[54b]

PENICILLIN ACYLASE (BACTERIAL)

705

trols carried out. As 6-APA runs slowly in the butan-l-ol/ethanol/water system, the top half of the chromatogram strip can be cut off after spraying and only the lower half laid on the B. subtilis plate. M a n y more strips can thus be handled allowing for replication and randomization of standards and unknowns. The above biochromatographic method is applicable also to measuring the rate of formation of 7-aminocephalosI)oranic acid from cephalosporins such as cephalothin which is also a substrate for the penicillin acylase of Escherichia coli. The l)rep,aration of the B. subtilis-seeded agar plate is e'~rrie(t out as follows : The spores of B. subtilis ATCC 6633 are prei)ared by inoculating Roux bottles containing 350 ml of Oxoid nutrient t)roth plus 2%, agar. The bottles are incubated for 5 days a t 3 7 ° before draihing off surphls liquid and incubating for a further 9 days. Sterile water ~50 mlt is then added to each bottle, and the spores are dislodged by shaking. The suspension so obtained is filtered through sterile glass wool to remove debris, and the filtrate is pasteurized at 70 ° for 1 hr before dispensing into small sterile glass bottles with screw caps for storage at 4 °. The stock sust)ension can be stored for a t least 6 months. A 300-ml volume of Oxoid blood agar base, 3.5% (CM55) is melted, cooled to 60% and inoculated with about 0.5 ml of a suitable dilution (about 1 in 40) of the stock B. subtilis spore suspension. The agar is then poured into a previously leveled tray or plate having a fiat glass base 33 X 40 cm to give a layer about 2 mm thick. The amount of spore inoculum should be such that the individual colonies of B. subtilis are only just touching one another and a 10 ~,g/ml solution of benzylpenicillin when placed in a 8-mm hole cut in the agar gives a zone of inhibition of about 30 mm after incubation. Too heavy an inoculum reduces the sensitivity of the assay.

[54b] Penicillin Acylase (Bacterial) By T. A. SAVIDGE and M. COLt: Penicillin acylases are widely distributed among bacteria and actinomycetes. 1,'-' Most of the published work is on the intracellular enzyme 1M. Cole, Process Biochem. 2, 35 (1967). : M. Cole, Biochem. J. 115, 733 (1969).

706

ANTIBIOTIC INACTIVATION AND MODIFICATION

[54b]

produced by Escherichia coli, and the preparation and properties of this enzyme are described in detail below. In addition and for comparison, we describe the published work 3-6 on the preparation and properties of the extracellular enzyme produced by the gram-positive organism Bacillus megaterium. A selection of other penicillin acylase-producing bacteria is listed in Table VI together with the summary of their properties.

Assay Assay procedures for penicillin acylase are described in a previous article. ~

T h e Penicillin Acylase of E s c h e r i c h i a c o i l Preparation Culture. The enzyme is produced by various strains, e.g., ATCC 9637, ATCC 11105, N C I B 8743,1 or N C I B 8743, selection A, a natural variant capable of growing well on a corr-steep liquor based medium. 2 For industrial use, various higher-yielding derivatives have been obtained by mutation and selection. Stock cultures are prepared from freeze-dried cultures by inoculating nutrient agar slopes and incubating at 27 ° for 24 hr. These slopes m a y be stored at 4 ° for up to 1 month. Workir~g slopes are prepared by subculturing the stock slopes onto the same medium and incubating for 24-48 hr at 270. 2 Fermentation. A most important constituent of the fermentation medium is phenylacetic acid, which is metabolized during the course of bacterial growth, increasing enzyme production by 5- to 10-fold. The nitrogen content of the medium m a y be provided by complex organic sources, such as corn-steep liquor (CSL)S yeast extract, ~ Casamino acids, ~ or by simple chemically defined sources, such as sodium glutamate. 1° Enzyme 3C. Chiang and R. E. Bennett, J. Bacteriol. 93, 302 (1967). U.S. Patent 3,144,395 (1964). 5U.S. Patent 3,446,705 (1969). 6D. Y. Ryu, C. F. Bruno, B. K. Lee, and K. Venkatasubramanian, in "Fermentation Technology Today" (G. Terui, ed.), p. 307. Society of Fermentation Technology, Japan, 1972. This volume [54a]. s British Patent 1,015,554 (1966). ' A. Szentirmai, Acta Microbiol. Acad. Sci. Hung. 12, 395 (1966). loD. A. Self, G. Kay, M. D. Lilly, and P. Dunnill, Biotechnol. Bioeng. 11, 337 (1969).

