World Journal of Microbiology & Biotechnology 12, 373-378
Cephalosporin C acylase and penicillin V acylase formation by Aeromonas sp. ACY 95 B.S. Deshpande, S. S. Ambedkar and J. G. Shewale* Aeromonas sp. ACY 95 produces constitutively and intracellularly a penicillin V acylase at an early stage of
fermentation (12 h) and a cephalosporin C acylase at a later stage (36 h). Some penicillins, cephalosporin C and their side chain moieties/analogues, phenoxyacetic acid, penicillin V and penicillin G, enhanced penicillin V acylase production while none of the test compounds affected cephalosporin C acylase production. Supplementation of the medium with some sugars and sugar derivatives repressed enzyme production to varying degrees. The studies on enzyme formation, induction and repression, and substrate profile suggest that the cephalosporin C acylase and penicillin V acylase are two distinct enzymes. Substrate specificity studies indicate that the Aeromonas sp. ACY 95 produces a true cephalosporin C acylase which unlike the enzymes reported hitherto hydrolyses cephalosporin C specifically. Key words: Aeromonas sp., 6-Aminopenicillanic acid, 7-Aminocephalosporanic acid, Cephalosporin C acylase, Penicillin V acylase.
The endeavour to isolate cultures producing cephalosporin C acylase began soon after the realisation of the clinical importance of semisynthetic cephalosporins derived from cephalosporin C via 7-aminocephalosporanic acid (7-ACA) (Demain et al. 1963; Walton 1964). Cephalosporin C acylase catalyses the hydrolysis of the linear amide bond present in cephalosporin C to generate 7-ACA and D-0c-aminoadipic acid. However, the one step enzymatic route has not yet been applied for the manufacture of 7-ACA. The processes presently used for the production of 7-ACA are through chemical (imino ether), two-step chemical-enzymatic (glyoxylic acid and glutaryl 7-ACA acylase) or two-step enzymatic (D-amino acid oxidase and glutaryt 7-ACA acylase) routes (Matsumoto I993). Efforts to isolate cephalosporin C acylase producers were intensified worldwide in the 1980s subsequent to the commercial application of immobilized penicillin acylase systems for the production of 6-aminopenicillanic acid (6-APA). Despite such efforts, only a few microbial cephalosporin C acylase producers
The authors are with Research and Development, Hindustan Antibiotics Limited, Pimpri, Pune 411018, India; fax: 91 212772327. *Corresponding author.
have been identified to date. This is attributed to either the D configuration of the side chain or to the location of the side chain amide bond between the delta-carboxyl group of a D-amino acid and the 7-amino group of 7-ACA (in contrast, enzymes hydrolysing the gamrna-carboxylic amide of glutaric acid have been well characterised) (Lowe 1989). The cephalosporin acylases reported in the literature are of two types: cephalosporin C acylases that catalyse the hydrolysis of both cephalosporin C and glutaryl 7-ACA and glutaryl 7-ACA acylases that catalyse the hydrolysis of glutaryl 7-ACA and some other derivatives of 7-ACA but not of cephalosporin C (Deshpande et at. 1994). The organisms that have been reported to produce cephalosporin C acylase are Arthrobacter oiscosus ATCC 53594, Aspergillus sp. MA-13, Bacillus megaterium ATCC 53667, Paecilomyces sp., Penicillium griseofulvin, Penicillium regulosm, Pseudomonas diminuta strains N 176 and V 22, Pseudomonas putida and Pseudomonas sp. SE83 (Niwa et al. 1977; Banyu 1984; Ichikawa et ai. 1987; Kawate et al. 1987; Matsuda et al. I987a; Vandamme 1988; Lein I988, 1989; Reyes et aI. 1990; Aramori et a]. 1991a, i99ib). Nevertheless, many of these reports are either in patents or in abstracts.
~) 1996 Rapid Science Publishers World Journal of Microbiology ~ Biotechnology, Vol I2, 1996
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B.S. Deshpande, S.S. Ambedkar and J.G. Shewale We report here the production of a specific cephalosporin C acylase and of a penicillin V acylase by Aeromonas sp. ACY 95.
Materials and Methods Organism and Growth Aeromonas sp. ACY 95 was grown in shake flask cultures using production medium containing (g/l): yeast extract, 5; peptone, 5; tri-sodium citrate 2H20, I0; di-sodium hydrogen phosphate 2HzO, 0.056; mono-sodium dihydrogen phosphate 2H20, 0.095 and trace minerals solution (g/l; ammonium molybdate 0.8; boric acid, 2; MnSO4.4H20,1.6; ZnSO4.7H20, 1.6; CuSO4.5H20, 0.15; KI, 0.45) 0.6 m[/l, pH 6.5. I00 ml of medium was used per 500 ml Er[enmeyer flask. Vegetative growth from a slant grown for 24 h at 28°C was suspended in 5 ml of sterile saline and I ml of this suspension was inoculated into 100 ml of medium. This seed culture was grown at 28°C with shaking (240 rev. min-i) for 24 h. 2 ml of the seed broth was transferred to i00 ml of production medium. The fermentation was carried out by incubating the flasks on a rotary shaker (240 rev. rain -1) at 28°C for 36 h. Cells were harvested by centrifugation at 8540 x g for I0 rain and washed once with saline.
Growth Growth was determined by drying the centrifuged cells at 80°C to a constant weight and is expressed as dry cell weight (DCW).
Cephalosporin C Acylase Activity The cells collected from I0 ml of broth were resuspended in 5.0 ml of 0.1 M Tris/HCl buffer, pH 9.0. 1 ml of this suspension was mixed with I ml of cephalosporin C K salt solution (20 mg/ ml) in 0.1 M Tris/HCl buffer, pH 9.0. The reaction mixture was incubated for 2 h at 40°C with gentle shaking. The reaction was terminated by the addition of 2 ml of 2 M acetate buffer, pH 3.0, centrifuged, and the 7-ACA present in the supematant was estimated by the p-dimethylaminobenzaldehyde method (Deshpande et al. 1993; Yatsimirskaya et al. 1995). Appropriate enzyme and substrate blanks were run. The formation of 7-ACA was monitored by HPLC analysis: column, C 8, 5 microns, 4 x 250 mm (Hewlett Packard); solvent system, 0.01 M phosphate buffer pH 7.0 and acetonitrile in the ratio of IOO to 0.5; flow rate 1.0 ml/min and detection wavelength 254 nm. The 7-ACA peak position was confirmed by coinjecting an authentic sample.
Penicillin V Acylase Activity The cells collected from 10 ml of broth were resuspended in 10 m[ of 0.I M phosphate buffer, pH 7.0. 0.2 ml of this suspension was mixed with 0.8 ml of 0.1 M phosphate buffer pH 7.0 and 1.0 ml of penicillin V K salt (40 mg/ml in 0.1 M phosphate buffer, pH 7.0). The reaction mixture was incubated at 40°C for 30 min with gentle shaking. The reaction was terminated by the addition of 2 m[ of 2 M acetate buffer, pH 3.0. The reaction mixture was centrifuged and 3 ml of the aliquot was processed as described earlier for estimation of 6-APA (Shewale et al. I987).
fl-Iactamase fl-[actamase activity was determined as described by Novik (1962).
