[54c] Penicillin acylase (fungal)

[54c] Penicillin acylase (fungal)

[54C] PENICILLIN ACYLASE (FUNGAL) 721 4.0 containing 1 M NaC1 (200 ml) and 0.1 M borate buffer pH 8.0 containing 1 M NaC1 (200 ml). The final produ...

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[54C]

PENICILLIN ACYLASE (FUNGAL)

721

4.0 containing 1 M NaC1 (200 ml) and 0.1 M borate buffer pH 8.0 containing 1 M NaC1 (200 ml). The final product, 36 g at an activity of 171.2 units/g, is stored damp at 4 ° and used as required; the enzyme activity is quite stable under these storage conditions. The properties of the enzyme may be altered by immobilization, depending upon the carrier and the method of attachment. For example, diffusional limitations may affect the apparent K,~ value. The binding of an enzyme to a charged support may alter the pH-activity profile especially at low ionic strengths; a shift in the pH optimum from 8.2 to 7.65 was reported for E. coli enzyme attached to triazinyl cellulose. -~; Attachment may alter the inhibition constants due to occlusion of allosteric centers or to repulsion effects in the case of charged supports. For example, with E. coli enzyme attached to triazinyl cellulose, the change in the K~ value for 6-APA from 7.1 mM for the free enzyme to 9.0 m M for the immobilized enzyme enabled a 90% conversion of 50 mM benzylpenicillin by the immobilized enzyrae to be achieved in about 80c~ of the time required using free enzyme with the same activity. ~s Immobilization of the B. megaterium enzyme onto bentonite increased the K~ for 6-APA from 26 to 250 mM and the K~ for phenylacetic acid from 450 to 620 mM. ~

[54c] Penicillin Acylase (Fungal) By

HL'BE~T VANDERHAEGHE

The "fungal penicillin acylase," which efficiently hydrolyzes phenoxymethylpenicillin, and also some other penicillins, is produced not only by filamentous fungi, but also by some actinomycetes and bacteria. The production by Penicillium chrysogenum and by F~sarium will be described below. Other producing microorganisms are: Aspergillus ochraceus, 1 Cephalosporium CMI 49137,1--3 Emericellopsis minima, 3 Epidermophyton floccosum, ~,~ Trichophyton mentagrophyta? ,~ Calonectria. ~ Nectria, ~ Pleurotus ostreatus(' Streptomyces lavendulae. ~ Strepto~Tyces M. Cole, Appl. Microbiol. 14, 98 (1966). : C. A. Claridge, J. R. Luttinger, and J. Lein, Proc. Soc. Exp. Biol. Med. 113, 1008 (1963). 3 M. Cole and G. N. Rolinson, Proc. Roy. Soc. Ser. B 154, 490 (1961). 4 j. Uri, G. Valu, and I. Bekesi, Naturu~issenscha]ten 51, 298 (1964). 5 I. C. I., British Patent 924,455 (1963), Chem. Abstr. 59, 2133 (1963). ~Biochemie Ges., Belg. Patent 615,659 (1962), Chem. Abstr. 58, 2819 (1963).

F. R. Batchelor, E. B. Chain, M. Richards, and G. N. Rolinson, Proc. Roy. Soc. Ser. B. 154, 522 (1961).

722

ANTIBIOTIC INACTIVATION AND MODIFICATION

[54c]

noursei, 8 Streptomyces erythreus, s Streptomyces netropsis, s and also Achromobacter sp. N C I B 94249 and Erwinia aroideae. ~° For other microorganisms, see the review by Cole. 1~ I t is likely that there exist differences between penicillin acylases produced by these microorganisms. Although phenoxymethylpenicillin is a good substrate, in several cases marked differences in pH optimum and substrate specificity are described.

