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Colorimetric assay of penicillin amidase activity using phenylacetyl-aminobenzoic acid as substrate
ANALYTICAL
103, 166- 169 (1980)
BIOCHEMISTRY
Calorimetric Assay of Penicillin Phenylacetyl-Aminobenzoic A. SZEWCZUK,
M. SIEWII+KI,
Amidase Activity Using Acid as Substrate AND R. S~OWI~~SKA’
Biochemical Laboratory, Institute of Immunology and Experimental Polish Academy of Sciences, Wroctaw, Poland
Therapy,
Received May 9, 1979 All three isomers of phenylacetyl-aminobenzoic acid are stable in buffer solutions and are suitable as substrates for the simple and sensitive assay of penicillin amidase activity in purified preparations, in bacterial cells as well as in immobilized preparations. By titration of the active center with phenylmethylsulfonyl fluoride it was demonstrated that phenylacetyl4-aminobenzoic acid as well as benzylpenicillin are hydrolyzed by the same enzyme. The new substrate was also used for rapid assay of the amidase activity in bacterial colonies. Both phenylacetyl-naphthylamides are very slowly hydrolyzed by the amidase.
Microbial penicillin amidase (EC3.5.1.11) is used in the production of 6-aminopenicillanic acid, some semisynthetic penicillins, and cephalosporins (1). For the amidase assay either penicillin (2,3) or synthetically obtained phenylacetyl-L-asparagine (4), 6nitro-3-phenylacetamido-benzoic acid (5), and 2-nitro-4-phenylacetamido-benzoic acid (6) are used as substrates. These methods are not very sensitive and sometimes not suitable for activity assay in crude enzyme preparations. In this communication the use of phenylacetyl-4-aminobenzoic acid and its isomers are stable substrates for the simple colorimetric assay of penicillin amidase activity is described. MATERIALS
AND METHODS
Anthranilic and 3-aminobenzoic acids and dicyclohexylcarbodiimide were purchased from BDH Chemical Ltd., Poole, England. 4-Aminobenzoic acid and naphthylamines were from POCh Gliwice, Poland. Phenylacetyl chloride (7) and phenylmethylsulfonyl fluoride (8) were synthesized. Penicillin I To whom correspondence
should be addressed.
OOO3-2697/80/05016644$02.00/0 Copyright All rights
0 1980 by Academic Press, Inc. of reproduction in any form reserved.
166
amidase isolated from Escherichia cofi and the enzyme immobilized by copolymerization with acrylamide in spherical beads were obtained as described recently (9). The amidase activity was determined using benzylpenicillin as substrate andp-dimethylaminobenzaldehyde (3). Phenylacetyl-4-aminobenzoic acid was obtained as follows: to 6.85 g (50 mmol) of 4-aminobenzoic acid in 2.5 ml 2 N NaOH and 25 ml acetone at O”C, 6.4 ml (50 mmol) of phenylacetyl chloride was added dropwise and pH was maintained at over 8 with 2 N NaOH. The crude product was completely precipitated with 2 N HCl and recrystallized twice by dissolving in 30 ml of 2 N NaOH; the dilution was with 100 ml of 20% ethanol and the acidification with 2 N HCl: mp 256259°C; lit. (10) 236°C. Yield: 7.7 g (60%). Similarly obtained were phenylacetyl-anthranilic and phenylacetyl-3-aminobenzoic acids. Their mp were 188- 19OWlit. 188”/1 l// and 261-264°C respectively. Anal. Calcd for CljH13N03: C, 70.58; H, 5.13; N, 5.45. Found for phenylacetyl-6 aminobenzoic acid: C, 70.73; H, 5.15; N, 5.55; for phenylacetyl-3-amino-benzoic acid: C, 69.98; H, 5.10; N, 5.36; for phenylacetyl-
ASSAY OF PENNICILLIN
