APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1979, p. 35-38 0099-2240/79/07-0035/04$02.00/0
Vol. 38, No. 1
Biochemical Properties of Penicillin Amidohydrolase from Micrococcus luteus D. H. NAM AND DEWEY D. Y. RYU* The Korea Advanced Institute of Science, POB 150, Chung-Ryang-Ri, Seoul, Korea
Received for publication 17 April 1979
Some biochemical properties of whole-cell penicillin amidohydrolase from Micrococcus luteus have been studied. This whole-cell enzyme showed its maximal activity at 36°C at pH 7.5. It was found that the activation energy of this enzyme was 8.03 kcal (ca. 33.6 kJ) per mol, and this amidohydrolase showed firstorder decay at 360C. The penicillin amidohydrolase was deactivated rapidly at temperatures above 500C during storage or preincubation for 24 h. The Michaelis constant, Ki, for penicillin G was determined as 2.26 mM, and the substrate inhibition constant, K"8, was 155 mM. The whole-cell penicillin amidohydrolase from M. luteus was capable of hydrolyzing penicillin G, penicillin V, ampicillin, and cephalexin, but not cephalosporin C and cloxacillin. This whole-cell enzyme also had synthetic activity for semisynthetic penicillins or cephalosporins from D-(-)-a-phenylglycine methyl ester and 6-a-aminopenicillanic acid or 7-amino-3deacetoxycephalosporanic acid. For cytoplasmic and cell debris fractions, the harvested whole cells were washed with 0.1 M phosphate buffer (pH 7.5) and then sonically disrupted for 15 min and centrifuged at 15,000 x g for 45 min in a refrigerated centrifuge. The supematant was used as cytoplasmic fraction, and the precipitated residue was collected as cell debris. Enzyme reaction. The whole-cell enzyme harvested from 10 ml of fermented broth catalyzed the hydrolytic reaction when added to 10 ml of substrate solution containing 10 mg of substrate (penicillin or cephalosporin derivatives) per 1 ml of 0.1 M phosphate buffer (pH 7.5). The same enzyme preparation also catalyzed the synthetic reaction when added to 10 ml of reaction solution containing 10 mg of 7-amino-3deacetoxycephalosporanic acid or 6-a-aminopenicillanic acid and 100 mg of D-(-)-a-phenylglycine methyl ester per 1 ml of 0.1 M phosphate buffer (pH 7.0). The resuspended whole-cell enzyme was mixed thoroughly with substrate solution, and was incubated at 36°C for 1 h. For quantitative determination of enzyme activity, a modified ninhydrin method was applied for the determination of the amount of 6-a-aminopenicillanic acid and 7-amino-3-deacetoxycephalosporanic acid (13, 9). After reaction, the whole-cell enzyme suspension was instantly treated with 0.5 ml of formic acid (98%) and centrifuged at 5,000 rpm for 20 min. The cell residue was discarded, and the supernatant was collected as the reaction mixture. One unit of penicillin amidohydrolase was defined as the amount of enzyme required to produce or convert 1 umol of 6-a-aminopenicillanic acid or 7-amino-3-deacetoxycephalosporanic acid per h. For kinetic study, potassium penicillin G solutions in 0.1 M phosphate buffer (pH 7.5) were prepared to give concentrations of 25, 20, 15, 10, 5, 2.5, 1, 0.5, and 0.25 mM. These were mixed with whole-cell enzyme
Recently, some microorganisms producing penicillin amidohydrolases with broad substrate specificity were reported (11-14). These amidohydrolases are capable of catalyzing not only the hydrolysis of penicillin G but also N-acylation of 6-aminopenicillanic acid or 7-amino-3-deacetoxycephalosporanic acid compounds, and they belong to ampicillin acylase according to Vandamme and Voets (19). We have studied the biochemical properties of the whole-cell penicillin amidohydrolase from this strain and reported the results in this paper. MATERIALS AND METHODS Enzyme preparation. Micrococcus luteus AHU 1427 (KY 3781) was used in this study. One milliliter of the enriched heavy culture was used to inoculate 100 ml of culture medium in a 500-ml Erlenmeyer flask with a cotton plug, and it was cultivated at 30°C for 48 h, aerobically, with shaking at 300 rpm. The culture medium was prepared by adjusting to pH 7.25 (this would be changed to pH 7.0 during autoclaving in the case of 500-ml flasks) and autoclaving at 120°C for 20 min. Throughout cultivation, no inducer such as phenylacetic acid was added. For the examination of synthetic enzyme activity,
four kinds of media reported previously (7, 14, 17, 18) were evaluated. The cell cultivation was carried out under the same conditions as those described above. After 48 h of cultivation, the fermented broth was harvested by centrifugation at 5,000 rpm for 20 min. The centrifuged microbial cake was suspended for washing in the same volume of 0.1 M phosphate buffer (pH 7.5) as the original broth volume, and was centrifuged again. The washed microbial pellet was used as the whole-cell enzyme. 35
36
APPL. ENVIRON. MICROBIOL.
