. In the preferred embodiment of the method according to the invention, a conductivity of l-4 m $ and a pH value of 5, o observed. The discontinuation of the solution of the enzyme the desired conductivity can be achieved by diluting; by desalination with mixed bed ion exchangers and by other common desalination processes. The best yield and enrichment in the subsequent elution, just enough bentonite is obtained as required for the 100% adsorption of the enzyme, which is determined by measurement the activity is checked in a sample of the supernatant. Among the preferred Conditions are about 2, o-5, o g bentonite / g dissolved protein. An even better one Purification is achieved when fractional precipitation is carried out by first add about 1/5 of the total amount of bentonite, almost exclusively inactive Foreign protein is adsorbed. After separating the added bentonite, then by adding the remaining amount the panicillin acylase adsorbed. To the Elution of the adsorbed penicillin acylase becomes o, l-l, o molar aqueous solutions inorganic or organic salts, e.g. phosphate, sulfate, acetate, at a pH value used by 6, o-9, o. Preferably, o, l-l, o molar solutions of Na or K acetate is used at pH 8.5 because it is divalent and divalent compared to salts trivalent anions preferentially penicillin acylase is eluted. Overall is through the adsorption on bentonite and elution of the enzyme a 4-6-fold enrichment in about 70-9o 96 yield achieved. The special advantages of this cleaning step lie in the simplicity of implementation on a technical scale, the volume reduction the solution containing the enzyme to 1 / lo of the crude extract and the recovery of a concentrated solution of the enriched enzyme, which makes further processing essential relieved.

The further purification of the penicillin acylase up to the pure enzyme protein takes place according to the process according to the invention by chromatography on suitable macroporous ion exchangers and subsequent crystallization. You can either by treatment with an anion exchanger or a cation exchanger win a solution of the partially purified enzyme, from which the pure enzyme is produced Can crystallize addition of a precipitating salt. Or you win in 2 steps by treatment with a cation exchanger and an anion exchanger in any Order a solution of the pure homogeneous enzyme, from which the enzyme then likewise can be crystallized.

Particularly suitable anion exchangers are those with basic groups substituted cellulose, e.g. aminoethyl cellulose, diethylaminoethyl cellulose (DEAE cellulose), Triethylaminoethyl cellulose (TEAE cellulose d corresponding cross-linked dextrans, e.g. DEAE-Sephadex W and QAE-Sephadex t The cleaning on these anion exchangers can be done at pH 6.5-9.0. Preferred the procedure will be in shape carried out on a column.

The enzyme is adsorbed from a dilute saline solution and through Lower the pH or increase the salt concentration in the elution buffer. Penicillin acylase activity is regularly in the first one to be eluted from the column Contains protein crops. The elution is preferably carried out with a concentration gradient from 0.01 to 0.1 M phosphate pH 7 0. The yield in this purification step is 30-90%.

Particularly suitable cation exchangers are those with acidic groups substituted cellulose, e.g. sulfoethyl cellulose, phospho-cellulsose, carboxymethyl cellulose, cross-linked dextrans, e.g.

SE-Sephadex, SP-Sephadex, CM-Sephadex and acrylamide gels, e.g.

CM-Bio-Gel or those containing carboxyl groups produced according to Biochemistry 0 4074 (1969) Polyacrylamide gels and macrotorous resin ion exchangers, e.g. Lewatit R SP-100, SP-120, CNPo The penicillin acylase is adsorbed onto the cation exchanger from 0.001-0.1 M saline solution at pH 3.5-6.0, preferably at pH 5.0 either by Adding the ion exchanger to the solution of the enzyme or by passing over the Enzyme solution through a column filled with the exchanger. Elution takes place either at a higher pH value of 6.0-8.5 or more advantageously at constant pH by increasing the salt concentration in stages or in the form of a linear gradient0 The preferred embodiment of the method according to the invention for the presen- tation of the pure penicillin acylase is elution at pH 5p0 through a linear gradient from 0.05-0.5 M sodium or potassium acetate. The penicillin acylase is eluted under these conditions as a sharp zone at about 0.2 M sodium acetate in 80-90% Yield.