anthranillic acid: C, 70.12; H, 5.21; N, 5.37. All three products were homogenous as shown by thin-layer chromatography-Polygram Sil-N-HR plates from Macherey-Nagel Company were used. Rf for phenylacetyl-C, -3-aminobenzoic, and -anthranilic acids in ethanol:chloroform:22.5% ammonia (53:30: 17) (v/v) were 0.53,0.58,0.70, respectively. Their spectra in the uv were similar to those of the corresponding aminobenzoic acids. N-phenylacetyl-aand -p-naphthylamides were obtained by condensation of phenylacetic acid (20 mmol) and naphthylamine (20 mmol) in tetrahydrofuran with dicyclohexylcarbodiimide (20 mmol) and recrystalized twice from acetone-petrol ether: yield about 40%; mp of cw-naphthylamide was 169- 170°C; lit. (12) 169°C and ofp-naphthylamide was 161-162°C; lit. (12) 161°C. Anal. Calcd for C,,H,,ON: C, 82.72; H, 5.79: N, 5.38. Found for N-phenylacetylcY-naphthylamide: C, 82.21; H,5.57; N, 5.21; for N-phenylacetyl+naphthylamide C, 81.91; H, 5.73; N, 5.38. Assay of penicillin amidase activity by the new method was performed as follows: 0.4 ml of 5 mM substrate (25.5 mg phenylacetylaminobenzoic acid dissolved in 0.5 ml 0.1 M Na,CO, and diluted to 20 ml with 80 mM phosphate buffer, pH 7.8) was mixed with 0.1 ml of the enzyme solution and incubated at 42°C for 10 min. The liberated aminobenzoic acid was calorimetrically measured by the procedure described earlier for yglutamyltranspeptidase assay (13). After incubation the substrate with the amidase 0.5 ml of freshly prepared 10 mM sodium nitrite in 0.25 M acetic acid was added and after 3-8 min at room temperature the solution was mixed with 1.5 ml of fresh 0.01% I-amino-8-hydroxy-naphthalene-3,6-disulfonic acid (H-acid)* in 0.66 M sodium carbonate. After at least 2 min the extinction of the red dye was measured at 535 nm against a blank. If the amidase activity was assayed in bacterial suspension or homogenate the 2 Abbreviations used: H-acid, I-amino-Shydroxynaphthalene-3,Gdisulfonic acid.
167
AMIDASE
liberated aminobenzoic acid was measured using the modified Bratton and Marshall procedure (14). Naphthylamine was also determined by the latter procedure when phenylacetyl-naphthylamides were used as substrates. RESULTS
Pure phenylacetyl-aminobenzoic acids do not give a color reaction in the described method. The extinction of the red dye obtained after diazotization and coupling with H-acid is directly proportional to the amount of free aminobenzoic acid. Good proportionality was also noted between the spectrophotometer reading and amidase concentration (Fig. 1). The activity of the amidase towards phenylacetyl-4-aminobenzoic acid is about five times lower than that toward penicillin but K, for these substrates are almost equal (Table 1). Phenylacetyl-anthranilic acid is hydrolyzed twice as fast as phenylacetyl-C aminobenzoic acid and the K, is much higher. The inhibitor constant (KJ for phenylacetate when using synthetic substrates is higher than with benzylpenicillin. To prove whether phenylacetyl-4-aminobenzoic acid is hydrolyzed by the penicillin amidase, samples of the amidase previously inhibited with various concentrations of phenylmethylsulfonyl fluoride were used for the activity assay. Almost the same depression in hydrolysis of benzylpenicillin and phenylacetyl-4-aminobenzoic acid was observed (Fig. 2). The amidase activities were assayed also in E. co/i cells, in homogenates, and in the immobilized enzyme. The ratio of the results obtained by the new method to that obtained by the classical method with benzylpenicillin was about 0.20. Phenylacetyl-aminobenzoic acids are relatively very stable in water solution. In phosphate buffer, pH 7.8, when 50 mM benzylpenicillin was incubated at 37°C it was suitable as a substrate for the amidase assay only for several hours. Incubation of phenylacetyl-4-aminobenzoate solution for 45 days
168
SZEWCZUK,
SIEWINSKI,