NAM AND RYU
and were incubated at 36°C for 10 min. The reaction rates during the 10 min were considered as initial enzyme reaction rates.
TABLE 1. Heat stability of whole-cell penicillin amidohydrolase at different temperaturesa Relative activity (% after 24 h of (~C)storage
Storage emp (°C) Storage temp
RESULTS AND DISCUSSION 100.0 Controlb 0 96.4 It was observed that 95.8% of total enzyme 95.2 10 activity was found in the cytoplasm fraction, and 20 93.7 the culture broth and cell debris contained insig88.5 30 nificant amounts of enzyme. Based on the results 36 54.5 of thin-layer chromatographic analysis of the 40 12.5 reaction mixture, it was concluded that M. luteus 50 0.0 has intracellular penicillin G amidohydrolase acThe intact whole-cell amidohydrolase penicillin tivity but no penicillinase activity. was incubated at each temperature for 24 h before Biochemical properties of penicillin ami- hydrolytic reaction. Control: 3.864 units/ml. Reaction dohydrolase. The whole-cell penicillin amido- conditions: 36°C; pH 7.5; 1 h; 10 mg/ml of substrate hydrolase from M. luteus showed an optimum solution. b Control refers to the initial activity before storage. temperature at 360C and an optimum pH at 7.5. From the experimental study on the effect of temperature on the initial reaction rate, the activation energy was estimated using an Arrhenius plot. As shown in Fig. 1, the activation energy was found to be 8.03 kcal (ca. 33.6 kJ) per mol. The penicillin amidohydrolase activity of the harvested whole-cell enzyme stored at various t .4 4temperatures was determined. The enzyme was completely deactivated at a storage temperature > A, A-AO exp (-0.025't) above 500C for 24 h (Table 1). The deactivation I-J INITIAL ENZYME ACTIVITY: 3.864 UNITS/ml of whole-cell enzyme showed first-order kinetics z during storage at 360C for 24 h (Fig. 2). The decay constant determined was 0.025 h-1. 3 The whole-cell penicillin amidohydrolase from M. luteus requires moderate reaction con0 5 10 15 20 25 ditions and is susceptible to thermal deactivaSTORAGE TIME (hr) tion. FIG. 2. Determination of deactivation constant of Kinetic study. The whole-cell enzyme kinetics were studied. The kinetic constants were the whole-cell penicillin amidohydrolase in hydrolytic reaction. The intact whole-cell penicillin amiestimated by Lineweaver-Burk plot from the dohydrolase was incubated at 36°C before hydrolytic
u~~~
w >
j
C
reaction. Initial activity: 3.864 units/ml. Reaction conditions; 36°C; pH 7.5; 1 h; 10 mg/ml of substrate solution.
