[55b]
CEPHALOSPORINACETYLESTERASE (Bacillus subt~lis)
731
minimized by addition of reducing agents, including 0.1 M sodium oxalate 4 and Na.~S.,04 (0.5 mg/ml).l After precipitation with (NH,)2S0~ and dialysis against 0.1 M Na~C_~O, the enzyme retains its activity for several months in solution at pH 7.0 and at 2 °. At 35 ° it is relatively unstable in this solution, and at 50 ° about 90% of its activity is lost in 30 rain. At 25 ° it is relatively stable between pH 5.0 and pH 8.25, but is rapidly inactivated at pH values of 4.0 or below. 4 Dialysis of the enzyme against water is accompanied by about 50% loss of activity.4 pH. The pH-activity curve with monoacetin as a substrate shows a maximum between pH 6.0 and 6.5. The activities at pH 5.5 and 7.0 are about 10% lower than that at the optimum pH. Kinetic Constants. Km values of 30 mM and 32 mM have been reported with triacetin as substrate, TM and values of 4.7 mM and 8.6 mM with cephalosporin C 1 and cephalothin, 6 respectively, as substrates. The value for V,,,~x with triacetin as substrate is 3.4 times that with cephalosporin C as substrate in the presence of the same amount of enzyme. 1 With one preparation of enzyme, the maximum rate of hydrolysis of cephalothin was 0.36 mg cephalothin per milligram of esterase per minute2 6B. J. Abbott, personal communication, 1969.
[55b] Cephalosporin Acetylesterase (Bacillus subtilis) By BERNARDJ. ABBOTTand DAVID S. FUKUDA H
H ,
+
O , / ~ N ~ 9 , ~ CH2--O-- ~ -- CHa
~
COOH
CH2OH COOH
7-Aminocephalosporanic acid, R~H O Cephalothin, R = t S ~ _ C H 2 ~ NH~
'
O
Lk
Cephalosporin C, R z H O O C - - C H - - ( C H 2 ) 3 - - C -
Enzymes that deacetylate cephalosporins are widespread in nature. Jeffery et al. 1 reported that citrus peel contains an enzyme that deacety1j. D'A. Jeffery, E. P. Abraham, and G. G. F. Newton, Biochem. J. 81, 591 (1961).
732
ANTIBIOTIC INACTIVATION AND MODIFICATION
[55b]
lates cephalosporin C and triacetin. A survey of microbial sources revealed that many bacteria and actinomycetes also are capable of deacetylating cephalosporins. 2,~ A similar enzyme activity has been found in mammalian tissues, where the enzyme appears to be most prevalent in the liver and kidney. ~ Although the enzyme is widespread, it has not been purified to homogeneity and very little is known about its physical characteristics or kinetic behavior. The data in this report, except where other documentation is cited, were derived from the authors' laboratory.
Assay Method
Principle. Cephalosporin esterase activity is readily assayed by titration with an automatic pH star. The reaction generates acetic acid which causes a drop in pH of the reaction mixture. A standardized KOH solution is automatically added to maintain the pH at a preset value (usually pH = 7.0). The amount of KOH added per unit of time is directly proportional to the reaction rate. The rate of KOH addition is automatically recorded on a moving chart, and the initial reaction rate can be determined from the slope of the line. Reagents 7-(Thiophene-2-acetamido)cephalosporanic acid (cephalothin), 5.0 mg/ml 7-Aminocephalosporanic acid (7-ACA), 2.5 mg/ml. These solutions are stable for 1 day at 4 °. Enzyme: 2.0-10.0 mg/ml of unbuffered crude esterase at pH -- 7. 0 KOH, standardized 0.004 N
Procedure. The reaction is followed using a Radiometer T T T l l automatic titrator, ABU l I T 0.25-ml burette and SBR3 recorder. The reaction vessel TTA31 contains 2.0 ml of substrate solution adjusted to pH -- 7.0 and is maintained at 25 ° with a water jacket. A stream of humidified nitrogen must be passed over the surface of the reaction mixture to exclude CO2. In the absence of N2, CO2 absorption lowers the pH and triggers the addition of the standardized KOH solution. At pH = 7.0 and 25% water attacks the fl-lactam ring causing hydrolysis 5 and release of protons. The nonenzymic hydrolysis, which is a firstorder reaction, is minimal at pH = 7.0, but it increases at both alkaline A. L. Demain, R. B. Walton, J. F. Newkirk, and I. M. Miller, Nature (London) 199, 909 (1963). 3U.S. Patent 3,304,236 (1967). 4 C. H. O'Callaghan and P. W. Muggleton, Biochem. J. 89, 304 (1963). 5 j. Konecny, E. Felber, and J. Gruner, J. Antibiotic. 36, 135 (1973).