[54b]

PENICILLIN ACYLASE (BACTERIAL)

707

production is greatest at incubation temperatures of between 24 ° and 30 ° and at rather low aeration rates. A particularly suitable fermentation medium is one based on CSL, the procedure being as follows. A working slope is flooded with saline solution, scraped, and 0.5-ml aliquots of the resulting cell suspension are used to inoculate 500-ml conical flasks closed with lint caps and containing 100 ml of medium consisting of CSL (usually supplied as a concentrated solution), 1.2% w/v on a dry solids basis, adjusted to pH 7.0 with NaOH and sterilized by autoclaving at 121 ° for 10 rain. The flasks are incubated on a rotary shaker (250 rpm) at 24 ° for 24 hr and then used to inoculate fermentation medium consisting of CSL 1.2% w/v dry solids; ammonium sulfate, 0.1% w/v; and phenylaeetic acid added as the ammoniun~, sodium, or potassium salt, 0.1% w/v. The pH is adjusted to 6.5 with NaOH, and aliquots (100 inl) are dispensed into 500-ml conical flasks closed with lint caps and sterilized at 121 ° for 10 rain. The flasks are inoculated with 1 ml of seed culture and incubated on a rotary shaker 1240 rpm, 1 inch radius circle) at 24 ° for 24 hr. For larger volumes a stirred fermentor is inoculated with seed culture (0.5% v/v) developed either in shaken flasks or, depending on the volume of the fermentor, from a second seed stage using the same medium in a stirred fermentor. The aeration and agitation rates during fermentation must be optimized for individual fermentor vessels, aeration rates of 0.4 to 0.9 v/v per minute having been reported. '-',11 Incubation at 24 ° is continued until the pH of the medium increases to 8.0, generally after around 20 hr; typical enzyme levels are around 0.3 units/ml2 ~ Higher enzyme levels have been reported to occur, '~ when the phenylacetate is added gradually from 8 to 21 hr after inoculation as a solution of the ammonium salt at pH 7.0. For a fermentation medimn volume of 600 liters, 600-ml aliquots of ammonium phenylacetate solution ,~re added hourly from 8 to 21 hr after inoculation to give a total addition equivalent to 0.35% w/v phenylacetic acid. The ammonium phcnylacetare solution is prepared by treating 25% w/v aqueous phenylacetic acid with concentrated aqueous ammonium hydroxide to pH 7.0 and sterilizing at 121 ° for 30 rain. The fermentation is terminated when the pH value rises to 8.0 after around 28 hr incubation. In circumstances where it is difficult to obtain corn-steep liquor, yeast extract (e.g., The English Grain Co. Ltd., Burton-on-Trent, Staffordshire) is a suitable alternative. Yeast extract (generally supplied at a solids content of 70%) is added to give a concentration in the medium of 2.5% w/v. With this medium, enzyme titers of 0.03 and 0.07 unit per ~'British Patent 1,250,069 (1971). ~2British Patent 1,261,711 (1972).