Assay Assay procedures for penicillin acylase are described in a previous article. TM P r o d u c t i o n and Purification PeniciUium chrysogenum Acylase

The penicillin acylase may be obtained from a penicillin-producing strain or from a mutant which has lost this property. 13 With the latter strain, the interpretation of the results is easier because no corrections must be made for endogenous {i-APA. The fermentation media described here TM differ somewhat from those used by other authors 13 for the same strain. It is, however, impossible t o tell which media give the highest yield, because the conditions of assay were different. Culture. ~ Penicillium chrysogenum mutant Wis. 49.408 (obtainable from the University of Wisconsin) is grown for 5 days at 26 ° on potato dextrose agar slants , containing (in g/liter): potato infusion, 200; dextrose, 20; and agar, 15. A spore suspension obtained from the agar slant is used to inoculate four 250-ml Erlenmeyer flasks, each containing 50 ml of the following medium (in g/liter): corn-steep powder, 25.0; sucrose, 20.0; CaC03 5.0; Na~S203.5H.,O, 0.2 adjusted to p H 5.5 with 10% N a O H before autoclaving. The flasks are incubated at 26 ° for 3 days on a rotary shaker operating at 150 rpm. These precultures are added to twenty 250-mi Erlenmeyer flasks (5 ml per flask) containing 50 ml of a synthetic medium 15 containing, additionally, 0.2% phenoxyacetic acid. The composition is (in g/liter): lactose, s I. Haupt and H. Thrum, Z. Allgem. Mikrobiol. 7, 343 (1967). o M. Cole, Nature (London) 203, 519 (1964). 1, E. J. Vandamme, J. P. Voets, and A. Dhaese, Ann. Inst. Pasteur 121, 435 (1971). 11M. Cole, Process Bioehem. 2, 35 (1967). 12This volume [54a]. is R. C. Erlckson and R. E. Bennett, Appl. Microbiol. 13, 738 ('1965). 14H. Vanderhaeghe, M. Claesen, A. Vlietinck, and G. Parmentier, Appl. Microbiol. 16, 1557 (1968). ~ F. G. Jarvis and M. J. Johnson, J. Bacteriol. 59, 51 (1950).

[54c]

PENICILLIN ACYLASE (FUNGAL)

723

30.0; dextrose, 10.0; ammonium lactate, 6.0; ammonium acetate, 3.5; KH2PO4, 3.0; Na2SO4, 0.5; MgSO4.TH._,O, 0.25; ZnSO~.7H_~O, 0.02; M n S Q . H 2 0 , 0.02; Fe(NH4)2(SO4)._,'6H20, 0.10; CuSO4"5H20, 0.005; CaCl.,-2H20, 0.05; and phenoxyacetic acid, 2.0. The sugars are sterilized separately and added aseptically to the sterile solution of the mineral components. The flasks are shaken at 26 ° for 2 days. The mycelium is collected on a Biichner funnel, washed with water, and then four times with 0.5 liter of cold (--30 to --40 °) acetone. The mycelium is kept in the air until the odor of acetone has disappeared. Either 60 g (46-82 g) of wet mycelium or 17 g (15-20 g) of dry mycelium are obtained from 20 Erlenmeyer flasks. The dry mycelimn may be kept for several months in a deep-freeze. Extraction. 1~ To remove the enzyme from the cells it is necessary to use salt solutions of sufficiently high concentration. Higher yields are obtained with 0.2 M sodium chloride than with 0.2 M sodium acetate, but there is no significant difference between wet and acetone-dried mycelium. Sonic treatment or disruption of the cells with mechanical methods does not improve the yield. The mycelium is shaken with 0.2 M sodium chloride (60 ml for 1.8 g of dried mycelium in a 250-ml flask) for 16 hr at 26 °. The contents of 10 flasks are filtered on a Biichner filter, and the filtrate (600 ml) is put in dialysis bags. After dialysis for 24 hr at 0-5 ° against l0 liters of distilled water, the operation is repeated for another 24 hr. The dialyzed filtrate is concentrated by submerging the dialysis bags in Carbowax G25000. When the volume is reduced to one-tenth, the dialysis bags are put in distilled water, and the solution is freeze-dried. The mycelium that has been extracted can be used for a second and eventually a third extraction. The activity of these extracts is sometimes similar to the first one, sometimes much lower. Only extracts of sufficiently high activity should be used. The activity of a freeze-dried extract is 0.0005 unit/mg. The unit is defined as the amount of enzyme which hydrolyzes 1 /~mole of phenoxymethylpenicillin per minute in 0.15 M phosphate buffer pH 7.5 at 37 °. The amount of 6-aminopenicillanic acid (6-APA) formed is determined by the t)iochromatographic assay, 12 but using Staphylococcus aureus instead of B. subtilis. Fusari~m Acylase