1.5-
C
1.0
A,
4
0.5-
0
z 0
c
0.0-
E * 8.028 KcoI/MOLE
-0.5 3.1
3.2
3.3
3.4
3.5
3.6
3.1
INVERSE ABSOLUTE TEMPERATURE, I/T (X 103)
FIG. 1. Determination of activation energy for the penicillin amidohydrolase reaction by Arrhenius graphical method (hydrolytic reaction).
reaction rates determined in the substrate concentration range of 0.25 to 25 mM. The values of Km and Vm determined were 2.66 mM penicillin G and 10.42 units, respectively (Fig. 3). The penicillin amidohydrolase activity was inhibited by high concentrations of substrate penicillin G. The substrate inhibition constant, Kij, was determined from plots of 1/V against (S). The Ki. value obtained was 155 mM penicillin G. Substrate specificity. Substrate specificity of M. luteus penicillin amidohydrolase was examined, and the results are summarized in Table 2. The degree of hydrolysis is scored from + to +++, based on relative amount of the hydrolytic product. A negative sign indicates that no hydrolytic product was detected.
PROPERTIES OF PENICILLIN AMIDOHYDROLASE
VOL. 38, 1979 6-
nocephalosporanic acid by acylase of Comaspecies (Y. Shibuya, T. Fujii, T. Yamaguchi, and T. Matsuda, Japanese patent no. 75101,584, 1975). For enzymatic hydrolysis of cephalosporin C, the use of two enzymes, D-amino acid oxidase and a certain acylase, must be required, and cephalosporin C cannot be cleaved by only one enzyme like penicillin amidohydrolase. Synthetic enzyme production. Penicillin amidohydrolase was found to be a reversible enzyme for a limited number of substrates, and it can synthesize semisynthetic penicillins or cephalosporins. Using M. luteus, the synthetic activity of enzyme for ampicillin and cephalexin was evaluated (Table 3). When using medium I, the highest enzyme acitivity was obtained as compared with the three other media evaluated. When medium III was used, slightly lower enzyme productivity was detected. However, the synthetic enzyme productivity was rather low in all cases studied, and further studies and improvements will be required for its practical application. Most penicillin amidohydrolases obtained from other microbial sources show product inhibition (the value of the inhibition constant Kip ranges about 1 to 25 mM) and relatively high Michaelis constants (Km values range about 2.5 to 10.3 mM) (13). The enzyme from M. luteus showed no product inhibition and a relatively low Km value (2.2 mM), although it showed substrate inhibition (the value of the inhibition constant, K,,, found was 155 mM). These results indicate that there are some advantages of using the enzyme from M. luteus, and it is perhaps one of the most promising strains for industrial application, once enzyme productivity and specific activity are increased to a practical level. monas
5-
2-
ENZYME LOADING *6.06 /Km
2.66
UNITS
mM
Vm * 10.42 UNITS
-0 5
37
0.
1.0
0.5 I/S
1.5
2.0
(mM-,)
FIG. 3. Lineweaver-Burk plot of initial hydrolytic reaction rate at different substrate concentrations.
TABLE 2. Substrate specificity of M. luteus penicillin amidohydrolase on /3-lactam antibioticsa Compound
Degree of hy-
drolysis
Rf value
0.80 0.77 0.30 0.76 0.23 6-APA 0.16 Cephalosporin C 0.22 7-ACA ++ 0.56 Cephalexin 0.15 7-ADCA Reaction conditions: 3600; pH 7.0; 6 h; 10 mg/ml of substrate solution. Symbols: +++, Hydrolytic product above 2 mg/ml; ++, hydrolytic product near 2 mg/ ml; +, hydrolytic product below 2 mg/ml; -, hydrolytic product was not detected. 6-APA, 6-a-Aminopenicillanic acid; 7-ACA, 7-aminocephalosporanic acid; 7ADCA, 7-amino-3-deacetoxycephalosporanic acid.