708

ANTIBIOTIC INACTIVATION AND MODIFICATION

[54b]

milligram of dry cells were obtained with NCIB 8743 and NCIB 8743 selection A, respectively. ~ Cell Harvest. For small volumes of fermentation broth, the cells may be collected as a paste by centrifugation at 13,000 g. Disc bowl centrifuges are preferable for larger volumesl:~; thus for ca. 500 liters, a Westfalia Disc Bowl Separator, Model SAOOH 205, may be used to produce a slurry at a cell concentration around 10 times that of the fermentor broth. Since the strain of E. coli can be of fecal origin, it may be desirable to kill the cells before harvesting by adding n-butyl acetate to the fermentor broth to a concentration of 1% (v/v), and stirring for 20 rain. Cell Disruption. Chemical methods for releasing enzyme from cells have been reported, TM but the following two mechanical methods are preferred. For laboratory-scale extractions, ultrasonic disintegration is carried out as follows. After resuspension of centrifuged cells in 0.1 M phosphate buffer, pH 7.8 and 4 °, a 25-ml aliquot is transferred to a chilled vessel, and the probe of an M.S.E. ultrasonicator (M.S.E., Crawley, Sussex, England) Model M158 is inserted to a depth of approximately 1 cm. The suspension is sonicated at 20 kc/sec and 100 W for 30 sec. After cooling for 30 sec, sonication is repeated in this fashion for a total of 3-5 rain depending upon the cell concentration. Larger volumes of cell slurry are best disrupted by passing the suspension through an industrial high pressure homogenizer. An example of a machine in common use for releasing intracellular enzymes is the Manton-Gaulin homogenizer (A.P.V. Co. Ltd., Crawley, Sussex, England), in which the suspended microorganisms are forced through a narrow slit between two steel faces. 13 It has been shown, 1~ that the release of fl-galactosidase by disruption of E. coli is dependent on pressure to the power 2.2. To release quantitatively the enzyme from 50 liters of concentrated cell suspension, the slurry is cooled to 4 ° and passed 4 times through a Manton-Gaulin Model 15M/SBA operated at a pressure of 500 kg/cm 2. After each pass the slurry is cooled to below 5 ° . On a small scale, cell debris is removed by centrifugation (e.g., Sharples Centrifuge Model 6P, Sharples Centrifuges Ltd., Camberley, Surrey, England), at. a speed of 15,000 rpm. The use of air-driven laboratory models should be avoided because of the danger of forming aerosols containing endotoxins. Tile large-scale removal of debris may prove difficult ~3S. E. C h a r m and C. C. Matteo, this series Vol. 22, p. 476. ~4Canadian P a t e n t 975,769 (1970). ~ P. P. Gray, P. Dunnill, and M. D. Lilly, in " F e r m e n t a t i o n Technology Today" (G. Terui, ed.), p. 347. Society of F e r m e n t a t i o n Technology, Japan, 1972.

[54bl

PENICILLIN ACYLASE (BACTERIAL)

709

because of the very small size of the solid particles having a small density differential with respect to the aqueous phase, but rotary vacuum filtration has been found to be satisfactory. ~5 Purification

A number of purification procedures have been reported but the following two methods I~','T are recommended. Crystalline enzyme has been obtained by the second method. M e t h o d A. TM After disruption of the E. coli cell slurry in tile MantonGaulin homogenizer and precipitation of nucleic acids with streptomycin sulfate (0.7% w/v), the enzyme is precipitated from the clarified solution by the slow addition of ammonium sulfate to give a 60% saturated solution. After leaving overnight at --5 ° the precipitate is centrifuged off in a Sharplcs 6P centrifuge, and the supernatant is discarded. The enzyme-containing precipitate is stored at 2 ° and processed further when required by redissolving it in 10 mM phosphate buffer, pH 7.0 and fraet.ionally precipitating with polyethylencglycol molecular weight 6000 (Shell Chenficals Ltd.). Tile fraction collected between 10 and 20% w/v polyethyleneglycol is redissolved in 10 mM phosphate buffer, pH 8.0, dialyzed against more of this buffer, and then passed down a column (45 }( 7.5 cm diameter) of DEAE-cellulose (Whatman grade DE-52~. The purified enzyme apl)ears in the first protein-containing fractions (the ~pecific activity of the most active fraction was 12.1 units/rag) which are pooled, dialyzed against 5 mM phosphate l)uffer, I)H 7.5, and freezedried. The powder is stored at --20°; all the isolation steps are carried out at .iround 5 °. The results obtained with this isolation procedure are shown in Table I. The apparent increase in total enzyme activity at the start of the polyethyleneglycol precipitation stage is because the hydroxylamine assay r which was used in the initial stages was replaced by the p-dimethylaminobenzaldehyde assay, ~ which, although less specific, is sufficiently sensitive to permit determination of the initial reaction rate. M e t h o d B. ~ After disruption of the cells, the 1)H of a 4-liter aliquot of ceil homogenate is adjusted to 5.0 with dilute sulfuric acid, and ttlc cell debris is removed by centrifugation. The pH of the supernatant is adjusted to 6.0 and fractionated by the addition of solid ammonium sulfate. The fraction precit)itating between 40 and 6 0 ~ saturation is collected and dissolved in 400 ml of 50 mM sodium acetate pH 5.0 and dialyzed against 20 volumes of the same buffer. The dialyzed solution ~'~K. Balasingham, D. Warburton, P. Dunnill, and M. D. Lilly, Biochim. Biopl~gs. Act(~ 276, 250 (1972). '~ C. Kutzbach and E. Rauenbusch, Hoppe Seyler's Z. Pliysiol. Chem. 354, 45 (1974).