Penicillin acylase has been found in the mycelia of Fusarium semirectum, ~,~ F. avenaceum TM and in the spores of Fusarium monili'~E. Waldschmidt-Leitz and G. Bretzel, Hoppe-Seyler's Z. Physiol. Chem. 337, 222 (1964), ~7E. Brandl, Hoppe-Seyler's Z. Physiol. Chem. 342, 86 (1965).

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ANTIBIOTIC INACTIVATION AND MODIFICATION

[54C]

]orme. ls,1~ With the last microorganism, no acylase activity is detected

in the mycelium nor in the fermentation broth. 2° The purification of the enzyme from F. s e m i t e c t u m is described below. 1~ Another purification of the enzyme from the same microorganism has also been described. 21 Although the specific activity of the latter preparation was lower, it was homogeneous when chromatographed on Sephadex G-150. This could be due to the fact that extraneous protein was tightly bound to the enzyme. No direct comparison of the two preparations has been made. Culture. F u s a r i u m a v e n a c e u m 14 and F. s e m i t e c t u m IG are grown on Jarvis-Johnson medium ~ containing 0.2% phenoxyacetic acid for 3 days at 26 or 28 °. For the first species, the preparation of the spore inoculum aDd the preculture is the same as described above for P e n i c i l l i u m chrysogenum, for the second no details are given. The different steps for growing the mycelium of F. s e m i t e c t u m in slightly different media have been given in another publication. 2~ The yield of acylase is apparently very similar. E x t r a c t i o n . ~6 The mycelium of F u s a r i u m s e m i t e c t u m BC805 is suspended in 0.2 M sodium acetate buffer at pH 6.0 (600 ml for 18 g of dry mycelium containing 18 units/g, and which is obtained from 1 liter of fermentation culture) and shaken for 15 hr. After filtration, the solution is alkalinized to pH 11.0 with NaOH, and the inactive precipitate is removed by centrifugation. The supernatant is immediately adjusted to p H 8.0 with acetic acid. The solution is treated three times with D E A E cellulose (10 g per 500 ml of solution). The clear solution, which is obtained by passing the liquid through a Sephadex G-25 column, is freezedried. From 190 g of mycelium containing 1905 units ~ (specific activity 0.01 unit/rag), 9.5 g of freeze-dried extract with a specific activity of 0.035 unit/mg (330 units, 17% yield) is obtained. F r a c t i o n a t i o n . ~ The extrac~ (9.5 g) is dissolved in 5 m M sodium citrate solution (100 ml/3 g) and while cooling to --10 °, acetone is added until a concentration of 44% is reached. After 20 min, the precipitate containing the enzyme is removed by centrifugation. After the precipitate is dry, 1.92 g of extract with a specific activity of 0.160 unit/mg 1~K. Singh, S. N. Seghal, and C. V~zina, Appl. Microbiol. 17, 643 (1969). 1~E. J. Vandamme, J. P. Voets, and C. Beyaert, Meded. Rijks]ac. Landbouwwetensch. Gent 36, 577 (1971). 2, E. J. Vandamme and J, P. Voets, Meded. Rijks]ac. Landbouwwetensch. Gent 37, 1185 (1972). 2~F. Baumann, R. Brunner, and M. RShr, Hoppe-Seyler's Z. Physiol. Chem. 352, 853 (1971). 22The unit is defined as the amount of enzyme that hydrolyzes 1 #mole of phenoxymethylpenicillin per minute at pH 7.5 and 32°. The amount of 6-APA was determined by iodometric assay of the aqueous phase after extraction of the residual penicillin in butylacetate from the acidified solution. See also footnote 17.