Esterase Activity Esterase (nonspecific) activity was determined by monitoring the
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World Journal of Microbiology & Biotechnology, Vol I2, I996
hydrolysis of p-nitrophenyl acetate at pH 7.8 and 40°C. The pnitrophenol liberated was measured at 405 nm under alkaline conditions. Cephalosporin C acylase and penicillin V acylase activities were expressed in International Units (IU). (1 IU is the amount of enzyme required for formation of I pmol of 7-ACA or 6-APA, respectively, per rain under the assay conditions).
Fractionation of Cephalosporin C Acylase and Penicillin V Acylase Washed cells (20 g) were suspended in I00 ml of 0.05 M phosphate buffer, pH 7.8 and homogenised. The extract collected after centrifugation was dialysed against 0.02 M phosphate buffer, pH 7.8 and 20 ml of the extract was loaded on the DEAE-Cellulose DE 52 column (1.4 x 8.5 cm) equilibrated with 0.02 M phosphate buffer, pH 7.8. The column was washed with 25 ml of equilibration buffer and eluted with equilibration buffer containing 0.2 M NaCI. Fractions of 2 m[ were collected.
Materials Penicillin G, penicillin V and cephalosporin C were from our production/pilot plant units. Glutary[ 7-ACA was prepared in our laboratory.
Results and Discussion During our screening programme for cephalosporin C acylase producers, we isolated approximately 50 bacterial and 20 fungal isolates from different sources. These isolates were tested for cephalosporin C acylase, glutaryl 7-ACA acylase, penicillin V acylase and penicillin G acylase. O n e isolate identified as Aeromonas sp. ACY 95, produced both cephalosporin C acylase and penicillin V acylase activities but no other isolate produced cephalosporin C acylase. All experiments were run in triplicate and the sets repeated at least twice with similar results. The pH, growth, cephalosporin C acylase and penicillin V acylase activities were monitored during growth in shake-flask cultures up to 72 h (Figure 1). Aeromonas sp. ACY 95 produced both cephalosporin C acylase and penicillin V acylase intracellularly but neither enzyme activity was detected in culture filtrates at any time during growth. Significantly, the production profiles of cephalosporin C acylase and penicillin V acylase were independent. Production of penicillin V acylase was observed in the early phase of the fermentation, the level reaching a maximum at 12 h, declining rapidly during the next 12 h and gradually during the rest of the period of fermentation. Formation of penicillin V acylase during the early part of the growth cycle indicates its close relation to nutritional aspects and primary metabolism. After 24 h the broth supematant was devoid of penicillin V acylase activity hence the possibility of leakage from the cells was ruled out. Thus, the penicillin V acylase formed was either degraded or inactivated intracellularly soon after cell growth was over. O n the other hand, formation of cephalosporin C acylase had a lag of g h, its activity increased with time
Aeromonas sp. acylases
I,--4
1-°I
0.8
250 ~"
~208 -
10
8
v ft.) A
0.6-
,,3
i,,--i
150 -
6
-
4
¢,J eU
r.j
.-.2
.+,-I
0.4-
rJt
0 1/1 C~
0.2-
"~
50
2
O
0 0.+
I
0
12
24
36
48
68
72
Ttme (h) Figure 1. The pH profile, growth and formation of cephalosporin C acylase and penicillin V acylase during the fermentation of Aeromonas sp. ACY 95. The culture was grown at 28°C and 240 rev. min ~ for 72 h on production medium. • - pH; C ) - - growth; • - - cephalosporin C acylase; • - - penicillin V acylase.
of fermentation, reached a maximum and remained unaltered from 36 to 48 h but declined thereafter (Figure 1). The results suggest association of cephalosporin C acylase with the secondary metabolism of the organism. In addition cephalosporin C acylase was not detected in the broth supernatant after 60 h during the decline in growth. Synthesis of glutaryl 7-ACA acylase by Pseudomonas sp. SY-77-1 has also been reported to occur during the pH increase in the medium (Shibuya et al. 1981). Cephalosporin C acylase production by P. griseofulvin and P. regulosm have been reported to increase during autolysis and with increasing pH (Reyes et al. 1990). The physiological role of penicillin and cephalosporin acylases has not been established although a possible role in the catabolism of carbon sources has been suggested (Matsuda et al. 1987a; Aramori et al. 1991b; Valle et al. I991). Reyes et al. (1990) have suggested that cephalosporin C acylase may have a role in the autolysis of filamentous fungi. Concentration of yeast extract and peptone each up to 15 g/l, individually or in combination, in the production medium did not alter the activities of the cephalosporin C acylase or of the penicillin V acylase. Cephalosporin C acylase formation by Escherichia coli JM 109pCCN 176 recombinant has been reported to increase by IOO% as a result of an increase in concentration of yeast extract from 5 to I5 g/l and Bactotryptone from 10 to 30 g/l (Aramori el al. 1991b).
Enzyme Repression
Various sugars and sugar derivatives promoted formation of cephalosporin C acylase and penicillin V acylase (Table 1). Among the different sugars tested: glucose and arabinose did not alter the formation of either cephalosporin C acylase or penicillin V acylase; fructose, sucrose, lactose, maltose and mannitol decreased the formation of penicillin V acylase but not of cephalosporin C acylase, while galacrose, cellobiose, mannose, raffinose, rhamnose, ribose, sorbitol, sorbose and trehalose decreased the formation of both cephalosporin C acylase and penicillin V acylase. Rhamnose and trehalose repressed the formation of cephalosporin C acylase by 82% and 8I%, respectively, whereas fructose repressed the formation of penicillin V acylase by 60%. Repressive effect of sugars on penicillin V acylase and penicillin G acylase by different microorganisms is well documented in the literature (Shewale & Sivaraman 1989; Deshpande et al. 1994). Relatively little is known about their effect on cephalosporin C acylase production. Glucose at 0.2%, has been reported to repress the production of cephalosporin C acylase by 78%, while the addition of fructose (0.2%) increases the production of cephalosporin C acylase by 81% in E. coli JM 109 pCCN I76 recombinant (Aramori et al. 1991b). Enzyme Induction
The effect of cephalosporin C, penicillin G and penicillin V
World Journal of Microbiology & Biotechnoloxy, Vol
I2,
199b
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B.S. Deshpande, S.S. Ambedkar and ].G. Shewale Table 1. Effect of sugars on the production of cephalosporln C acylase and penicillin V acylase by Aeromonas sp. ACY 95. Sugar
Growth
(2 g/I)
(DCW g/I)
Nil Glucose Fructose Sucrose
Lactose G a lactos e Maltose
Arabinose Cellobiose Mannose Raffinose Rhamnose
Ribose Sorbitol Mannitol Sorbose Trehalose
Penicillin V Cephalosporin C acylase acylase (lUll) (lUll)
3.14
90
2.83 4.11 2.92 2.78
87 36 63 72
1.00 1.04 0.95 1.08 1.06
2.74
69
0.79
2.88 4.01 3.01 3.01 2.95 3.05 3.08 2.89 3.95 3.01 2.92
75 84 63 63 54 48 54 42 40 60 66
1.06 1.06 0.86
0.59 0.54 0.18 0.40 0.50 1.10 0.63 0.19
Aeromonas sp. ACY95 was grown at 28°C and 240 rev. m i n - ' for 36 h on production medium supplemented with the indicated sugars.