Penicillin G Penicillin V Ampicillin Cloxacillin
+++ + ++
The M. luteus enzyme could hydrolyze penicillin G, ampicillin, and cephalexin, and it deacylated penicillin V only slightly. Cloxacillin was not cleaved. This enzyme could not deacylate cephalosporin C. These results are consistent with the results of other investigators (4-6, 8, 12, 15, 16). That is, this enzyme can deacylate the simple alkyl or a-aminoalkyl side chain from /3lactam antibiotics, but not the bulky group or the side chain containing dipolar ions. Recently, it was reported that D-amino acid oxidase of some origins oxidatively deaminates the D-a-aminoadipyl side chain of cephalosporin C to yield 7-/B-(oxoadipamido)-cephalosporanic acid (10). This compound is known to be oxidatively decarboxylated with ease to 7-,8-(4-carboxybutanamido)-cephalosporanic acid, which can be easily deacylated enzymatically to 7-ami-
TABLE 3. Synthetic activity of M. luteus penicillin amidohydrolase on ,B-lactam antibioticsa Synthetic activity Medium
Cephalexin Ampicillin 0.12 0.51 0.00 0.17 0.03 0.15 0.06 0.35
Reference
foredia media for
(10) I (11) II (6) III (12) IV a Substrate solution: 100 mg of D-(-)-a-phenylglycine methyl ester hydrochloride and 10 mg of 7-amino3-deacetoxycephalosporanic acid or 6-a-aminopenicillanic acid in 1 ml of phosphate buffer (pH 7.0). Reaction conditions: 360C; pH 7.0; 1 h; 10 mg/mi of substrate solution. b Expressed as micromoles per hour per milliliter of broth.
38
NAM AND RYU
LITERATURE CITED 1. Alicino, J. F. 1961. lodometric assay of natural and synthetic penicillins, 6-aminopenicillanic acid and cephalosporin C. Anal. Chem. 33:648-649. 2. Bomstein, J., and W. G. Evans. 1965. Automated colorimetric determination of 6-aminopenicillanic-acid in fermentation media. Anal. Chem. 37:576-578. 3. Boxer, G. E., and P. M. Everett. 1949. Colorimetric determination of benzylpenicillin. Anal. Chem. 21:670673. 4. Claridge, C. A., J. R. Luttinger, and J. Lein. 1963. Specificity of penicillin amidases. Proc. Soc. Exp. Biol. Med. 113:1008-1012. 5. Demain, A. L., R. B. Walton, J. F. Newkirk, and I. M. Miller. 1963. Microbial degradation of cephalosporin C. Nature (London) 199:909-910. 6. Erickson, R. C., and R. E. Bennett. 1965. Penicillin acylase activity of Penicillium chrysogenum. Appl. Microbiol. 13:738-742. 7. Fujii, T., K. Matsumoto, and T. Watanabe. 1976. Enzymatic synthesis of cephalexin. Proc. Biochem. 11:2124. 8. Huang, H. T., T. A. Sato, and G. M. Shull. 1963. Distribution of substrate specificity of benzylpenicillin acylase. Appl. Microbiol. 11:1-6. 9. Marrelli, L. P. 1968. Colorimetric method for determination of 7-aminocephalosporanic acid (7-ACA) and related compounds. J. Pharm. Sci. 57:2172-2173. 10. Mazzeo, P., and A. Romeo. 1972. Enzymic and chemical transformation of the side chain of cephalosporin c. J. Chem. Soc. Perkin Trans. 1, p. 2532. 11. Nara, T., M. Misawa, R. Okachi, and M. Yamamoto.