From the use of anion exchangers alone or in combination solutions of the highly enriched peniclin acylase obtained with a cation exchanger the enzyme becomes through precipitation with a suitable salt, preferably Brought ammonium sulfate to crystallize. The addition of the salt can be considered concentrated Solution or in solid form. Very dilute solutions of the enzyme are used beforehand conveniently concentrated by known methods, e.g. by membrane filtration or by precipitation with ammonium sulfate to 70% saturation and redissolving the Precipitation in a smaller volume. Crystallization takes place depending on the protein concentration from 45-55% saturated ammonium sulfate solution, preferably at a pH of 6.0-7.0 and room temperature within a few days.

Crystals that form quickly have the shape of splintered ones under the microscope Needles. With slow crystallization one obtains very regular tables (fig 1: Copy of a picture at 200x magnification). The solution of the crystallized Enzyme gives only 1 sharp band in disk electrophoresis (7% gel, pH 8.9) (Fig 2).

The pure crystalline penicillin acylase is characterized by the following properties characterized: specificity: cleavage of benzylpenicillin, N-phenacetyl-L-asparagine, 6-Nitro-3 (N-phenacetyl) aminobenzoic acid and other amides of phenylacetic acid.

Molecular weight: 71000 + 2000 from the sedimentation equilibrium with one partial specific volume of v = 0.665 ml / g; 70,000 t 5,000 from thin-layer gel filtration Sedimentation coefficient: S20, w = 2.56 # 0.035 at pH 7.4 Uv spectrum (Figure 3): in phosphate buffer pH 7.0 (A) and in 0.1 N NaOH (B) CD spectrum: pH 4-10, (FIG 4) Conditions: <250 nm: 0.085 mg / ml, d = 0.1 cm; > 250 nm: 0.85 mg / ml, d = 1 cm [Amino acid analysis} Isoelectric point: 6.8 + 0.2 (determined by electrofocusing) SephadextY: registered trademark of Pharmacia Fine Chemicals AB, Uppsala Biogel R: registered trademark of Bio-Rad Laboratories lewatit R: registered Trademark of Farbenfabriken Bayer AG Example 1 a) The zur Implementation of the method according to the invention required crude extract from E. coli cells is obtained by making a Kuleur of E. Coli ATCC 21 728 by centrifugation Concentrated to a Schlann and the Zellon according to known mechanical methods, optionally with the addition of 3% methyl isobutyl ketone, opens up.

b) 610 liters of a crude extract obtained in this way, containing 1.3.

106 U, according to the invention with deionized water to 2100 Ltr. Pre-diluted, adjusted to pH 5.0 with sulfuric acid and centrifuged. In the clear The supernatant was found to be 1.42.106 U with a specific activity of 0.421 U / mgO sub After the pH had been set to 5.0, 13.8 kg of bentonite (type B from Erbslöh) were stirred in and separated after 30 minutes in a centrifuge. The separated bentonite was eluted with 90 liters of 0.5 M sodium acetate solution, pH 8.0, and filtered off. Obtain were 92.5 liters, containing 1.12.106E (86%) with a specific activity of 1.64 U / mg.

c) 1 liter of the bentonite eluate obtained according to b0 was against 0.0001 Dialyzed potassium phosphate buffer pH 7.0: 8250 U 1.8 U / mg.

The enzyme solution was made of DEAE-Sephadex through a column 10 × 15 cm A-50 given in the same buffer. This was followed by a linear gradient eluted from 2 liters of 0.01 and 0.1 M buffer pH 7.0 each. There were fractions of 25 ml caught. Penioillin acylase was found in fractions 110-170; 1.2 l 6760 U (83%) 6 U / mg.

d) The solution from c) was obtained by adding solid ammonium sulfate (472 ct / liter) precipitated, centrifuged off the bodice and dissolved a in water Part of these solutions contain 4700 E, with ammonium sulfate to 46% saturation offset, centrifuged until clear and stored at room temperature.