did not change the amidase assay but the content of free 4-aminobenzoic acid was increased from 0.02 to 0.6%. The proposed method was also applied as a rapid test for the detection of penicillin amidase in bacterial strains. In this test paper disks (Whatman No. 1) saturated with 5 mM phenylacetyl-4-aminobenzoic acid in 50 mM phosphate buffer, pH 7.8, were used. They were contacted for 30 s with well-separated bacterial colonies grown on normal agar plates with addition of 0.5% phenylacetate as the amidase inducer. Then the paper disks were incubated at 37°C for 10 min, dried, and sprayed with 10 mM sodium nitrite in 0.25 M acetic acid and then with 0.01% H-acid in 0.5 M sodium carbonate and dried again. Red spots prove amidase activity in the bacterial colonies. The intensities of the spots measured densitometrically are correlated with the amidase activity in the colonies. Both synthesized phenylacetyl-naphthylamides are insoluble in water and sparingly soluble in 5% dimethylformamide at pH 7.8 (0.06 pmol/ml). In the latter solvent (Y-and p-naphthylamides were hydrolyzed by the amidase 660 and 200 times slower than benzylpenicillin.
AND SLOWINSKA TABLE
1
COMPARISON PHENYLACETYL-AMINOBENZOIC ACIDS WITH BENZYLPENIC~LLIN AS SUBSTRATES FOR THE AMIDASE
(mM)
Ki @W for phenylacetate
21
0.18
3.0
37
0.40
5.3
44 100
0.95 0.20
4.5 2.0
Relative activity*
Substrate Phenylacetyl-C aminobenzoic acid Phenylacetyl-3aminobenzoic acid Phenylacetylanthranilic acid Benzylpenicillin
K,
* Measured with 24 mM substrate at pH 7.8.
buffers at pH 6. They are suitable as substrates for simple assay of penicillin amidase activity and enzymically liberated aminobenzoic acid can be transformed into stable red dye after diazotization and coupling with H-acid. The proposed method is more sensitive than some other known calorimetric methods. The molar extinction coefficient of the dye formed from p-aminobenzoic acid is 36,000 (13) whereas this constant for 6aminopenicillanic acid, liberated from peni-
DISCUSSION
Phenylacetyl-aminobenzoic acids can be easily synthesized and are well soluble in
8 0
I finok
FIG. 1. Dependence of extinction on the enzyme concentration. The amidase from E. coli and phenylacetyl-4-aminobenzoic acid were used in the assay procedure described under Materials and Methods.
2 concentration
3 of
L PMSF
g 5
6
/ lo+M/
FIG. 2. Effect of phenylmethylsulfonyl fluoride (PMSF) on the amidase activity. Enzyme was incubated at pH 6.0 and 25°C for 30 min (15) with various concentrations of PMSF and then the amidase activity with phenylacetyl-4-aminobenzoic acid (0) and benzylpenicillin (0) was tested.
ASSAY OF PENNICILLIN
cillin, after coupling with p-dimethylaminobenzaldehyde is 1,500 and for 6-nitro-3phenylacetamido-benzoic acid liberated from chromogenic substrate is 9,090 (5). The described phenylacetyl-aminobenzoic acids are stable in buffer solutions and therefore can be used in special projects, i.e., for the investigation of the operation stability of immobilized penicillin amidase. Another application of the proposed method is as a rapid test for the detection of penicillin amidase production by bacterial strains. This is based on using paper disks saturated with the described substrate; red spots prove amidase activity. These spots are stable after drying the paper disks and their intensity can be measured densitometrically. Thus the test for detection of amidase is suitable also for the quantitive measurement of enzyme activity in a single bacterial colony and may be useful in the selection of bacterial producers of penicillin amidase. ACKNOWLEDGMENTS The authors express their thanks to Dr. Jadwiga Wieczorek for her kind help in paper disk detection of penicillin amidase in bacterial colonies, and to Mrs. Danuta Lang for technical assistance.