710

ANTIBIOTIC INACTIVATION A N D MODIFICATION

•-3 .~.

6666

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6

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[54b]

[54b]

PENICILLIN ACYLASE (BACTERIAL)

711

is applied to a column (90 X 5 cm) of SE-Sephadex C-50 and equilibrated with the same buffer (chromatography is carried out in a constant temperature room at 20% and the fractions are cooled to 5-10°). Elution is carried out with a linear gradient composed of equal volume {5 liters) of 0.07 and 0.25 M sodium acetate, pH 5.0, added at a rate of 150 ml/hr. After a breakthrough peak, the acylase is eluted between 4.2 and 5.0 liters at a buffer concentration of approximately 0.15 M. The enzymecontaining fractions are concentrated to 130 ml under vacuum, slight amounts of precipitate being removed by centrifugation. The solution is then dialyzed against 10 mM potassium phosphate pH 7.0 and applied to a column (40 X 2.5 cm) of DEAE-Sephadex A-50 in the same buffer. The enzyme is eluted with a linear gradient composed of equal volumes (1 liter) of 10 mM and 0.1 M phosphate, pH 7.0, the acylase occurring in the first protein peak. The enzyme-containing fractions are combined and brought to 1 mM EDTA; the enzyme is precipitated with ammonium sulfate, which is added to give 70% saturation. The precipitate is recovered by centrifugation and dissolved in a minimum volume of ice-cold 45% saturated ammonium sulfate, pH 6.0. A slight turbidity is centrifuged off, and the enzyme is induced to crystallize by storage at room temperature for a few days. The crystals are recovered by centrifugation and suspended in 50% saturated ammonium sulfate. The overall yield of crystalline enzyme is 25%, and the total purification is close to 200fold, the most important step being gradient elution from the cation exchanger (purification factor: 13) The Penicillin Acylase of Bacillus rnega teriurn Preparation Culture. Bacillus megaterium, ATCC 14945 is maintained as lyophilized cell suspensions or on meat extract agar slopes. ~ Fermentation. ~ A seed medium consisting of enzyme-hydrolyzed casein (Amber Lab. Inc., Milwaukee, Wisconsin), 4%; glucose 0.5%, and Ucon antifoam LB 625 (Dow Chemical Co., Midland, Michigan), 0.05% is adjusted to pH 7.0 before and after sterilization at 121 ° for 30 rain. It is then inoculated and incubated with aeration and agitation at 30 ° for 24 hr. This seed is used to inoculate at a concentration of 3% (v/v), fermentation medium having the same composition as the seed medium with the exception of enzyme-hydrolyzed casein at 3% instead of 4%. After incubation with aeration and agitation (0.2 HP/100 gal) at 30 ° for 8 hr, 0.15% (w/v) phenylacetic acid is added and incubation is continued for about 70 hr. An enzyme titer of 0.22 unit per milliliter of cul-