[55b]
CEPHALOSPORINACETYLESTERASE (Bacillus subtilis)
733
and acidic pH values. Nonenzymic hydrolysis is also accelerated at higher temperatures. The rate of nonenzymic hydrolysis of the substrate is measured by determining the rate of KOH addition in the absence of enzyme. The reaction is then started by adding 100 ~l of enzyme solution, and the total rate of KOH addition is determined. The reaction is linear for at least 15 rain. A convenient chart speed for the recorder is 0.5 cm/min. The chart contains 100 divisions, and each division corresponds to the addition of 10 nmoles of titrant. The delivery of 0.25 ml from the burette causes full-scale deflection of the recorder pen and corresponds to the hydrolysis of 1 ~mole of substrate in the 2.0-ml reaction vessel. Units and Specific Activity. A unit may be defined as the amount of enzyme that hydrolyzes 10 nmoles of cephalothin per minute at 25 °, pH = 7.0, at a cephalothin concentration of 5 mg/ml. Specific activity may be expressed as units per milligram of protein.
Preparation of the Enzyme
Cultivation of the Microo,rganism. The enzyme is prevalent in strains of Bacillus subtilis. Strain WRRL-B-558 (from the Western Regional Research Laboratory) is cultivated in trypticase soy broth on a rotary shaker at 30 °. During cultivation the pH declines to 6.2 and then begins to rise. After 22 hr, when the pH is about 7.8, the cells are harvested by centrifugation. The enzyme may be recovered from either the culture supernatant or from the sedimented biomass. About 75% of the total esterase activity is associated with the biomass fraction. Partial Preparation of the Enzyme. Although most of the enzyme is fo~md with the biomass, the specific activity of a crude biomass preparation is sinfilar to that obtained from a culture supernatant. Isolation of the esterasc from the supernatant obviates the need for a sonication and nucleic acid removal step. The enzyme is recovered from the supernatant by saturation with ammonium sulfate. The ammonium sulfate is removed by dialysis against distilled water and the salt-free enzyme may be stored after lyophilization. A 2- to 3-fold purificatioll can be achieved by recovering the protein fraction that precipitates between 50% and 80% of ammonium sulfate saturation. Additional purification can be obtained by stirring 2.0 mg/ml of salt-free unbuffered esterase preparation with 4.0 mg/ml bentonite at pH -- 6.5 and 25 °. After 3 hr the bentonite is removed by centrifugation. The supernatant contains about 75% of the initial esterase activity and exhibits a 2.5-fold increase in specific activity. Further purification (~40-fold~ is possible with Sephadex G-200 chromatography. Using a calibrated Sephadex column, it was determined
734
ANTIBIOTIC INACTIVATION A N D
MODIFICATION
[55]
that the cephalosporin acetylesterase has a molecular weight of about 190,000.
Properties Stability. The enzyme is extremely stable. It may be stored in an unbuffered solution at 25 ° and pH - 7.0 for 3 weeks with little or no loss of activity. Sodium azide (0.02~) must be added to prevent the growth of microorganisms. At 80 ° all activity is lost within 5 rain; however, the activity is retained after 10 rain at 80 ° if heating occurs in the presence of 1.0 M phosphate buffer. The salt-free enzyme loses 50~ of its activity in 5 min at 70 °, but no activity is lost at 60 ° after 1 hr. pH and Temperature Optima. The enzyme has a narrow pH optimum at pH -- 7.0, and the optimum temperature is between 40 ° and 50 °. Substrates and Inhibitors. In addition to cephalosporins, the enzyme will hydrolyze mono- and triacetin, a-naphthyl acetate, and glucose pentaacetate. The enzyme does not hydrolyze casein, acetanilide, p-nitrophenylacetate, p-nitrophenylsulfate, and it is not inhibited by arsenilic acid or bis (p-nitrophenyl) phosphate. Kinetic Properties. The enzyme exhibits Michaelis-Menten kinetics with cephalosporins and triacetin. The Michaelis constants measured with these substrates are cephalothin, 8.3 raM; 7-ACA, 2.8 raM; triacetin, 0.46 raM. The products of 7-ACA hydrolysis (acetate q- deacetyl 7-ACA) are weak competitive inhibitors of the reaction. The Ki values are 5 )< 10.2 M for acetate and 3.6 X 10-2 M for deacetyl 7-ACA.
[56] Chloramphenicol Hydrolase By
CH20H I H2N-C-H I H- C-OH
CHzOH CI2CHCOHN- C-H !