169
AMIDASE
REFERENCES 1. Vandamme, E. J., and Voets, J. P. (1974) Advan. A@. Microbiof. 17, 3 11-369. 2. Cole, M., Savidge, T., and Vanderhaegde, H. (1975) in Methods in Enzymology (Hash, J. H., ed.), Vol. 43, pp. 698-705, Academic Press, New York, 1975. 3. Nys, P. S., Savitskaya, E. M., and Kolygina, T. S. (1973) Antibiotiki 18, 270-273. 4. Bauer, K., Kaufmann, W., and Ludwig, S. A. (1971) Z. Physiol. Chem. 352, 1723-1724. 5. Kutzbach, C., and Rauenbusch, E. (1974) Z. Physiol.
Chem.
355, 45-53.
6. Nys, P. S., Kolygina, T. S., and Garaev, M. M. (1977) Antibiofiki, 22, 211-216. 7. Baker, W., Ollis, W. 0.. and Poole, V. D. (1949) J. Chem. Sot. 307-314. 8. Gold, A. M., and Fahmey, D. (1964)Biochemistr?/ 3, 783-791. 9. Szewczuk, A., Ziomek, E., Mordarski, M., Siewinski, M., and Weiczorek, J. (1979) Biotechno/. Bioeng. 21, 1543-1552. 10. Cole, M. (I%9) Biochem. J. 115, 741-745. 11. Diesbach, H., Gross, J., andTschanner, W. (1951) Helv. Chim. Acta 34, lOSO- 1060. 12. Tsatsas, G., and Delaby, R. (1956) Ann. Pharm. Fr. 14, 621-635. M. (1978) 13. Szewczuk, A., Wellman-Bednawska, Clin. Chim. Acra 84, 19-26. 14. Goldberg, J. A., and Rutenburg, A. M. (1958) Cancer 11, 283-291. 15. Shvyadas, V. K., Margolin, A. L., Sherstyuk, S. F., Klyosov, A. A., and Berezin, I. V. (1977) Bioorgan.
Kchimia
(Moscow)
3, 546-554.
103, 166- 169 (1980)
BIOCHEMISTRY
Calorimetric Assay of Penicillin Phenylacetyl-Aminobenzoic A. SZEWCZUK,
M. SIEWII+KI,
Amidase Activity Using Acid as Substrate AND R. S~OWI~~SKA’
Biochemical Laboratory, Institute of Immunology and Experimental Polish Academy of Sciences, Wroctaw, Poland
Therapy,
Received May 9, 1979 All three isomers of phenylacetyl-aminobenzoic acid are stable in buffer solutions and are suitable as substrates for the simple and sensitive assay of penicillin amidase activity in purified preparations, in bacterial cells as well as in immobilized preparations. By titration of the active center with phenylmethylsulfonyl fluoride it was demonstrated that phenylacetyl4-aminobenzoic acid as well as benzylpenicillin are hydrolyzed by the same enzyme. The new substrate was also used for rapid assay of the amidase activity in bacterial colonies. Both phenylacetyl-naphthylamides are very slowly hydrolyzed by the amidase.
Microbial penicillin amidase (EC3.5.1.11) is used in the production of 6-aminopenicillanic acid, some semisynthetic penicillins, and cephalosporins (1). For the amidase assay either penicillin (2,3) or synthetically obtained phenylacetyl-L-asparagine (4), 6nitro-3-phenylacetamido-benzoic acid (5), and 2-nitro-4-phenylacetamido-benzoic acid (6) are used as substrates. These methods are not very sensitive and sometimes not suitable for activity assay in crude enzyme preparations. In this communication the use of phenylacetyl-4-aminobenzoic acid and its isomers are stable substrates for the simple colorimetric assay of penicillin amidase activity is described. MATERIALS
AND METHODS
Anthranilic and 3-aminobenzoic acids and dicyclohexylcarbodiimide were purchased from BDH Chemical Ltd., Poole, England. 4-Aminobenzoic acid and naphthylamines were from POCh Gliwice, Poland. Phenylacetyl chloride (7) and phenylmethylsulfonyl fluoride (8) were synthesized. Penicillin I To whom correspondence
should be addressed.