Table 2. Effect of inducers on formation of cephalosporin C acylase and penicillin V acylase by Aeromonas sp. ACY 95. Compound (0.5 g/I)
Growth (DCW Oil)
Nil Adipic acid DL-ct-amino adipic acid
Glutaric acid Phenoxyacetic acid Phenylacetic acid Penicillin V Penicillin G Cephalosporin C
PenicillinV acylase (lUll)
Cephalosporin C acylase (lUll)
3.23 3.26
76 60
1.0 0.8
3.28 3.64 3.01 3.08 3.25 2.92 3.26
85 82 200 60 123 102 75
1.0 0.9 0.8 1.1 0.8 1.1 1.0
Aeromonas sp. ACY 95 was grown at 28°C and 240 rev. m i n - ' for 36 h on production medium supplemented with the indicated compounds.
and their side chain moieties or side chain analogues on enzyme production was studied (Table 2). Both cephalosporin C acylase and penicillin V acylase were produced constitutively. None of the compounds studied altered cephalosporin C acylase production significantly (> 20%). However, phenoxyacetic acid, penicillin V and penicillin G enhanced the formation of penicillin V acylase by 163%, 61% and 34%, respectively. Phenylacetic acid, phenoxyacetic acid, penicillin G, penicillin V and their analogues have been reported to induce the formation of penicillin G acylase and penicillin V acylase in
3 7~
WorldJournalofMicrobiology& Biotechnology,Vol I2, 1996
some microorganisms (Shewale & Sivaraman I989). Glutaryl 7-ACA acylases are induced by glutaric acid in Pseudomonas SY-77-1 and its mutants C36 and GK 16 (Ichikawa et al. 1981a; Shibuya et al. 1981). Separation of Cephalosporin C Acylase and Penicillin V Acylase
Cephalosporin C acylase and penicillin V acylase activities were separated by chromatography on DEAE-Cellulose DE52. Cephalosporin C acylase was bound to the anion exchanger whereas penicillin V acylase was collected along with unadsorbed proteins. The elution of cephalosporin C acylase was achieved with 0.2 M NaCl. The specific activity of cephalosporin C acylase and penicillin V acylase in the peak fractions was increased by a factor 19.6 and 2.5 respectively. Enzymic Properties and Substrate Specificity
The optimum pH for cephalosporin C acylase and penicillin V acylase activities from Aeromonas sp. ACY 95 were 9.0 and 7.0, respectively. Optimum pH of cephalosporin C acylase from P. diminuta strains N 176 and V 22 and their recombinants has been reported to be 9.0 (Aramori et al. I991b). Glutaryl 7-ACA acylase from Pseudomonas mutants GK16 and C36 exhibit catalytic activity between pH 6.5 and 10.0 (Ichikawa et al. 1981b). The whole cells and cell homogenate were assayed for fl-lactamase activity towards cephalosporin C, penicillin V and penicillin G and nonspecific esterase activity towards p-nitrophenyl acetate. None of these activities were detected either in whole cells or in the cell homogenate. Pseudomonas SY-77-1 a natural isolate producing glutaryl 7-ACA acylase, produces fl-lactamase (Shibuya et al. 1981). Deacetylesterase activity was detected in broths of P. griseofulvin and P. regulosm that produce cephalosporin C acylase (Reyes et al. 1990). The substrate profile of whole cells and partially purified cephalosporin C acylase and penicillin V acylase was studied using cephalosporin C, glutaryl 7-ACA, penicillin V and penicillin G at equimolar concentrations. Only cephalosporin C acylase and penicillin V acylase activities were observed and glutaryl 7-ACA and penicillin G were not accepted as substrates by the whole cells. The partially purified cephalosporin C acylase and penicillin V acylase preparations were highly specific for cephalosporin C and penicillin V, respectively and none of the other fl-lactam substrates tested were hydrolysed. The growth profile, induction and repression pattern and substrate specificity studies indicate that cephalosporin C Acylase and penicillin V acylase produced by Aeromonas sp. ACY 95 are two distinct enzymes. Thus, Aeromonas sp. ACY 95 produces an enzyme that hydrolyses cephalosporin C specifically. To our knowledge this is the first report of a microorganism producing an enzyme that is active on cephalosporin C but not on glutaryl 7-ACA.
Aeromonas sp. acylases Table 3. Comparison of the activity of cephalosporin C acylase from different sources towards cephalosporin C and glutaryl 7ACA as substrates Organism
Relative activity Cephalosporin C
Aeromonas sp. ACY 95
Glutaryl 7-ACA
Reference a
Present
100.0
0
P. diminuta N 176
2.4
100
1
E. coli JM109 pCCN 176-1
3.7
100
1
P. diminuta V 22
3.2
100
1
E. coli JM109 pCCV 22-1
8.5
100
1
Pseudomonas sp. SE 83 Acylase II
5.0
100
2
a 1, Aramori et al. 1991b; 2, M a t s u d a et al. 1987a.
The substrate profiles of the cephalosporin C acylase preparations reported in the literature are compared in Table 3. It is important to note that the enzyme preparations referred to as cephalosporin C acylase, whose substrate profiles have been studied, are glutary[ 7-ACA acylases exhibiting cephalosporin C acylase activity, the relative activity towards cephalosporin C being less than 10% of that towards glutaryl 7-ACA (Matsuda et al. 1987a; Vandamme 1988; Aramori et al. I991b; Isogai & Fukagawa 1991; Kumar et al. 1993; Deshpande et a]. 1994). These enzyme preparations are referred to as cephalosporin C acylase mainly because of their ability to hydrolyse cephalosporin C, though at low rates. The cephalosporin C acylase from Aeromonas sp. ACY 95 did not accept glutaryl 7-ACA as substrate and hydrolysed cephalosporin C specifically. Thus, the cephalosporin C acylase produced by Aeromonas sp. ACY 95 is a cephalosporin C acylase in the true sense. This study opens a new avenue for the production of 7-ACA.
Acknowledgements We thank Dr. S.R. Naik, General Manager for his encouragement during the course of this work and Mr. A.G. Nirgudkar, Mr. S.S. Marathe and Miss. M.M. Damame for the HPLC analyses. We also thank Dr. C. SivaRaman for his critical reading and comments.