712

ANTIBIOTIC INACTIVATION AND MODIFICATION

[54b]

ture fluid has been reported. 3 The whole broth is then treated with a floceulant, 0.5% (v/v) Primafloc C-3 (Rohm & Haas Co. Philadelphia, Pennsylvania) and 0.2% v/v toluene. The pH is adjusted to 7.0-7.5, the cells are removed by centrifugation as described for the E. coli enzyme and the enzyme-containing supernatant is retained. Extraction and Purification 3 The supernatant liquid is acidified with dilute acetic acid to pH 6.4, and the enzyme is adsorbed onto acid-washed Celite (No. 545, JohnsManville Co., New York) added at a ratio of 15 g/liter. The suspension is stirred for 2 hr, during which time the pH is maintained at 6.4 by further addition of acetic acid; the Celite-enzyme complex is collected by filtration and then slurried in 24% (w/v) ammonium sulfate in 0.1 M Tris buffer adjusted to pH 8.4 with NH40H and transferred to a glass column. Enzyme is eluted from the Celite column by further addition of the ammonium sulfate solution, and the eluate is concentrated under vacuum at 40 ° . The protein gradually precipitates from the solution, concentration being stopped as soon as ammonium sulfate begins to crystallize. The precipitated protein is separated by filtration and then resuspended in 50 mM phosphate buffer, pH 7.0 and dialyzed for 24 hr against a 50-fold volume of 25 mM phosphate buffer, pH 6.4. Further purification is achieved by fractionation on a column (47 X 4.7 cm) containing carboxymethylcellulose, impurities being eluted at a flow rate of 100 ml/hr with 25 mM phosphate buffer, pH 6.4, and the enzyme with 0.1 M phosphate buffer, pH 6.5. By repeating the Celite adsorption at a ratio of 75 g/liter and ammonium sulfate precipitation, the enzyme is purified still more, as indicated in Table II. Properties o] the Enzymes The properties of the enzymes produced by E. coli and B. megaterium are given below. Those for other bacteria and actinomycetes are given in Table VI. Physical Characteristics. The crystalline E. coli enzyme forms regular rectangular plates (ca. 150 X 80 ~m) having a specific activity of 48 units/mg, a molecular weight of 71,000 ± 2000 by sedimentation equilibrium (cf. the B. megaterium enzyme, 120,000 determined by the same method~), and 70,000 ± 5000 by thin-layer gel filtration. The isoelectric point determined by gel electrofocusing is 6.8 ± 0.2. For partially purified enzyme preparations, enzyme is visualized by staining the gel with a

[54b]

PENICILLIN ACYLASE (BACTERIAL)

713

TABLE II PURIFICATION OF PFNICILLIN ACYbASE FROM Bacillus megaterium

Fraction Crude broth supernatant fluid First-cycle Celite adsorption and ammonium sulfate precipitation Carboxymethylcellulose column fractionation Second-cycle Celite adsorption and ammonimn sulfate precipitation

Total Total Specific Volume activity ~ protein activity Yield (ml) (kilounits) ( m g ) (units/rag) (%) 34,000 58

7.6 5.25

23,000 340

0.33 15.5

101) 69

565

3.9

160

24.6

52

10

2.55

81

31.5

33

" Unit of enzyme activity is defined as the amount of enzyme required to produce i tmmle 6-aminopenicillanic acid from benzylpenicillin per minute in 0.1 M borate buffer (pH 8.7) at 37°.