Crystallization began within a week. The crystals were centrifuged off, dissolved in 0.01 M phosphate buffer and against the same buffer dialyzed: 3300 U (70 56) with 12.45 U / mgO Example 2 60 liters of one according to the example 1 a) prepared digested crude extract were adjusted to pH 5.0 and centrifuged. The supernatant clear solution contained 155,000 U of the specific Activity 0.3 U / mg. The addition of mixed-bed ion exchangers resulted in up to 1.0 MS desalinated. 200 g of bentonite (type SF, Fa.

Serva) added, centrifuged and discarded; then there were others 800 g added, centrifuged and with 20 Ltr.

0.5 M sodium acetate pH 8.0 eluted. Result: 110,000 U, 1.5 U / mg.

The enzyme solution was dialyzed against 0.5 M sodium acetate buffer pH 5.0 and on an 11x95 cm1 column filled with SE-Sephadex C-50 in 0.05 M sodium acetate pH 5.0, given. The column was with a linear gradient of 15 Ltr. 0.07 and 0.25 M sodium acetate pH 5.0 eluted. Penicillin acylase was found in the eluate of 24-27 I; tr. Result: 88,000 U (80 56), 4.2 U / mg.

29,000 U of this solution were against 0.01 M potassium phosphate buffer pH 7.0 dialyzed and applied to a column 11x25 cm DEAE-Sephadex A-50 in 0.01 M potassium phosphate pH 7.0 given. Elution took place with a gradient of 0.01 to 0.1 M potassium phosphate pH 7.0, penicillin acylase was eluted towards the end of the gradient. Result: 21,000 E (73 56); 11.2 U / mg.

Part of this solution, containing 6000 U, was made with solid ammonium sulfate brought to approx. 45 56 saturation. Large crystals formed after about 2 weeks on the vessel wall. After further 2 weeks at room temperature the crystals are centrifuged off and an aliquot is dissolved in water.

Yield: 4260 U (71 56), 13.1 U / mg.

Example 3 Containing a bentonite eluate produced according to Example 1 4100 U at 2.0 U / mg was dialyzed against 0.05 M sodium acetate pH 5.0 and over given a column 2.5 x 25 cm with Lewatit SP-100-Na + form. The elution took place with a gradient of 0.05-0.5 M sodium acetate pH 5.0. The active fractions contained 2020 E (56 56), 6.6 U / mg.

The enzyme solution was adjusted to pH 6.5 with NaOH and added of solid ammonium sulfate to 48 56 saturation brought the pure enzyme to crystallization. 1250 U (62 56), 12.1 U / mg.

Claims (5)

Claims 1) Pure, crystalline penicillin acylase from Escherichia coli 2) procedure for the production of pure, crystalline penicillin acylase, characterized in that that you get a crude extract from E. coli cells, which in addition to penicillin acylase still contains cell debris containing E. coli cells to a pH of 3.5 to 5.5, preferably 5.0, adjusts and centrifuges the extract, the clear solution obtained as a supernatant the penicillin acylase treated with aluminum silicates, preferably bentonite, the adsorbate is separated and eluted, the enriched clear solution obtained the penicillin acylase chromatographed on macroporous ion exchangers, and off the solution of the partially purified penicillin acylase obtained in this way is the pure enzyme by adding ammonium sulfate, optionally dissolved in water, for crystallization brings. 3) Method according to claim 2, characterized in that as Ion exchanger uses an anion exchanger. 4) Method according to claim 2, characterized in that as Ion exchanger used a cation exchanger. 5) Method according to claim 2, characterized in that as Ion exchanger an anion exchanger and a cation exchanger one behind the other used in any order. L e r s e i t e