References Aramori, 1., Fukagawa, M., Tsumura, M., lwami, M., Yokota, Y., Kojo, H., Kohsaka, M., Ueda, Y. & Imanaka, H. I991a Isolation
of soil strains producing new cephalosporin acylases. Journal of Fermentation and Bioengineering. 72, 227-231. Aramori, I., Fukagawa, M., Tsumura, M., lwami, M., Isogai, T., Ono, H., Ishitani, Y., Kojo, H., Kohsaka, M., Ueda, Y. & Imanaka, H. 199Ib Cloning and nuc|eotide sequencing of new glutaryl 7-ACA and cephalosporin C acylase genes from Pseudomonas strains. Journal of Fermentation and Bioengineering 72, 232-243. Banyu Pharmaceutical Company Limited 1984 Preparation of 7ACA using Paecilomycessp. Japanese Patent No. 155219. Demain, A.L., Walton, R.B., Newrirk, J.F., Millar, I.M. 1963 Microbial degradation of cephalosporin C. Nature 199, 909--910. Deshpande, B.S., Ambedkar, S.S. & Shewale, J.G. 1993 Comparative evaluation of determination of fl-lactam intermediates by p-dirnethylaminobenzaldehyde. Hindustan Antibiotics Bulletin 35, 195-198. Deshpande, B.S., Ambedkar, S.S., Sudhakaran, V.K. & Shewale, J.G. 1994 Molecular biology of ]~-lactam acylases. World Journal of Microbiology and Biotechnology 10, 129-138. Goi, H., Niwa, T., Nojiri, C., Miyado, S., Seki, M. & Yamada, Y. I978 7-aminocephalosporanic acid and its derivatives. Japan Kokai Tokkyo Koho Japanese Patent 78 94093. Ichikawa, S., Mural, Y., Yamamoto, S., Shibuya, Y., Fujii, T., Komatsu, K. & Kodaira, R. 198Ia The isolation and properties of Pseudomonas mutants with an enhanced productivity of 7 fl(4-carboxybutanamido)-cephalosporanic acid acylase. Agricultural and Biological Chemistry 45, 2225-2229. Ichikawa, S., Shibuya, Y., Matsumoto, K., Fujii, T., Komatsu, K. & Kodaira, R. i981b Purification and properties of 7 ]~-(4 carboxy butanamido)-cephalosporanic acid acylase produced by mutants derived from Pseudomonas. Agricultural and Biological Chemistry 45, 2231-2236. Ichikawa, S., Yamamoto, K. & Matsuyama, K. 1987 Manufacture of cephalosporin C acylase. Japan Kokai Tokkyo Koho Japanese Patent 87 48379. Isogai, T. & Fukagawa, M. 1991 Direct production of 7-aminocephalosporanic acid and 7-aminodeacetyl cephalosporanic acid by recombinant Acremonium ckrysogenum. Aetinomycetologia 5, 102-111. Kawate, S., Fukuo, T. & Kunito, K. I987 Cephalosporin C acylase. Part I. Isolation and cultivation of cephalosporin C acylase producing microorganism. Technol Rep Kausai University 29, 77-94. Kumar, K.K., Sudhakaran, V.K., Deshpande, B.S., Ambedkar, S.S. & Shewale, J.G. 1993 Cephalosporin acylases: Enzyme production, structure and application in the production of 7-ACA. Hindustan Antibiotics Bulletin 35, 111-125. Lein, I. 1988 One step conversion of cephalosporin C to 7-amino cephalosporanic acid with cepbalosporin C amidase of Arthrobatter viscosus. European Patent 283218. Lein, J. 1989 One step conversion of cephalosporin C and derivatives to 7-ACA derivatives. European Patent 322032. Lowe, D.A. 1989 Immobilized fl-lactam acylases. Developments in Industrial Microbiolology 30, 121-130. Matsuda, A., Matsuyama, K., Yamamoto, K., Ichikawa, S. & Komatsu, K-I. 1987a Cloning and characterization of the genes for two distinct cephalosporin acylases from a Pseudomonas strain. Journal of Bacteriology 169, 5815-5821. Matsuda, A., Toma, K. & Komatsu, K-I. 1987b Nucleotide sequences of the genes for two distinct cephalosporin acylases from a Pseudomonas strain. Journal of Bacteriology 169, 5821 5826. Matsumoto, K. 1993 Production of 6-APA, 7-ACA and 7-ADCA by immobilized penicillin and cephalosporin amidases. Bioprocess Technology 16, 67-88.
Wvrld Journal of; Microbir;logy & Biotechnology, Vo! I2, ~996
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B,S. Deshpande, S.S. Ambedkar and J,G. Shewale Niwa, T., Nojiri, C., Goi, H., Miyado, S., Kai, F., Seki, S., Yamada, Y. & Niida, T. 1977 7-Amino cepham compounds using mold fungi. German Often 2723463. Novik, R.P. I962 Micro-Iodometric assay for penicillinase. Biochemical Journal 83, 236--240. Reyes, F., Martinez, N.J., Alfonso, C., Cob-Patino, J.L. & Solivery, J. 1990 Cephalosporin C acylase in the autolysis of filamentous fungi. Journal of Pharmacy and Pharmacology 42, I28-131. Shewale, J.G. & Sivaraman, H. 1989 Penicillin acylases: Enzyme production and its application in the manufacture of 6-APA Process Biochemistry 24, 146--154. Shewale, J.G., Kumar, K.K. & Ambekar, G.R. 1987 Evaluation of determination of 6-APA by p-dimethylaminobenzaldehyde. Biotechnology Techniques 1, 69-72. Shibuya, Y., Matsumoto, K. & Fujii, T. 1981 Isolation and properties of 7-fl-(4-carboxybutanamido) cephalosporanic acid acylase-producing bacteria. Agricultural and Biological Chemistry 45, 1561-I567. Valle, F., Balbus, P., Merino, E. & Bolivar, F. 1991 The role of
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penicillin amidases in nature and in industry. Trends in Biochemical Sciences 16, 36--40. Vandamme, E.J. 1988 Immobilized biocatalysts and antibiotic production: Biochemical, genetical and biotechnical aspects. In: Bioreactor Immobilized Enzymes and Cells: Fundamentalsand Applications, ed Moo-Yung, M. pp. 261-286. New York: Elsevier Applied Sciences. Walton, R.B. 1964 Search for microorganisms producing cephalosporin C amidase. Developments in Industrial Microbiology 5, 349-353. Yatsimirskaya, N.T., Sosnovskaya, I.N. & Yatsimirsky, A.K. 1995 Spectrophotometric determination of 6-aminopenicillanic and 7aminocephalosporanic acids as the Schiff's bases with p-dimethylaminobenzaldehyde in the presence of sodium dodecyl sulphate micelles. Analytical Biochemistry 229, 249-255.