novel substrate, 6-nitro-3-phenylacetamidobenzoic acid which is cleaved to form a yellow product, 6-nitro-3-aminobenzoic acidY pH and Temperature Optima. The optimum temperature for the initial rates of reaction using benzylpenicillin as substrate is around 55 ° for tile E. coli'-' and 45 ° for the B. megaterium 3 enzymes. Both enzymes are unstable at these temperatures however and 37 ° is generally chosen. Tilt- ot)timmn pH values for the two enzymes are about 8.2 for E. coli and about 8.5 for B. megaterium, but at these values, benzylpenicillin can be rather unstable and slightly lower values, e.g., 7.8, are used. Kinetics and Inhibitors. Both of the enzymes described above are inhibited by the reaction products produced by the hydrolysis of benzylpenicillin. ~,'" Phenylacetie acid competitively inhibits tile two enzymes, although as the kinetic data in Table I I I show, the inhibitory effect on the B. megaterium enzyme is very much less than on E. coli acylase. Both enzymes are noncompetitively inhibited by 6-APA and again, the effect is greater with the E. coli enzyme. A further difference between tile two enzymes is that the E. coli acylase only is inhibited by substrate. The K,, values of the two enzymes are also shown in Table I I I ; this constant has been determined for the enzymes obtained from various sources, and these are smmnarized in Table IV. Although the conditions of temperature and pH under which these values were determined m a y differ, the very high value for E. coli cells suggests considerable diffusional restrictions across the cell wall. Nevertheless the use of the cell-

714

ANTIBIOTIC INACTIVATION AND MODIFICATION

[54b]

TABLE I l l COMPARISON OF KINETIC CONSTANTS OF PENICILLIN ACYLASES PRODUC~'D BY Escherichia coli AND Bacillus megaterium Parameter

E. coli NCIB 8743A~

B. megaterium ATCC 14945b

Vm,x (~moles 6-APAd/minute/mg) Km (mM) Ki, 6-APA, (mM) Ki, phenylaeetic acid (mM) K., benzylpenicillin (mM)

7.1 0.67 7.1 4.8 270

1.1 c 4.5 26 450 Not inhibited

a Kinetic constants obtained at pH 8.0 and 37 °. a Kinetic constants obtained at pH 8.7 and 37 °. Calculated from graphical data of C. Chiang and R. E. Bennett [J. Bacteriol. 93, 302 (1967)]. a 6-APA, 6-aminopenicillanicacid. TABLE IV COMPARISON OF Era VALUES OF PENICILLIN ACYLASE FROM DIFFERENT SOURCES Organism Escherichia coli ATCC 9637 E. coli NCIB 8743A Crystalline enzyme of Escherichia coli ATCC 11105 Whole cells of E. coli NCIB 8743A Bacillus megaterium Streptomyces lavendulae Fusarium semitectum

Km Value (mM)

Reference

7.7 0.67 0.02

a b c

30.0 4.5 10.3 2.5

/

a D. A. Self, G. Kay, M. D. Lilly, and P. Dunnill, Biotechnol. Bioeng. 11, 337 (1969). a K. Balasingham, D. Warburton, P. Dunnill, and M. D. Lilly, Biochim. Biophys. Acta 276, 250 (1972). c C. Kutzbach and E. Rauenbusch, Hoppe Seyler's Z. Physiol. Chem. $54, 45 (1974). d M. Cole, Biochem. J. 116, 733 (1969). e C. Chiang and R. E. Bennett, J. Bacteriol. 93, 302 (1967). / F . R. Batchelor, E. B. Chain, M. Richards, and G. N. Rolinson, Proc. Roy. Soc. Ser. B 154~, 522 (1961). g E. Brandl, Hoppe-Seyler's, Z. Physiol. Chem. 342, 86 (1965). b o u n d e n z y m e for the c o m m e r c i a l p r o d u c t i o n of 6 - A P A is still widely practiced. T h e k i n e t i c d a t a for the E . coli 18 a n d t h e B. m e g a t e r i u m ~ e n z y m e s h a v e been used i n derived r a t e e q u a t i o n s d e s c r i b i a g the h y d r o l y s i s of lSD. Warburton, P. Dunnill, and M. D. Lilly, Biotechnol. Bioeng. 15, 13 (1973).