APPL. ENVIRON. MICROBIOL. 1971. Enzymatic synthesis of D(-)-a-aminobenzylpenicillin. Agric. Biol. Chem. 33:1676-1682. 12. Nueesch, J., J. Gruner, F. Knuesel, and H. T. Treichler. 1967. Cephalosporin C- and 7-aminocephalosporanic acid degrading enzymes from microorganisms. Pathol. Microbiol. 30:880-889. 13. Ryu, D., L. F. Bruno, B. K. Lee, and K. Venkat. 1972. Microbial penicillin amidohydrolase and the performance of a continuous enzyme reactor system, p. 307-314. In G. Terui (ed.), Proc. IV IFS: Fennentation technology today. Society of Fermentation Technology, Osaka. 14. Shimizu, M., T. Masuike, H. Fujita, K. Kimura, R. Okachi, and T. Nara. 1975. Search for microorganisms producing cephalosporin acylase and enzymatic synthesis of cephalosporins. Agric. Biol. Chem. 39:1225-1232. 15. Shimizu, M., R. Okachi, K. Kimura, and T. Nara. 1975. Purification and properties of penicillin acylase from Kluyvera citrophila. Agric. Biol. Chem. 39:16611655. 16. Sjoberg, B., L. Nathorst-Westfelt, and B. Ortengren. 1967. Enzymatic hydrolysis of some penicillins and cephalosporins by E. coli acylase. Acta Chem. Scand. 21:547-551. 17. Takahashi, T., Y. Yamazaki, and K. Kato. 1974. Substrate specificity of an a-amino acid ester hydrolase produced by Acetobacter turbidans (ATCC 9325). Biochem. J. 137:497-593. 18. Takahashi, T., Y. Yamazaki, K. Kato, and M. Isono. 1972. Enzymatic synthesis of cephalosporins J. Am. Chem. Soc. 94:4035-4037. 19. Vandamme, E. J., and J. P. Voets. 1974. Microbial penicillin acylases. Adv. Appl. Microbiol. 17:311-369.
Vol. 38, No. 1
Biochemical Properties of Penicillin Amidohydrolase from Micrococcus luteus D. H. NAM AND DEWEY D. Y. RYU* The Korea Advanced Institute of Science, POB 150, Chung-Ryang-Ri, Seoul, Korea
Received for publication 17 April 1979
Some biochemical properties of whole-cell penicillin amidohydrolase from Micrococcus luteus have been studied. This whole-cell enzyme showed its maximal activity at 36°C at pH 7.5. It was found that the activation energy of this enzyme was 8.03 kcal (ca. 33.6 kJ) per mol, and this amidohydrolase showed firstorder decay at 360C. The penicillin amidohydrolase was deactivated rapidly at temperatures above 500C during storage or preincubation for 24 h. The Michaelis constant, Ki, for penicillin G was determined as 2.26 mM, and the substrate inhibition constant, K"8, was 155 mM. The whole-cell penicillin amidohydrolase from M. luteus was capable of hydrolyzing penicillin G, penicillin V, ampicillin, and cephalexin, but not cephalosporin C and cloxacillin. This whole-cell enzyme also had synthetic activity for semisynthetic penicillins or cephalosporins from D-(-)-a-phenylglycine methyl ester and 6-a-aminopenicillanic acid or 7-amino-3deacetoxycephalosporanic acid. For cytoplasmic and cell debris fractions, the harvested whole cells were washed with 0.1 M phosphate buffer (pH 7.5) and then sonically disrupted for 15 min and centrifuged at 15,000 x g for 45 min in a refrigerated centrifuge. The supematant was used as cytoplasmic fraction, and the precipitated residue was collected as cell debris. Enzyme reaction. The whole-cell enzyme harvested from 10 ml of fermented broth catalyzed the hydrolytic reaction when added to 10 ml of substrate solution containing 10 mg of substrate (penicillin or cephalosporin derivatives) per 1 ml of 0.1 M phosphate buffer (pH 7.5). The same enzyme preparation also catalyzed the synthetic reaction when added to 10 ml of reaction solution containing 10 mg of 7-amino-3deacetoxycephalosporanic acid or 6-a-aminopenicillanic acid and 100 mg of D-(-)-a-phenylglycine methyl ester per 1 ml of 0.1 M phosphate buffer (pH 7.0). The resuspended whole-cell enzyme was mixed thoroughly with substrate solution, and was incubated at 36°C for 1 h. For quantitative determination of enzyme activity, a modified ninhydrin method was applied for the determination of the amount of 6-a-aminopenicillanic acid and 7-amino-3-deacetoxycephalosporanic acid (13, 9). After reaction, the whole-cell enzyme suspension was instantly treated with 0.5 ml of formic acid (98%) and centrifuged at 5,000 rpm for 20 min. The cell residue was discarded, and the supernatant was collected as the reaction mixture. One unit of penicillin amidohydrolase was defined as the amount of enzyme required to produce or convert 1 umol of 6-a-aminopenicillanic acid or 7-amino-3-deacetoxycephalosporanic acid per h. For kinetic study, potassium penicillin G solutions in 0.1 M phosphate buffer (pH 7.5) were prepared to give concentrations of 25, 20, 15, 10, 5, 2.5, 1, 0.5, and 0.25 mM. These were mixed with whole-cell enzyme