(Received as revised 29 January 1996; accepted 2 February 1996)
Cephalosporin C acylase and penicillin V acylase formation by Aeromonas sp. ACY 95 B.S. Deshpande, S. S. Ambedkar and J. G. Shewale* Aeromonas sp. ACY 95 produces constitutively and intracellularly a penicillin V acylase at an early stage of
fermentation (12 h) and a cephalosporin C acylase at a later stage (36 h). Some penicillins, cephalosporin C and their side chain moieties/analogues, phenoxyacetic acid, penicillin V and penicillin G, enhanced penicillin V acylase production while none of the test compounds affected cephalosporin C acylase production. Supplementation of the medium with some sugars and sugar derivatives repressed enzyme production to varying degrees. The studies on enzyme formation, induction and repression, and substrate profile suggest that the cephalosporin C acylase and penicillin V acylase are two distinct enzymes. Substrate specificity studies indicate that the Aeromonas sp. ACY 95 produces a true cephalosporin C acylase which unlike the enzymes reported hitherto hydrolyses cephalosporin C specifically. Key words: Aeromonas sp., 6-Aminopenicillanic acid, 7-Aminocephalosporanic acid, Cephalosporin C acylase, Penicillin V acylase.
The endeavour to isolate cultures producing cephalosporin C acylase began soon after the realisation of the clinical importance of semisynthetic cephalosporins derived from cephalosporin C via 7-aminocephalosporanic acid (7-ACA) (Demain et al. 1963; Walton 1964). Cephalosporin C acylase catalyses the hydrolysis of the linear amide bond present in cephalosporin C to generate 7-ACA and D-0c-aminoadipic acid. However, the one step enzymatic route has not yet been applied for the manufacture of 7-ACA. The processes presently used for the production of 7-ACA are through chemical (imino ether), two-step chemical-enzymatic (glyoxylic acid and glutaryl 7-ACA acylase) or two-step enzymatic (D-amino acid oxidase and glutaryt 7-ACA acylase) routes (Matsumoto I993). Efforts to isolate cephalosporin C acylase producers were intensified worldwide in the 1980s subsequent to the commercial application of immobilized penicillin acylase systems for the production of 6-aminopenicillanic acid (6-APA). Despite such efforts, only a few microbial cephalosporin C acylase producers
The authors are with Research and Development, Hindustan Antibiotics Limited, Pimpri, Pune 411018, India; fax: 91 212772327. *Corresponding author.
have been identified to date. This is attributed to either the D configuration of the side chain or to the location of the side chain amide bond between the delta-carboxyl group of a D-amino acid and the 7-amino group of 7-ACA (in contrast, enzymes hydrolysing the gamrna-carboxylic amide of glutaric acid have been well characterised) (Lowe 1989). The cephalosporin acylases reported in the literature are of two types: cephalosporin C acylases that catalyse the hydrolysis of both cephalosporin C and glutaryl 7-ACA and glutaryl 7-ACA acylases that catalyse the hydrolysis of glutaryl 7-ACA and some other derivatives of 7-ACA but not of cephalosporin C (Deshpande et at. 1994). The organisms that have been reported to produce cephalosporin C acylase are Arthrobacter oiscosus ATCC 53594, Aspergillus sp. MA-13, Bacillus megaterium ATCC 53667, Paecilomyces sp., Penicillium griseofulvin, Penicillium regulosm, Pseudomonas diminuta strains N 176 and V 22, Pseudomonas putida and Pseudomonas sp. SE83 (Niwa et al. 1977; Banyu 1984; Ichikawa et ai. 1987; Kawate et al. 1987; Matsuda et al. I987a; Vandamme 1988; Lein I988, 1989; Reyes et aI. 1990; Aramori et a]. 1991a, i99ib). Nevertheless, many of these reports are either in patents or in abstracts.
~) 1996 Rapid Science Publishers World Journal of Microbiology ~ Biotechnology, Vol I2, 1996
3 73
B.S. Deshpande, S.S. Ambedkar and J.G. Shewale We report here the production of a specific cephalosporin C acylase and of a penicillin V acylase by Aeromonas sp. ACY 95.
Materials and Methods Organism and Growth Aeromonas sp. ACY 95 was grown in shake flask cultures using production medium containing (g/l): yeast extract, 5; peptone, 5; tri-sodium citrate 2H20, I0; di-sodium hydrogen phosphate 2HzO, 0.056; mono-sodium dihydrogen phosphate 2H20, 0.095 and trace minerals solution (g/l; ammonium molybdate 0.8; boric acid, 2; MnSO4.4H20,1.6; ZnSO4.7H20, 1.6; CuSO4.5H20, 0.15; KI, 0.45) 0.6 m[/l, pH 6.5. I00 ml of medium was used per 500 ml Er[enmeyer flask. Vegetative growth from a slant grown for 24 h at 28°C was suspended in 5 ml of sterile saline and I ml of this suspension was inoculated into 100 ml of medium. This seed culture was grown at 28°C with shaking (240 rev. min-i) for 24 h. 2 ml of the seed broth was transferred to i00 ml of production medium. The fermentation was carried out by incubating the flasks on a rotary shaker (240 rev. rain -1) at 28°C for 36 h. Cells were harvested by centrifugation at 8540 x g for I0 rain and washed once with saline.
Growth Growth was determined by drying the centrifuged cells at 80°C to a constant weight and is expressed as dry cell weight (DCW).
Cephalosporin C Acylase Activity The cells collected from I0 ml of broth were resuspended in 5.0 ml of 0.1 M Tris/HCl buffer, pH 9.0. 1 ml of this suspension was mixed with I ml of cephalosporin C K salt solution (20 mg/ ml) in 0.1 M Tris/HCl buffer, pH 9.0. The reaction mixture was incubated for 2 h at 40°C with gentle shaking. The reaction was terminated by the addition of 2 ml of 2 M acetate buffer, pH 3.0, centrifuged, and the 7-ACA present in the supematant was estimated by the p-dimethylaminobenzaldehyde method (Deshpande et al. 1993; Yatsimirskaya et al. 1995). Appropriate enzyme and substrate blanks were run. The formation of 7-ACA was monitored by HPLC analysis: column, C 8, 5 microns, 4 x 250 mm (Hewlett Packard); solvent system, 0.01 M phosphate buffer pH 7.0 and acetonitrile in the ratio of IOO to 0.5; flow rate 1.0 ml/min and detection wavelength 254 nm. The 7-ACA peak position was confirmed by coinjecting an authentic sample.
Penicillin V Acylase Activity The cells collected from 10 ml of broth were resuspended in 10 m[ of 0.I M phosphate buffer, pH 7.0. 0.2 ml of this suspension was mixed with 0.8 ml of 0.1 M phosphate buffer pH 7.0 and 1.0 ml of penicillin V K salt (40 mg/ml in 0.1 M phosphate buffer, pH 7.0). The reaction mixture was incubated at 40°C for 30 min with gentle shaking. The reaction was terminated by the addition of 2 m[ of 2 M acetate buffer, pH 3.0. The reaction mixture was centrifuged and 3 ml of the aliquot was processed as described earlier for estimation of 6-APA (Shewale et al. I987).
fl-Iactamase fl-[actamase activity was determined as described by Novik (1962).