[54b]

PENICILLIN ACYLASE (BACTERIAL)

715

benzylpenicillin to 6-APA. These equations have been used to predict satisfactorily, the progress of the reaction in batch and continuous stirred tank reactors. Substrate Profile--Hydrolytic Direction. The relative rates of deacylation of various penicillins by the cell bound acylase of E. coli 2 and by the extracellular acylase of B. megaterium ~ are shown in Table V. Both TABLE V SUBSTRATE PROFILES FOR THE ACYLASES OF Escherichia coil AND

Bacillus megaterium Relative Penicillins a n d other substrates

r a t e s of hydrolysis

Cell-bound acylase of Cell-free acylase of E. coli N C I B 8743 B. megaterium A T C C 14945

P enicillins Benzylp-HydroxybenzylDL-a-HydroxybenzylD-a-Aminobenzyla-Carboxybenzyl2-Furyhnethyl2-ThienylmethylPhenoxymethyl~-Phenoxyethyln-PropoxymethylIsobutoxymethyln - H e p t ylA2-Pentenyl AllylmercaptomethylEthylthiomethylPhenyl2,6-Dimethoxyphenyl5- M e t hyl-3-phenyl-4-isox azolyl-

100 (6.3 ~:moles/hr) 150 88 50 < 5 91 80 5.5 < 5 40 29 < 5 ---< 5 < 5 --

100 (38 ~ m o l e s / h r ) --

---3.4 4.0

0 16.3 10.5 10. 5 .... 0 0

Other substrates Phenylacetamide N-Methylphenylacetamide N-Phenylacetylglycine N-Phenylacetyl-DL-leucine N-Phenylacetyl-DL-alanine N-Pheiiylacetyl-D-~-aminop h e n y l a c e t i c acid N- Phenylacetyt-L-c~-aminop h e n y l a c e t i e acid Benzylpenicilloic acid

109 -182 73 -0

23 !0 -15 --

139

---

22

--

716

ANTIBIOTIC INACTIVATION AND MODIFICATION

[54b]

enzymes readily hydrolyze benzylpenicillin and more slowly hydrolyze alkylpenieillins. The substrate profiles for the penicillin acylases of other organisms are shown in Table VI. For the E. coli enzyme it would seem that aeyl groups of the size of phenylacetyl are optimal and a-substitution with amino or hydroxy is tolerated, but not carboxy. The structure of the acyl side chain is the main factor controlling enzyme activity. Derivatives at the 3-earboxy group of benzylpenicillin such as amides and esters are good substrates giving 6-APA amides and 6-APA esters. 2 Cephalosporins with suitable acyl side chains, such as cephalothin (2thienylmethylcephalosporin), are good substrates, yielding the nucleus 7-aminocephalosporanic acid (7-ACA2), however the naturally occurring substance cephalosporin C (~-aminoadipylcephalosporin) is not a substrate29 Both enzymes are able to hydrolyze acyl groups from compounds other than penicillins and cephalosporins as shown in Table V. The B. megaterium enzyme carries out this reaction less readily than the E. coli enzyme. The enzyme from E. coli is stereospecific, only phenylacetyl-Lamino acids being hydrolyzed at a significant rate. Details of this reaction and others involving nonpenicillin substrates have been published. 2°-22 The substrate profile of the crystalline enzyme1~ confirms that a single enzyme is responsible for the deacylation of both penicillins and nonpenicillins. Substrate P r o f i l e - S y n t h e t i c Direction. The penicillin acylase of E. coli is reversible. 2~,24 At optimum pH values using the enzyme from E. coli NCIB 8743, the rate of synthesis of benzylpenicillin from 6-APA and phenylacetic acid (pH 5.0) is about 12% of the rate of hydrolysis of benzylpenicillin (pH 8.5). The best carboxylic acids for reactions in the synthetic direction are phenylacetic acids, with or without hydroxy and amino substituents in the ring. However a-hydroxyphenylacetic acid is a poor substrate while phenylacetic acids substituted with a-amino, a-carboxy, or a-methoxy are not substrates. The best alkyl carboxylic acid is 3-hexenoic acid. The use of derivatives of a-aminophenylacetic acid such as amide, N-glyeyl, or methyl ester, enables the D-a aminophenylacetyl group to be readily lgB. Sjoberg, L. Nathorst-Westfelt, and B. Ortengren, Acta Chem. Scand. 21, 547 (1967). 20M. Cole, Biochem. J. 115, 747 (1969). 21G. Lucente, A. Romeo, and D. Rossi, Experie~tia 21, 317 (1965). 22A. Romeo, G. Lucente, D. Rossi, and G. Zanotti, Tetrahedon Lett. 21, 1799 (1971). 23W. Kaufmann, K. Bauer, H. A. Offe, in Antimicrob. Ag. Ann. 1960, 1 (1961). 24G. N. Rolinson, F. R. Batchelor, D. Butterworth, J. Cameron-Wood, M. Cole, G. C. Eustace, M. V. Hart, M. Richards, and E. B. Chain, Nature (London) 187, 236 (1960).