OOO3-2697/80/05016644$02.00/0 Copyright All rights
0 1980 by Academic Press, Inc. of reproduction in any form reserved.
166
amidase isolated from Escherichia cofi and the enzyme immobilized by copolymerization with acrylamide in spherical beads were obtained as described recently (9). The amidase activity was determined using benzylpenicillin as substrate andp-dimethylaminobenzaldehyde (3). Phenylacetyl-4-aminobenzoic acid was obtained as follows: to 6.85 g (50 mmol) of 4-aminobenzoic acid in 2.5 ml 2 N NaOH and 25 ml acetone at O”C, 6.4 ml (50 mmol) of phenylacetyl chloride was added dropwise and pH was maintained at over 8 with 2 N NaOH. The crude product was completely precipitated with 2 N HCl and recrystallized twice by dissolving in 30 ml of 2 N NaOH; the dilution was with 100 ml of 20% ethanol and the acidification with 2 N HCl: mp 256259°C; lit. (10) 236°C. Yield: 7.7 g (60%). Similarly obtained were phenylacetyl-anthranilic and phenylacetyl-3-aminobenzoic acids. Their mp were 188- 19OWlit. 188”/1 l// and 261-264°C respectively. Anal. Calcd for CljH13N03: C, 70.58; H, 5.13; N, 5.45. Found for phenylacetyl-6 aminobenzoic acid: C, 70.73; H, 5.15; N, 5.55; for phenylacetyl-3-amino-benzoic acid: C, 69.98; H, 5.10; N, 5.36; for phenylacetyl-
ASSAY OF PENNICILLIN
anthranillic acid: C, 70.12; H, 5.21; N, 5.37. All three products were homogenous as shown by thin-layer chromatography-Polygram Sil-N-HR plates from Macherey-Nagel Company were used. Rf for phenylacetyl-C, -3-aminobenzoic, and -anthranilic acids in ethanol:chloroform:22.5% ammonia (53:30: 17) (v/v) were 0.53,0.58,0.70, respectively. Their spectra in the uv were similar to those of the corresponding aminobenzoic acids. N-phenylacetyl-aand -p-naphthylamides were obtained by condensation of phenylacetic acid (20 mmol) and naphthylamine (20 mmol) in tetrahydrofuran with dicyclohexylcarbodiimide (20 mmol) and recrystalized twice from acetone-petrol ether: yield about 40%; mp of cw-naphthylamide was 169- 170°C; lit. (12) 169°C and ofp-naphthylamide was 161-162°C; lit. (12) 161°C. Anal. Calcd for C,,H,,ON: C, 82.72; H, 5.79: N, 5.38. Found for N-phenylacetylcY-naphthylamide: C, 82.21; H,5.57; N, 5.21; for N-phenylacetyl+naphthylamide C, 81.91; H, 5.73; N, 5.38. Assay of penicillin amidase activity by the new method was performed as follows: 0.4 ml of 5 mM substrate (25.5 mg phenylacetylaminobenzoic acid dissolved in 0.5 ml 0.1 M Na,CO, and diluted to 20 ml with 80 mM phosphate buffer, pH 7.8) was mixed with 0.1 ml of the enzyme solution and incubated at 42°C for 10 min. The liberated aminobenzoic acid was calorimetrically measured by the procedure described earlier for yglutamyltranspeptidase assay (13). After incubation the substrate with the amidase 0.5 ml of freshly prepared 10 mM sodium nitrite in 0.25 M acetic acid was added and after 3-8 min at room temperature the solution was mixed with 1.5 ml of fresh 0.01% I-amino-8-hydroxy-naphthalene-3,6-disulfonic acid (H-acid)* in 0.66 M sodium carbonate. After at least 2 min the extinction of the red dye was measured at 535 nm against a blank. If the amidase activity was assayed in bacterial suspension or homogenate the 2 Abbreviations used: H-acid, I-amino-Shydroxynaphthalene-3,Gdisulfonic acid.