Esterase Activity Esterase (nonspecific) activity was determined by monitoring the
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World Journal of Microbiology & Biotechnology, Vol I2, I996
hydrolysis of p-nitrophenyl acetate at pH 7.8 and 40°C. The pnitrophenol liberated was measured at 405 nm under alkaline conditions. Cephalosporin C acylase and penicillin V acylase activities were expressed in International Units (IU). (1 IU is the amount of enzyme required for formation of I pmol of 7-ACA or 6-APA, respectively, per rain under the assay conditions).
Fractionation of Cephalosporin C Acylase and Penicillin V Acylase Washed cells (20 g) were suspended in I00 ml of 0.05 M phosphate buffer, pH 7.8 and homogenised. The extract collected after centrifugation was dialysed against 0.02 M phosphate buffer, pH 7.8 and 20 ml of the extract was loaded on the DEAE-Cellulose DE 52 column (1.4 x 8.5 cm) equilibrated with 0.02 M phosphate buffer, pH 7.8. The column was washed with 25 ml of equilibration buffer and eluted with equilibration buffer containing 0.2 M NaCI. Fractions of 2 m[ were collected.
Materials Penicillin G, penicillin V and cephalosporin C were from our production/pilot plant units. Glutary[ 7-ACA was prepared in our laboratory.
Results and Discussion During our screening programme for cephalosporin C acylase producers, we isolated approximately 50 bacterial and 20 fungal isolates from different sources. These isolates were tested for cephalosporin C acylase, glutaryl 7-ACA acylase, penicillin V acylase and penicillin G acylase. O n e isolate identified as Aeromonas sp. ACY 95, produced both cephalosporin C acylase and penicillin V acylase activities but no other isolate produced cephalosporin C acylase. All experiments were run in triplicate and the sets repeated at least twice with similar results. The pH, growth, cephalosporin C acylase and penicillin V acylase activities were monitored during growth in shake-flask cultures up to 72 h (Figure 1). Aeromonas sp. ACY 95 produced both cephalosporin C acylase and penicillin V acylase intracellularly but neither enzyme activity was detected in culture filtrates at any time during growth. Significantly, the production profiles of cephalosporin C acylase and penicillin V acylase were independent. Production of penicillin V acylase was observed in the early phase of the fermentation, the level reaching a maximum at 12 h, declining rapidly during the next 12 h and gradually during the rest of the period of fermentation. Formation of penicillin V acylase during the early part of the growth cycle indicates its close relation to nutritional aspects and primary metabolism. After 24 h the broth supematant was devoid of penicillin V acylase activity hence the possibility of leakage from the cells was ruled out. Thus, the penicillin V acylase formed was either degraded or inactivated intracellularly soon after cell growth was over. O n the other hand, formation of cephalosporin C acylase had a lag of g h, its activity increased with time
Aeromonas sp. acylases
I,--4
1-°I
0.8
250 ~"
~208 -
10
8
v ft.) A
0.6-
,,3
i,,--i
150 -
6
-
4
¢,J eU
r.j
.-.2
.+,-I
0.4-
rJt
0 1/1 C~
0.2-
"~
50
2
O
0 0.+
I
0
12
24
36
48
68
72
Ttme (h) Figure 1. The pH profile, growth and formation of cephalosporin C acylase and penicillin V acylase during the fermentation of Aeromonas sp. ACY 95. The culture was grown at 28°C and 240 rev. min ~ for 72 h on production medium. • - pH; C ) - - growth; • - - cephalosporin C acylase; • - - penicillin V acylase.
of fermentation, reached a maximum and remained unaltered from 36 to 48 h but declined thereafter (Figure 1). The results suggest association of cephalosporin C acylase with the secondary metabolism of the organism. In addition cephalosporin C acylase was not detected in the broth supernatant after 60 h during the decline in growth. Synthesis of glutaryl 7-ACA acylase by Pseudomonas sp. SY-77-1 has also been reported to occur during the pH increase in the medium (Shibuya et al. 1981). Cephalosporin C acylase production by P. griseofulvin and P. regulosm have been reported to increase during autolysis and with increasing pH (Reyes et al. 1990). The physiological role of penicillin and cephalosporin acylases has not been established although a possible role in the catabolism of carbon sources has been suggested (Matsuda et al. 1987a; Aramori et al. 1991b; Valle et al. I991). Reyes et al. (1990) have suggested that cephalosporin C acylase may have a role in the autolysis of filamentous fungi. Concentration of yeast extract and peptone each up to 15 g/l, individually or in combination, in the production medium did not alter the activities of the cephalosporin C acylase or of the penicillin V acylase. Cephalosporin C acylase formation by Escherichia coli JM 109pCCN 176 recombinant has been reported to increase by IOO% as a result of an increase in concentration of yeast extract from 5 to I5 g/l and Bactotryptone from 10 to 30 g/l (Aramori el al. 1991b).
Enzyme Repression
Various sugars and sugar derivatives promoted formation of cephalosporin C acylase and penicillin V acylase (Table 1). Among the different sugars tested: glucose and arabinose did not alter the formation of either cephalosporin C acylase or penicillin V acylase; fructose, sucrose, lactose, maltose and mannitol decreased the formation of penicillin V acylase but not of cephalosporin C acylase, while galacrose, cellobiose, mannose, raffinose, rhamnose, ribose, sorbitol, sorbose and trehalose decreased the formation of both cephalosporin C acylase and penicillin V acylase. Rhamnose and trehalose repressed the formation of cephalosporin C acylase by 82% and 8I%, respectively, whereas fructose repressed the formation of penicillin V acylase by 60%. Repressive effect of sugars on penicillin V acylase and penicillin G acylase by different microorganisms is well documented in the literature (Shewale & Sivaraman 1989; Deshpande et al. 1994). Relatively little is known about their effect on cephalosporin C acylase production. Glucose at 0.2%, has been reported to repress the production of cephalosporin C acylase by 78%, while the addition of fructose (0.2%) increases the production of cephalosporin C acylase by 81% in E. coli JM 109 pCCN I76 recombinant (Aramori et al. 1991b). Enzyme Induction
The effect of cephalosporin C, penicillin G and penicillin V
World Journal of Microbiology & Biotechnoloxy, Vol
I2,
199b
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B.S. Deshpande, S.S. Ambedkar and ].G. Shewale Table 1. Effect of sugars on the production of cephalosporln C acylase and penicillin V acylase by Aeromonas sp. ACY 95. Sugar
Growth
(2 g/I)
(DCW g/I)
Nil Glucose Fructose Sucrose
Lactose G a lactos e Maltose
Arabinose Cellobiose Mannose Raffinose Rhamnose
Ribose Sorbitol Mannitol Sorbose Trehalose
Penicillin V Cephalosporin C acylase acylase (lUll) (lUll)
3.14
90
2.83 4.11 2.92 2.78
87 36 63 72
1.00 1.04 0.95 1.08 1.06
2.74
69
0.79
2.88 4.01 3.01 3.01 2.95 3.05 3.08 2.89 3.95 3.01 2.92
75 84 63 63 54 48 54 42 40 60 66
1.06 1.06 0.86
0.59 0.54 0.18 0.40 0.50 1.10 0.63 0.19
Aeromonas sp. ACY95 was grown at 28°C and 240 rev. m i n - ' for 36 h on production medium supplemented with the indicated sugars.