[54(::]

PENICILLIN ACYLASE (FUNGAL)

725

is obtained. The operation is repeated, but the concentration of acetone is raised only to 37%. This yields 254 mg of extract with a specific activity of 1.10 unit/mg (280 units, 15%). Final Purification. TM The enzyme preparation (180 rag) is dissolved in 10 ml of 0.2 M sodium citrate buffer pH 4.8 and adsorbed on an Amberlite 1RC-50 column and eluted with the same buffer, the pH gradually being increased to 5.1. The elution is monitored by measuring the extinction at 280 nm and assaying the enzyme. The column chromatography is repeated. The volume of the eluate is reduced to 10 ml by freeze-drying, and salt is removed by passing this solution through a column of Sephadex G-25. After elution with water and lyophilization, 17 mg of a product with a specific activity 3.02 units/rag is obtained.

Properties Stability. A solution of the enzyme preparation from Fusarium semitectum is stable for at least 24 hr at 32 ° and pH 6.0 to 10.0. Loss of activity is observed at pH 4.2.16 The extracellular enzyme of Streptomyces lavendulae seems to be less stable. Only 67% of the original activity is present after storing the sterile culture filtrate at 35 ° and pH 8.0 for 24 hrY Chemical and Physical Properties. Examination of the purified preparation from Fusarium semitectum in the ultracentrifuge indicates that the product is homogeneous, with a sedimentation constant of 7.76 S. The molecular weight is estimated to be in the range of 65,000, but a precise determination of the diffusion constant has not been performed because of the limited amount of product. This acylase contains two atoms of zinc per molecule. TM A molecular weight of 62,000 is inferred for the acylase of Erwinia aroideae from thin-layer chromatography on Sephadex G-100 gelY° Activators and Inhibitors. No activators have been described, but the Fusarium enzyme is inactivated when zinc is complexed with 8-hydroxyquinoline. The activity is fully restored by addition of ZnSO4, and partially with MnSO,, MgS04, COSO4, and FeS0t. TM In this connection, it should be noted that Szentirmai ~ reported the inhibition of acylase production in Escherichia coli by 130 ~g/ml of 8-hydroxyquinoline. Optimum pH and Temperature. It is difficult to determine optimum pH and temperature for this enzyme because the substrate (phenoxymethylpenicillin) is rapidly destroyed at alkaline pH and high temperature. The optimum pH for Penicillium acylase has not been determined, but there is a marked increase of the rate of hydrolysis when the pH ='A. Szentirmai, Appl. Microbiol. 12, 185 (1964).

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ANTIBIOTIC INACTIVATION AND MODIFICATION

[54C]