167
AMIDASE
liberated aminobenzoic acid was measured using the modified Bratton and Marshall procedure (14). Naphthylamine was also determined by the latter procedure when phenylacetyl-naphthylamides were used as substrates. RESULTS
Pure phenylacetyl-aminobenzoic acids do not give a color reaction in the described method. The extinction of the red dye obtained after diazotization and coupling with H-acid is directly proportional to the amount of free aminobenzoic acid. Good proportionality was also noted between the spectrophotometer reading and amidase concentration (Fig. 1). The activity of the amidase towards phenylacetyl-4-aminobenzoic acid is about five times lower than that toward penicillin but K, for these substrates are almost equal (Table 1). Phenylacetyl-anthranilic acid is hydrolyzed twice as fast as phenylacetyl-C aminobenzoic acid and the K, is much higher. The inhibitor constant (KJ for phenylacetate when using synthetic substrates is higher than with benzylpenicillin. To prove whether phenylacetyl-4-aminobenzoic acid is hydrolyzed by the penicillin amidase, samples of the amidase previously inhibited with various concentrations of phenylmethylsulfonyl fluoride were used for the activity assay. Almost the same depression in hydrolysis of benzylpenicillin and phenylacetyl-4-aminobenzoic acid was observed (Fig. 2). The amidase activities were assayed also in E. co/i cells, in homogenates, and in the immobilized enzyme. The ratio of the results obtained by the new method to that obtained by the classical method with benzylpenicillin was about 0.20. Phenylacetyl-aminobenzoic acids are relatively very stable in water solution. In phosphate buffer, pH 7.8, when 50 mM benzylpenicillin was incubated at 37°C it was suitable as a substrate for the amidase assay only for several hours. Incubation of phenylacetyl-4-aminobenzoate solution for 45 days
168
SZEWCZUK,
SIEWINSKI,
did not change the amidase assay but the content of free 4-aminobenzoic acid was increased from 0.02 to 0.6%. The proposed method was also applied as a rapid test for the detection of penicillin amidase in bacterial strains. In this test paper disks (Whatman No. 1) saturated with 5 mM phenylacetyl-4-aminobenzoic acid in 50 mM phosphate buffer, pH 7.8, were used. They were contacted for 30 s with well-separated bacterial colonies grown on normal agar plates with addition of 0.5% phenylacetate as the amidase inducer. Then the paper disks were incubated at 37°C for 10 min, dried, and sprayed with 10 mM sodium nitrite in 0.25 M acetic acid and then with 0.01% H-acid in 0.5 M sodium carbonate and dried again. Red spots prove amidase activity in the bacterial colonies. The intensities of the spots measured densitometrically are correlated with the amidase activity in the colonies. Both synthesized phenylacetyl-naphthylamides are insoluble in water and sparingly soluble in 5% dimethylformamide at pH 7.8 (0.06 pmol/ml). In the latter solvent (Y-and p-naphthylamides were hydrolyzed by the amidase 660 and 200 times slower than benzylpenicillin.
AND SLOWINSKA TABLE
1
COMPARISON PHENYLACETYL-AMINOBENZOIC ACIDS WITH BENZYLPENIC~LLIN AS SUBSTRATES FOR THE AMIDASE
(mM)
Ki @W for phenylacetate
21
0.18
3.0
37
0.40
5.3
44 100
0.95 0.20
4.5 2.0
Relative activity*
Substrate Phenylacetyl-C aminobenzoic acid Phenylacetyl-3aminobenzoic acid Phenylacetylanthranilic acid Benzylpenicillin
K,
* Measured with 24 mM substrate at pH 7.8.
buffers at pH 6. They are suitable as substrates for simple assay of penicillin amidase activity and enzymically liberated aminobenzoic acid can be transformed into stable red dye after diazotization and coupling with H-acid. The proposed method is more sensitive than some other known calorimetric methods. The molar extinction coefficient of the dye formed from p-aminobenzoic acid is 36,000 (13) whereas this constant for 6aminopenicillanic acid, liberated from peni-
DISCUSSION
Phenylacetyl-aminobenzoic acids can be easily synthesized and are well soluble in
8 0
I finok
FIG. 1. Dependence of extinction on the enzyme concentration. The amidase from E. coli and phenylacetyl-4-aminobenzoic acid were used in the assay procedure described under Materials and Methods.