Table 2. Effect of inducers on formation of cephalosporin C acylase and penicillin V acylase by Aeromonas sp. ACY 95. Compound (0.5 g/I)
Growth (DCW Oil)
Nil Adipic acid DL-ct-amino adipic acid
Glutaric acid Phenoxyacetic acid Phenylacetic acid Penicillin V Penicillin G Cephalosporin C
PenicillinV acylase (lUll)
Cephalosporin C acylase (lUll)
3.23 3.26
76 60
1.0 0.8
3.28 3.64 3.01 3.08 3.25 2.92 3.26
85 82 200 60 123 102 75
1.0 0.9 0.8 1.1 0.8 1.1 1.0
Aeromonas sp. ACY 95 was grown at 28°C and 240 rev. m i n - ' for 36 h on production medium supplemented with the indicated compounds.
and their side chain moieties or side chain analogues on enzyme production was studied (Table 2). Both cephalosporin C acylase and penicillin V acylase were produced constitutively. None of the compounds studied altered cephalosporin C acylase production significantly (> 20%). However, phenoxyacetic acid, penicillin V and penicillin G enhanced the formation of penicillin V acylase by 163%, 61% and 34%, respectively. Phenylacetic acid, phenoxyacetic acid, penicillin G, penicillin V and their analogues have been reported to induce the formation of penicillin G acylase and penicillin V acylase in
3 7~
WorldJournalofMicrobiology& Biotechnology,Vol I2, 1996
some microorganisms (Shewale & Sivaraman I989). Glutaryl 7-ACA acylases are induced by glutaric acid in Pseudomonas SY-77-1 and its mutants C36 and GK 16 (Ichikawa et al. 1981a; Shibuya et al. 1981). Separation of Cephalosporin C Acylase and Penicillin V Acylase
Cephalosporin C acylase and penicillin V acylase activities were separated by chromatography on DEAE-Cellulose DE52. Cephalosporin C acylase was bound to the anion exchanger whereas penicillin V acylase was collected along with unadsorbed proteins. The elution of cephalosporin C acylase was achieved with 0.2 M NaCl. The specific activity of cephalosporin C acylase and penicillin V acylase in the peak fractions was increased by a factor 19.6 and 2.5 respectively. Enzymic Properties and Substrate Specificity
The optimum pH for cephalosporin C acylase and penicillin V acylase activities from Aeromonas sp. ACY 95 were 9.0 and 7.0, respectively. Optimum pH of cephalosporin C acylase from P. diminuta strains N 176 and V 22 and their recombinants has been reported to be 9.0 (Aramori et al. I991b). Glutaryl 7-ACA acylase from Pseudomonas mutants GK16 and C36 exhibit catalytic activity between pH 6.5 and 10.0 (Ichikawa et al. 1981b). The whole cells and cell homogenate were assayed for fl-lactamase activity towards cephalosporin C, penicillin V and penicillin G and nonspecific esterase activity towards p-nitrophenyl acetate. None of these activities were detected either in whole cells or in the cell homogenate. Pseudomonas SY-77-1 a natural isolate producing glutaryl 7-ACA acylase, produces fl-lactamase (Shibuya et al. 1981). Deacetylesterase activity was detected in broths of P. griseofulvin and P. regulosm that produce cephalosporin C acylase (Reyes et al. 1990). The substrate profile of whole cells and partially purified cephalosporin C acylase and penicillin V acylase was studied using cephalosporin C, glutaryl 7-ACA, penicillin V and penicillin G at equimolar concentrations. Only cephalosporin C acylase and penicillin V acylase activities were observed and glutaryl 7-ACA and penicillin G were not accepted as substrates by the whole cells. The partially purified cephalosporin C acylase and penicillin V acylase preparations were highly specific for cephalosporin C and penicillin V, respectively and none of the other fl-lactam substrates tested were hydrolysed. The growth profile, induction and repression pattern and substrate specificity studies indicate that cephalosporin C Acylase and penicillin V acylase produced by Aeromonas sp. ACY 95 are two distinct enzymes. Thus, Aeromonas sp. ACY 95 produces an enzyme that hydrolyses cephalosporin C specifically. To our knowledge this is the first report of a microorganism producing an enzyme that is active on cephalosporin C but not on glutaryl 7-ACA.
Aeromonas sp. acylases Table 3. Comparison of the activity of cephalosporin C acylase from different sources towards cephalosporin C and glutaryl 7ACA as substrates Organism
Relative activity Cephalosporin C
Aeromonas sp. ACY 95
Glutaryl 7-ACA
Reference a
Present
100.0
0
P. diminuta N 176
2.4
100
1
E. coli JM109 pCCN 176-1
3.7
100
1
P. diminuta V 22
3.2
100
1
E. coli JM109 pCCV 22-1
8.5
100
1
Pseudomonas sp. SE 83 Acylase II
5.0
100
2
a 1, Aramori et al. 1991b; 2, M a t s u d a et al. 1987a.
The substrate profiles of the cephalosporin C acylase preparations reported in the literature are compared in Table 3. It is important to note that the enzyme preparations referred to as cephalosporin C acylase, whose substrate profiles have been studied, are glutary[ 7-ACA acylases exhibiting cephalosporin C acylase activity, the relative activity towards cephalosporin C being less than 10% of that towards glutaryl 7-ACA (Matsuda et al. 1987a; Vandamme 1988; Aramori et al. I991b; Isogai & Fukagawa 1991; Kumar et al. 1993; Deshpande et a]. 1994). These enzyme preparations are referred to as cephalosporin C acylase mainly because of their ability to hydrolyse cephalosporin C, though at low rates. The cephalosporin C acylase from Aeromonas sp. ACY 95 did not accept glutaryl 7-ACA as substrate and hydrolysed cephalosporin C specifically. Thus, the cephalosporin C acylase produced by Aeromonas sp. ACY 95 is a cephalosporin C acylase in the true sense. This study opens a new avenue for the production of 7-ACA.
Acknowledgements We thank Dr. S.R. Naik, General Manager for his encouragement during the course of this work and Mr. A.G. Nirgudkar, Mr. S.S. Marathe and Miss. M.M. Damame for the HPLC analyses. We also thank Dr. C. SivaRaman for his critical reading and comments.