[54b]

PENICILLIN ACYLASE (BACTERIAL)

717

transferred to 6-APA to give ampicillin. 23,-~5 The rate of synthesis of benzylpenicillin and a-hydroxybenzylpenicillin is also increased by such means, but not a-carboxybenzylpenicillin. The accumulation of penicillin in the above synthetic reactions has an inhibitory effect on the reaction which thus comes to equilibrium. Use of a molar excess of carboxylic acid or its derivative shifts the equilibrium in favor of a greater conversion of 6-APA into penicillin. 25 If the pH is allowed to drift upward during synthetic reactions, product penicillin hydrolyzes to 6-APA. The optimum pH for synthesis is between 5.0 and 7.0, depending on substrates and duration of reaction. 2~ By the use of derivatives of carboxylic acids, the substrate range of penicillin acylase can be much extended; for example, phenoxyacetic acid does not support the synthesis of phenoxymethylpenicillin but N-phenoxyacetylglycine does? ° The acyl transferase reaction is stereospecific, penicillins being synthesized at a much greater rate by transfer of an acyl group from an L-amino acid than from a D-amino acid. x° Immobilized Enzyme. The technique of immobilizing enzymes in general with either soluble, or more usually insoluble inert carriers has been reported extensively.2s The main advantage of immobilized enzyme is greater stability, enabling the enzyme to be re-used many times. It has been reported in the literature (',~; that some of these immobilization techniques have been applied to the penicillin acylases produced by E. coli and B. megaterium, but a particularly useful method employing a commercially available carrier is that based on the attachment of proteins to water-insoluble polysaccharide polymers or derivatives thereof activated by reaction with cyanogen halides. 2s To immobilize E. coli enzyme, the following procedure may be used. Cyanogen bromide-activated Sepharose 4B (Pharmacia Co.) (15 g) i,~ suspended in 10 mM HC1 (3 liters) and stirred for 15 min. The gel is recovered on a glass filter and resuspended in a solution of partially purified penicillin acylase in 0.1 M borate buffer, pH 8.75, containing 0.5 M NaC1 (200 ml). The specific activity of the enzyme solution is 6.33 units of protein per milligram, and the total activity is 7407 units. The enzyme and carrier are gently stirred at room temperature for 18 hr. The gel is recovered and resuspended in 1.0 M ethanolamine pH 8.0 (500 ml) and stirred for 2 hr to block any remaining active groups. Ionically bound enzyme is removed by washing the gel three times alternately with 0.1 M acetate buffer pH M. Cole, Biochem. J. 115, 757 (1969). :s O. Zaborsky, "Immobilized Enzymes." Chem. Rubber Publ. Co., Cleveland, Ohio, 1973. ~ D. Warburton, K. Balasingham, P. Dunnill, and M. D. Lilly Biochirn. Biophys. Acta 284, 278 (1972). 2~R. Axen, J. Porath, and S. Ernb~ck, Nature (London) 214, 1302 (1967).

718

ANTIBIOTIC INACTIVATION AND MODIFICATION

[54b]

Z

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[54b] PENICILLIN ACYLASE (BACTERIAL) 705 trols carried out. As 6-APA runs slowly in the butan-l-ol/ethanol/water system, the top half of the chroma...
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