2 concentration
3 of
L PMSF
g 5
6
/ lo+M/
FIG. 2. Effect of phenylmethylsulfonyl fluoride (PMSF) on the amidase activity. Enzyme was incubated at pH 6.0 and 25°C for 30 min (15) with various concentrations of PMSF and then the amidase activity with phenylacetyl-4-aminobenzoic acid (0) and benzylpenicillin (0) was tested.
ASSAY OF PENNICILLIN
cillin, after coupling with p-dimethylaminobenzaldehyde is 1,500 and for 6-nitro-3phenylacetamido-benzoic acid liberated from chromogenic substrate is 9,090 (5). The described phenylacetyl-aminobenzoic acids are stable in buffer solutions and therefore can be used in special projects, i.e., for the investigation of the operation stability of immobilized penicillin amidase. Another application of the proposed method is as a rapid test for the detection of penicillin amidase production by bacterial strains. This is based on using paper disks saturated with the described substrate; red spots prove amidase activity. These spots are stable after drying the paper disks and their intensity can be measured densitometrically. Thus the test for detection of amidase is suitable also for the quantitive measurement of enzyme activity in a single bacterial colony and may be useful in the selection of bacterial producers of penicillin amidase. ACKNOWLEDGMENTS The authors express their thanks to Dr. Jadwiga Wieczorek for her kind help in paper disk detection of penicillin amidase in bacterial colonies, and to Mrs. Danuta Lang for technical assistance.
169
AMIDASE
REFERENCES 1. Vandamme, E. J., and Voets, J. P. (1974) Advan. A@. Microbiof. 17, 3 11-369. 2. Cole, M., Savidge, T., and Vanderhaegde, H. (1975) in Methods in Enzymology (Hash, J. H., ed.), Vol. 43, pp. 698-705, Academic Press, New York, 1975. 3. Nys, P. S., Savitskaya, E. M., and Kolygina, T. S. (1973) Antibiotiki 18, 270-273. 4. Bauer, K., Kaufmann, W., and Ludwig, S. A. (1971) Z. Physiol. Chem. 352, 1723-1724. 5. Kutzbach, C., and Rauenbusch, E. (1974) Z. Physiol.
Chem.
355, 45-53.
6. Nys, P. S., Kolygina, T. S., and Garaev, M. M. (1977) Antibiofiki, 22, 211-216. 7. Baker, W., Ollis, W. 0.. and Poole, V. D. (1949) J. Chem. Sot. 307-314. 8. Gold, A. M., and Fahmey, D. (1964)Biochemistr?/ 3, 783-791. 9. Szewczuk, A., Ziomek, E., Mordarski, M., Siewinski, M., and Weiczorek, J. (1979) Biotechno/. Bioeng. 21, 1543-1552. 10. Cole, M. (I%9) Biochem. J. 115, 741-745. 11. Diesbach, H., Gross, J., andTschanner, W. (1951) Helv. Chim. Acta 34, lOSO- 1060. 12. Tsatsas, G., and Delaby, R. (1956) Ann. Pharm. Fr. 14, 621-635. M. (1978) 13. Szewczuk, A., Wellman-Bednawska, Clin. Chim. Acra 84, 19-26. 14. Goldberg, J. A., and Rutenburg, A. M. (1958) Cancer 11, 283-291. 15. Shvyadas, V. K., Margolin, A. L., Sherstyuk, S. F., Klyosov, A. A., and Berezin, I. V. (1977) Bioorgan.
Kchimia
(Moscow)
3, 546-554.