is raised from 6.5 to 8.5.13,~4 The reaction rate at 35 ° is 1.5 times that observed at 300.13 For the Fusarium enzyme, the rate of hydrolysis of phenoxymethylpenicillin increases from 25 ° to 370,14,1' but the effect levels off from 40 ° to 500.17 The optimum pH is between 7.0 and 8.0, with a broad maximum at 7.5 for the acylase of F. semitectum, 1°,1~ F. avenaceum 14 and F. monili]orme. ~8,19 The optimum pH of acylase of Streptomyces lavendulae is 10.0, and the highest rate is observed at 50 ° when measured for a short period of time (30 rain).8 For Erwinia aroideae a pH optimum of 4.5-5.5 is observed with phenoxymethylpenicillin as substrate, and a pH optimum of 8.0 for cloxacillin and methicillin. 11 It may be concluded from these data that the acylases produced by these microorganisms are not identical, although all hydrolyze phenoxymethylpenicillin much better than benzylpenicillin. This conclusion is supported by the observations on the substrate specificity (see below). Kinetic Properties. All Km values are high, indicating a low affinity of penicillin acylase for the substrate. For Fusarium enzyme the following Km values have been reported for the cleavage of phenoxymethylpenicillin: 2.5-2.8 mM (without indication of pH or temperature),17 4.75 mM at pH 7.5 and 370, 21 and 5.75 mM at pH 8 and 28 ° using Fusarium moniliforme spores. 19 No Km has been published for the hydrolysis of penicillin by Penicillium chrysogenum acylase, but Km values are 1.1 to 1.4 mM for the deacylation of some phenoxyacetylamino acids. 2~ In this publication, ~4 however, there are indications that this enzyme is different from that responsible for the cleavage of penicillin. The extracellular acylase of Streptomyces lavendulae has a Km of 10.3 mM for the hydrolysis of phenoxymethylpenicillin at pH 8.7 and 340. 8 Substrate Specificity. Only semiquantitative data are available concerning the rate of hydrolysis of different penicillins by "fungal acylase." They are given in the table for the acylases of Penicillium chrysogenum and Fusarium aveneceum. Penicillin N, T M isopenicillin N, 14 methicillin, 2 and cephalosporin 2 are not hydrolyzed by Penicillium chrysagenum acylase. The enzyme of Streptomyces lavendulae rapidly deacylates penicillin V, penicillin H2F, and penicillin K, but very slowly deacylates penicillin G 7 (for Erwinia aroideae, see under "Optimum pH"). It has been reported that several phenoxyacetylamino acids are hydrolyzed by Penicillium chrysogenum, but the enzyme seems to be different from that responsible for the deacylation of penicillins. 24 An enzyme 24R. Brunner, M. RShr, and M. Zinner, Monatsh. Chem. 97, 952 (1966).

[54c1

PENICILLIN ACYLASE (FUNGAL)

727

~C ¢D

¢D '-~

,,-1

--I+-FI -F

I

I

I

+ ÷ l J l ÷ + + ÷

©

c~

.< m¢*

U

• ~

~'~

o°~-~-~j~l~j

~"~

728

A N T I B I O T IINACTIVATION C AND MODIFICATION

[55a]

preparation obtained by extraction of Fusarium semitectum with 0.2 M sodium chloride deacylates phenoxymethylpenicillin but not phenoxyacetylamino acids. 2l The converse was observed for an extract prepared in a different way from the same microorganism. ~5 ~F. Baumann, M. RShr, and R. Brunner, Zentr. Bakteriol. Abt. I, Re]. 229, 351 (1972).

[55a] Cephalosporin Acetylesterase (Citrus) By E. P. ABRAHAMand PATRIClA FAWCETT +

This enzyme hydrolyzcs cephalosporin C [(I), R = ()~C.CH(NHa) (CH2) 3C0] to deacetylcephalosporin C.X It also hydrolyzes other cephalosporins containing an O-acetyl group to the corresponding deacetyleephalosporins (II) 2 and 7-aminocephalosporanic acid (I, R = H) to 7-aminodeacetylcephalosporanic acid (7-aminocephalosporadesic acid), a H H RNH [ ~

H H R N t t ~ + HOAe

+ H20

O

CHzOAe CO2- M+

(I)

0 ~/

~ "CH:~OH CO~- M+

(n)

Assay Methods The assay is based on measurement of the acid liberated during hydrolysis of an acetyl ester. Cephalosporin C or triacetin may be used as a substrate. Manometric. The rate of hydrolysis at 30 ° is determined in a Warburg respirometer at pH 7.0 by measurement of the liberation of CO2 from NaHCO3. With cephalosporin C as substrate, the main compartment of. each vessel contains 1.7 ml of 0.15 M NaC1, 0.3 ml of esterase solution, and 0.5 ml of 43 mM N a H C Q ; the side bulb contains 0.1 ml of 43 mM NaHCO~ and 0.4 ml of a solution of cephalosporin C Na salt (40 mg). With triacetin as substrate the main compartment contains 85 mg of triacetin in 2 ml of 0.15 M NaC1 and 0.5 ml of 43 mM NaHCO~; the side 1j. D'A. Jeffery, E. P. Abraham, and G. G. F. Newton, Biochem. J. 81, 591 (1961). "~U.S. Patent 3,459,746 (1969). 3U.K. Patent 1,066,347 (1967).