Recently, some microorganisms producing penicillin amidohydrolases with broad substrate specificity were reported (11-14). These amidohydrolases are capable of catalyzing not only the hydrolysis of penicillin G but also N-acylation of 6-aminopenicillanic acid or 7-amino-3-deacetoxycephalosporanic acid compounds, and they belong to ampicillin acylase according to Vandamme and Voets (19). We have studied the biochemical properties of the whole-cell penicillin amidohydrolase from this strain and reported the results in this paper. MATERIALS AND METHODS Enzyme preparation. Micrococcus luteus AHU 1427 (KY 3781) was used in this study. One milliliter of the enriched heavy culture was used to inoculate 100 ml of culture medium in a 500-ml Erlenmeyer flask with a cotton plug, and it was cultivated at 30°C for 48 h, aerobically, with shaking at 300 rpm. The culture medium was prepared by adjusting to pH 7.25 (this would be changed to pH 7.0 during autoclaving in the case of 500-ml flasks) and autoclaving at 120°C for 20 min. Throughout cultivation, no inducer such as phenylacetic acid was added. For the examination of synthetic enzyme activity,
four kinds of media reported previously (7, 14, 17, 18) were evaluated. The cell cultivation was carried out under the same conditions as those described above. After 48 h of cultivation, the fermented broth was harvested by centrifugation at 5,000 rpm for 20 min. The centrifuged microbial cake was suspended for washing in the same volume of 0.1 M phosphate buffer (pH 7.5) as the original broth volume, and was centrifuged again. The washed microbial pellet was used as the whole-cell enzyme. 35
36
APPL. ENVIRON. MICROBIOL.
NAM AND RYU
and were incubated at 36°C for 10 min. The reaction rates during the 10 min were considered as initial enzyme reaction rates.
TABLE 1. Heat stability of whole-cell penicillin amidohydrolase at different temperaturesa Relative activity (% after 24 h of (~C)storage
Storage emp (°C) Storage temp
RESULTS AND DISCUSSION 100.0 Controlb 0 96.4 It was observed that 95.8% of total enzyme 95.2 10 activity was found in the cytoplasm fraction, and 20 93.7 the culture broth and cell debris contained insig88.5 30 nificant amounts of enzyme. Based on the results 36 54.5 of thin-layer chromatographic analysis of the 40 12.5 reaction mixture, it was concluded that M. luteus 50 0.0 has intracellular penicillin G amidohydrolase acThe intact whole-cell amidohydrolase penicillin tivity but no penicillinase activity. was incubated at each temperature for 24 h before Biochemical properties of penicillin ami- hydrolytic reaction. Control: 3.864 units/ml. Reaction dohydrolase. The whole-cell penicillin amido- conditions: 36°C; pH 7.5; 1 h; 10 mg/ml of substrate hydrolase from M. luteus showed an optimum solution. b Control refers to the initial activity before storage. temperature at 360C and an optimum pH at 7.5. From the experimental study on the effect of temperature on the initial reaction rate, the activation energy was estimated using an Arrhenius plot. As shown in Fig. 1, the activation energy was found to be 8.03 kcal (ca. 33.6 kJ) per mol. The penicillin amidohydrolase activity of the harvested whole-cell enzyme stored at various t .4 4temperatures was determined. The enzyme was completely deactivated at a storage temperature > A, A-AO exp (-0.025't) above 500C for 24 h (Table 1). The deactivation I-J INITIAL ENZYME ACTIVITY: 3.864 UNITS/ml of whole-cell enzyme showed first-order kinetics z during storage at 360C for 24 h (Fig. 2). The decay constant determined was 0.025 h-1. 3 The whole-cell penicillin amidohydrolase from M. luteus requires moderate reaction con0 5 10 15 20 25 ditions and is susceptible to thermal deactivaSTORAGE TIME (hr) tion. FIG. 2. Determination of deactivation constant of Kinetic study. The whole-cell enzyme kinetics were studied. The kinetic constants were the whole-cell penicillin amidohydrolase in hydrolytic reaction. The intact whole-cell penicillin amiestimated by Lineweaver-Burk plot from the dohydrolase was incubated at 36°C before hydrolytic
u~~~
w >
j
C
reaction. Initial activity: 3.864 units/ml. Reaction conditions; 36°C; pH 7.5; 1 h; 10 mg/ml of substrate solution.