H-C-OH
0
L. C. VINING
HzO
NO2
CHLORAMPHENICOL
Cl2CHCOOH +
0 NOz
p-NITROPHENYLSERINOL
CEPHALOSPORINACETYLESTERASE (Bacillus subt~lis)
731
minimized by addition of reducing agents, including 0.1 M sodium oxalate 4 and Na.~S.,04 (0.5 mg/ml).l After precipitation with (NH,)2S0~ and dialysis against 0.1 M Na~C_~O, the enzyme retains its activity for several months in solution at pH 7.0 and at 2 °. At 35 ° it is relatively unstable in this solution, and at 50 ° about 90% of its activity is lost in 30 rain. At 25 ° it is relatively stable between pH 5.0 and pH 8.25, but is rapidly inactivated at pH values of 4.0 or below. 4 Dialysis of the enzyme against water is accompanied by about 50% loss of activity.4 pH. The pH-activity curve with monoacetin as a substrate shows a maximum between pH 6.0 and 6.5. The activities at pH 5.5 and 7.0 are about 10% lower than that at the optimum pH. Kinetic Constants. Km values of 30 mM and 32 mM have been reported with triacetin as substrate, TM and values of 4.7 mM and 8.6 mM with cephalosporin C 1 and cephalothin, 6 respectively, as substrates. The value for V,,,~x with triacetin as substrate is 3.4 times that with cephalosporin C as substrate in the presence of the same amount of enzyme. 1 With one preparation of enzyme, the maximum rate of hydrolysis of cephalothin was 0.36 mg cephalothin per milligram of esterase per minute2 6B. J. Abbott, personal communication, 1969.
[55b] Cephalosporin Acetylesterase (Bacillus subtilis) By BERNARDJ. ABBOTTand DAVID S. FUKUDA H
H ,
+
O , / ~ N ~ 9 , ~ CH2--O-- ~ -- CHa
~
COOH
CH2OH COOH
7-Aminocephalosporanic acid, R~H O Cephalothin, R = t S ~ _ C H 2 ~ NH~
'
O
Lk
Cephalosporin C, R z H O O C - - C H - - ( C H 2 ) 3 - - C -
Enzymes that deacetylate cephalosporins are widespread in nature. Jeffery et al. 1 reported that citrus peel contains an enzyme that deacety1j. D'A. Jeffery, E. P. Abraham, and G. G. F. Newton, Biochem. J. 81, 591 (1961).
732
ANTIBIOTIC INACTIVATION AND MODIFICATION
[55b]
lates cephalosporin C and triacetin. A survey of microbial sources revealed that many bacteria and actinomycetes also are capable of deacetylating cephalosporins. 2,~ A similar enzyme activity has been found in mammalian tissues, where the enzyme appears to be most prevalent in the liver and kidney. ~ Although the enzyme is widespread, it has not been purified to homogeneity and very little is known about its physical characteristics or kinetic behavior. The data in this report, except where other documentation is cited, were derived from the authors' laboratory.
Assay Method
Principle. Cephalosporin esterase activity is readily assayed by titration with an automatic pH star. The reaction generates acetic acid which causes a drop in pH of the reaction mixture. A standardized KOH solution is automatically added to maintain the pH at a preset value (usually pH = 7.0). The amount of KOH added per unit of time is directly proportional to the reaction rate. The rate of KOH addition is automatically recorded on a moving chart, and the initial reaction rate can be determined from the slope of the line. Reagents 7-(Thiophene-2-acetamido)cephalosporanic acid (cephalothin), 5.0 mg/ml 7-Aminocephalosporanic acid (7-ACA), 2.5 mg/ml. These solutions are stable for 1 day at 4 °. Enzyme: 2.0-10.0 mg/ml of unbuffered crude esterase at pH -- 7. 0 KOH, standardized 0.004 N
Procedure. The reaction is followed using a Radiometer T T T l l automatic titrator, ABU l I T 0.25-ml burette and SBR3 recorder. The reaction vessel TTA31 contains 2.0 ml of substrate solution adjusted to pH -- 7.0 and is maintained at 25 ° with a water jacket. A stream of humidified nitrogen must be passed over the surface of the reaction mixture to exclude CO2. In the absence of N2, CO2 absorption lowers the pH and triggers the addition of the standardized KOH solution. At pH = 7.0 and 25% water attacks the fl-lactam ring causing hydrolysis 5 and release of protons. The nonenzymic hydrolysis, which is a firstorder reaction, is minimal at pH = 7.0, but it increases at both alkaline A. L. Demain, R. B. Walton, J. F. Newkirk, and I. M. Miller, Nature (London) 199, 909 (1963). 3U.S. Patent 3,304,236 (1967). 4 C. H. O'Callaghan and P. W. Muggleton, Biochem. J. 89, 304 (1963). 5 j. Konecny, E. Felber, and J. Gruner, J. Antibiotic. 36, 135 (1973).