1.5-
C
1.0
A,
4
0.5-
0
z 0
c
0.0-
E * 8.028 KcoI/MOLE
-0.5 3.1
3.2
3.3
3.4
3.5
3.6
3.1
INVERSE ABSOLUTE TEMPERATURE, I/T (X 103)
FIG. 1. Determination of activation energy for the penicillin amidohydrolase reaction by Arrhenius graphical method (hydrolytic reaction).
reaction rates determined in the substrate concentration range of 0.25 to 25 mM. The values of Km and Vm determined were 2.66 mM penicillin G and 10.42 units, respectively (Fig. 3). The penicillin amidohydrolase activity was inhibited by high concentrations of substrate penicillin G. The substrate inhibition constant, Kij, was determined from plots of 1/V against (S). The Ki. value obtained was 155 mM penicillin G. Substrate specificity. Substrate specificity of M. luteus penicillin amidohydrolase was examined, and the results are summarized in Table 2. The degree of hydrolysis is scored from + to +++, based on relative amount of the hydrolytic product. A negative sign indicates that no hydrolytic product was detected.
PROPERTIES OF PENICILLIN AMIDOHYDROLASE
VOL. 38, 1979 6-
nocephalosporanic acid by acylase of Comaspecies (Y. Shibuya, T. Fujii, T. Yamaguchi, and T. Matsuda, Japanese patent no. 75101,584, 1975). For enzymatic hydrolysis of cephalosporin C, the use of two enzymes, D-amino acid oxidase and a certain acylase, must be required, and cephalosporin C cannot be cleaved by only one enzyme like penicillin amidohydrolase. Synthetic enzyme production. Penicillin amidohydrolase was found to be a reversible enzyme for a limited number of substrates, and it can synthesize semisynthetic penicillins or cephalosporins. Using M. luteus, the synthetic activity of enzyme for ampicillin and cephalexin was evaluated (Table 3). When using medium I, the highest enzyme acitivity was obtained as compared with the three other media evaluated. When medium III was used, slightly lower enzyme productivity was detected. However, the synthetic enzyme productivity was rather low in all cases studied, and further studies and improvements will be required for its practical application. Most penicillin amidohydrolases obtained from other microbial sources show product inhibition (the value of the inhibition constant Kip ranges about 1 to 25 mM) and relatively high Michaelis constants (Km values range about 2.5 to 10.3 mM) (13). The enzyme from M. luteus showed no product inhibition and a relatively low Km value (2.2 mM), although it showed substrate inhibition (the value of the inhibition constant, K,,, found was 155 mM). These results indicate that there are some advantages of using the enzyme from M. luteus, and it is perhaps one of the most promising strains for industrial application, once enzyme productivity and specific activity are increased to a practical level. monas
5-
2-
ENZYME LOADING *6.06 /Km
2.66
UNITS
mM
Vm * 10.42 UNITS
-0 5
37
0.
1.0
0.5 I/S
1.5
2.0
(mM-,)
FIG. 3. Lineweaver-Burk plot of initial hydrolytic reaction rate at different substrate concentrations.
TABLE 2. Substrate specificity of M. luteus penicillin amidohydrolase on /3-lactam antibioticsa Compound
Degree of hy-
drolysis
Rf value
0.80 0.77 0.30 0.76 0.23 6-APA 0.16 Cephalosporin C 0.22 7-ACA ++ 0.56 Cephalexin 0.15 7-ADCA Reaction conditions: 3600; pH 7.0; 6 h; 10 mg/ml of substrate solution. Symbols: +++, Hydrolytic product above 2 mg/ml; ++, hydrolytic product near 2 mg/ ml; +, hydrolytic product below 2 mg/ml; -, hydrolytic product was not detected. 6-APA, 6-a-Aminopenicillanic acid; 7-ACA, 7-aminocephalosporanic acid; 7ADCA, 7-amino-3-deacetoxycephalosporanic acid.
Penicillin G Penicillin V Ampicillin Cloxacillin
+++ + ++
The M. luteus enzyme could hydrolyze penicillin G, ampicillin, and cephalexin, and it deacylated penicillin V only slightly. Cloxacillin was not cleaved. This enzyme could not deacylate cephalosporin C. These results are consistent with the results of other investigators (4-6, 8, 12, 15, 16). That is, this enzyme can deacylate the simple alkyl or a-aminoalkyl side chain from /3lactam antibiotics, but not the bulky group or the side chain containing dipolar ions. Recently, it was reported that D-amino acid oxidase of some origins oxidatively deaminates the D-a-aminoadipyl side chain of cephalosporin C to yield 7-/B-(oxoadipamido)-cephalosporanic acid (10). This compound is known to be oxidatively decarboxylated with ease to 7-,8-(4-carboxybutanamido)-cephalosporanic acid, which can be easily deacylated enzymatically to 7-ami-
TABLE 3. Synthetic activity of M. luteus penicillin amidohydrolase on ,B-lactam antibioticsa Synthetic activity Medium
Cephalexin Ampicillin 0.12 0.51 0.00 0.17 0.03 0.15 0.06 0.35
Reference
foredia media for
(10) I (11) II (6) III (12) IV a Substrate solution: 100 mg of D-(-)-a-phenylglycine methyl ester hydrochloride and 10 mg of 7-amino3-deacetoxycephalosporanic acid or 6-a-aminopenicillanic acid in 1 ml of phosphate buffer (pH 7.0). Reaction conditions: 360C; pH 7.0; 1 h; 10 mg/mi of substrate solution. b Expressed as micromoles per hour per milliliter of broth.
38
NAM AND RYU
LITERATURE CITED 1. Alicino, J. F. 1961. lodometric assay of natural and synthetic penicillins, 6-aminopenicillanic acid and cephalosporin C. Anal. Chem. 33:648-649. 2. Bomstein, J., and W. G. Evans. 1965. Automated colorimetric determination of 6-aminopenicillanic-acid in fermentation media. Anal. Chem. 37:576-578. 3. Boxer, G. E., and P. M. Everett. 1949. Colorimetric determination of benzylpenicillin. Anal. Chem. 21:670673. 4. Claridge, C. A., J. R. Luttinger, and J. Lein. 1963. Specificity of penicillin amidases. Proc. Soc. Exp. Biol. Med. 113:1008-1012. 5. Demain, A. L., R. B. Walton, J. F. Newkirk, and I. M. Miller. 1963. Microbial degradation of cephalosporin C. Nature (London) 199:909-910. 6. Erickson, R. C., and R. E. Bennett. 1965. Penicillin acylase activity of Penicillium chrysogenum. Appl. Microbiol. 13:738-742. 7. Fujii, T., K. Matsumoto, and T. Watanabe. 1976. Enzymatic synthesis of cephalexin. Proc. Biochem. 11:2124. 8. Huang, H. T., T. A. Sato, and G. M. Shull. 1963. Distribution of substrate specificity of benzylpenicillin acylase. Appl. Microbiol. 11:1-6. 9. Marrelli, L. P. 1968. Colorimetric method for determination of 7-aminocephalosporanic acid (7-ACA) and related compounds. J. Pharm. Sci. 57:2172-2173. 10. Mazzeo, P., and A. Romeo. 1972. Enzymic and chemical transformation of the side chain of cephalosporin c. J. Chem. Soc. Perkin Trans. 1, p. 2532. 11. Nara, T., M. Misawa, R. Okachi, and M. Yamamoto.
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