[55b]
CEPHALOSPORINACETYLESTERASE (Bacillus subtilis)
733
and acidic pH values. Nonenzymic hydrolysis is also accelerated at higher temperatures. The rate of nonenzymic hydrolysis of the substrate is measured by determining the rate of KOH addition in the absence of enzyme. The reaction is then started by adding 100 ~l of enzyme solution, and the total rate of KOH addition is determined. The reaction is linear for at least 15 rain. A convenient chart speed for the recorder is 0.5 cm/min. The chart contains 100 divisions, and each division corresponds to the addition of 10 nmoles of titrant. The delivery of 0.25 ml from the burette causes full-scale deflection of the recorder pen and corresponds to the hydrolysis of 1 ~mole of substrate in the 2.0-ml reaction vessel. Units and Specific Activity. A unit may be defined as the amount of enzyme that hydrolyzes 10 nmoles of cephalothin per minute at 25 °, pH = 7.0, at a cephalothin concentration of 5 mg/ml. Specific activity may be expressed as units per milligram of protein.
Preparation of the Enzyme
Cultivation of the Microo,rganism. The enzyme is prevalent in strains of Bacillus subtilis. Strain WRRL-B-558 (from the Western Regional Research Laboratory) is cultivated in trypticase soy broth on a rotary shaker at 30 °. During cultivation the pH declines to 6.2 and then begins to rise. After 22 hr, when the pH is about 7.8, the cells are harvested by centrifugation. The enzyme may be recovered from either the culture supernatant or from the sedimented biomass. About 75% of the total esterase activity is associated with the biomass fraction. Partial Preparation of the Enzyme. Although most of the enzyme is fo~md with the biomass, the specific activity of a crude biomass preparation is sinfilar to that obtained from a culture supernatant. Isolation of the esterasc from the supernatant obviates the need for a sonication and nucleic acid removal step. The enzyme is recovered from the supernatant by saturation with ammonium sulfate. The ammonium sulfate is removed by dialysis against distilled water and the salt-free enzyme may be stored after lyophilization. A 2- to 3-fold purificatioll can be achieved by recovering the protein fraction that precipitates between 50% and 80% of ammonium sulfate saturation. Additional purification can be obtained by stirring 2.0 mg/ml of salt-free unbuffered esterase preparation with 4.0 mg/ml bentonite at pH -- 6.5 and 25 °. After 3 hr the bentonite is removed by centrifugation. The supernatant contains about 75% of the initial esterase activity and exhibits a 2.5-fold increase in specific activity. Further purification (~40-fold~ is possible with Sephadex G-200 chromatography. Using a calibrated Sephadex column, it was determined
734
ANTIBIOTIC INACTIVATION A N D
MODIFICATION
[55]
that the cephalosporin acetylesterase has a molecular weight of about 190,000.
Properties Stability. The enzyme is extremely stable. It may be stored in an unbuffered solution at 25 ° and pH - 7.0 for 3 weeks with little or no loss of activity. Sodium azide (0.02~) must be added to prevent the growth of microorganisms. At 80 ° all activity is lost within 5 rain; however, the activity is retained after 10 rain at 80 ° if heating occurs in the presence of 1.0 M phosphate buffer. The salt-free enzyme loses 50~ of its activity in 5 min at 70 °, but no activity is lost at 60 ° after 1 hr. pH and Temperature Optima. The enzyme has a narrow pH optimum at pH -- 7.0, and the optimum temperature is between 40 ° and 50 °. Substrates and Inhibitors. In addition to cephalosporins, the enzyme will hydrolyze mono- and triacetin, a-naphthyl acetate, and glucose pentaacetate. The enzyme does not hydrolyze casein, acetanilide, p-nitrophenylacetate, p-nitrophenylsulfate, and it is not inhibited by arsenilic acid or bis (p-nitrophenyl) phosphate. Kinetic Properties. The enzyme exhibits Michaelis-Menten kinetics with cephalosporins and triacetin. The Michaelis constants measured with these substrates are cephalothin, 8.3 raM; 7-ACA, 2.8 raM; triacetin, 0.46 raM. The products of 7-ACA hydrolysis (acetate q- deacetyl 7-ACA) are weak competitive inhibitors of the reaction. The Ki values are 5 )< 10.2 M for acetate and 3.6 X 10-2 M for deacetyl 7-ACA.
[56] Chloramphenicol Hydrolase By
CH20H I H2N-C-H I H- C-OH
CHzOH CI2CHCOHN- C-H !
H-C-OH
0
L. C. VINING
HzO
NO2
CHLORAMPHENICOL
Cl2CHCOOH +
0 NOz
p-NITROPHENYLSERINOL