References Aramori, 1., Fukagawa, M., Tsumura, M., lwami, M., Yokota, Y., Kojo, H., Kohsaka, M., Ueda, Y. & Imanaka, H. I991a Isolation
of soil strains producing new cephalosporin acylases. Journal of Fermentation and Bioengineering. 72, 227-231. Aramori, I., Fukagawa, M., Tsumura, M., lwami, M., Isogai, T., Ono, H., Ishitani, Y., Kojo, H., Kohsaka, M., Ueda, Y. & Imanaka, H. 199Ib Cloning and nuc|eotide sequencing of new glutaryl 7-ACA and cephalosporin C acylase genes from Pseudomonas strains. Journal of Fermentation and Bioengineering 72, 232-243. Banyu Pharmaceutical Company Limited 1984 Preparation of 7ACA using Paecilomycessp. Japanese Patent No. 155219. Demain, A.L., Walton, R.B., Newrirk, J.F., Millar, I.M. 1963 Microbial degradation of cephalosporin C. Nature 199, 909--910. Deshpande, B.S., Ambedkar, S.S. & Shewale, J.G. 1993 Comparative evaluation of determination of fl-lactam intermediates by p-dirnethylaminobenzaldehyde. Hindustan Antibiotics Bulletin 35, 195-198. Deshpande, B.S., Ambedkar, S.S., Sudhakaran, V.K. & Shewale, J.G. 1994 Molecular biology of ]~-lactam acylases. World Journal of Microbiology and Biotechnology 10, 129-138. Goi, H., Niwa, T., Nojiri, C., Miyado, S., Seki, M. & Yamada, Y. I978 7-aminocephalosporanic acid and its derivatives. Japan Kokai Tokkyo Koho Japanese Patent 78 94093. Ichikawa, S., Mural, Y., Yamamoto, S., Shibuya, Y., Fujii, T., Komatsu, K. & Kodaira, R. 198Ia The isolation and properties of Pseudomonas mutants with an enhanced productivity of 7 fl(4-carboxybutanamido)-cephalosporanic acid acylase. Agricultural and Biological Chemistry 45, 2225-2229. Ichikawa, S., Shibuya, Y., Matsumoto, K., Fujii, T., Komatsu, K. & Kodaira, R. i981b Purification and properties of 7 ]~-(4 carboxy butanamido)-cephalosporanic acid acylase produced by mutants derived from Pseudomonas. Agricultural and Biological Chemistry 45, 2231-2236. Ichikawa, S., Yamamoto, K. & Matsuyama, K. 1987 Manufacture of cephalosporin C acylase. Japan Kokai Tokkyo Koho Japanese Patent 87 48379. Isogai, T. & Fukagawa, M. 1991 Direct production of 7-aminocephalosporanic acid and 7-aminodeacetyl cephalosporanic acid by recombinant Acremonium ckrysogenum. Aetinomycetologia 5, 102-111. Kawate, S., Fukuo, T. & Kunito, K. I987 Cephalosporin C acylase. Part I. Isolation and cultivation of cephalosporin C acylase producing microorganism. Technol Rep Kausai University 29, 77-94. Kumar, K.K., Sudhakaran, V.K., Deshpande, B.S., Ambedkar, S.S. & Shewale, J.G. 1993 Cephalosporin acylases: Enzyme production, structure and application in the production of 7-ACA. Hindustan Antibiotics Bulletin 35, 111-125. Lein, I. 1988 One step conversion of cephalosporin C to 7-amino cephalosporanic acid with cepbalosporin C amidase of Arthrobatter viscosus. European Patent 283218. Lein, J. 1989 One step conversion of cephalosporin C and derivatives to 7-ACA derivatives. European Patent 322032. Lowe, D.A. 1989 Immobilized fl-lactam acylases. Developments in Industrial Microbiolology 30, 121-130. Matsuda, A., Matsuyama, K., Yamamoto, K., Ichikawa, S. & Komatsu, K-I. 1987a Cloning and characterization of the genes for two distinct cephalosporin acylases from a Pseudomonas strain. Journal of Bacteriology 169, 5815-5821. Matsuda, A., Toma, K. & Komatsu, K-I. 1987b Nucleotide sequences of the genes for two distinct cephalosporin acylases from a Pseudomonas strain. Journal of Bacteriology 169, 5821 5826. Matsumoto, K. 1993 Production of 6-APA, 7-ACA and 7-ADCA by immobilized penicillin and cephalosporin amidases. Bioprocess Technology 16, 67-88.
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B,S. Deshpande, S.S. Ambedkar and J,G. Shewale Niwa, T., Nojiri, C., Goi, H., Miyado, S., Kai, F., Seki, S., Yamada, Y. & Niida, T. 1977 7-Amino cepham compounds using mold fungi. German Often 2723463. Novik, R.P. I962 Micro-Iodometric assay for penicillinase. Biochemical Journal 83, 236--240. Reyes, F., Martinez, N.J., Alfonso, C., Cob-Patino, J.L. & Solivery, J. 1990 Cephalosporin C acylase in the autolysis of filamentous fungi. Journal of Pharmacy and Pharmacology 42, I28-131. Shewale, J.G. & Sivaraman, H. 1989 Penicillin acylases: Enzyme production and its application in the manufacture of 6-APA Process Biochemistry 24, 146--154. Shewale, J.G., Kumar, K.K. & Ambekar, G.R. 1987 Evaluation of determination of 6-APA by p-dimethylaminobenzaldehyde. Biotechnology Techniques 1, 69-72. Shibuya, Y., Matsumoto, K. & Fujii, T. 1981 Isolation and properties of 7-fl-(4-carboxybutanamido) cephalosporanic acid acylase-producing bacteria. Agricultural and Biological Chemistry 45, 1561-I567. Valle, F., Balbus, P., Merino, E. & Bolivar, F. 1991 The role of
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penicillin amidases in nature and in industry. Trends in Biochemical Sciences 16, 36--40. Vandamme, E.J. 1988 Immobilized biocatalysts and antibiotic production: Biochemical, genetical and biotechnical aspects. In: Bioreactor Immobilized Enzymes and Cells: Fundamentalsand Applications, ed Moo-Yung, M. pp. 261-286. New York: Elsevier Applied Sciences. Walton, R.B. 1964 Search for microorganisms producing cephalosporin C amidase. Developments in Industrial Microbiology 5, 349-353. Yatsimirskaya, N.T., Sosnovskaya, I.N. & Yatsimirsky, A.K. 1995 Spectrophotometric determination of 6-aminopenicillanic and 7aminocephalosporanic acids as the Schiff's bases with p-dimethylaminobenzaldehyde in the presence of sodium dodecyl sulphate micelles. Analytical Biochemistry 229, 249-255.
(Received as revised 29 January 1996; accepted 2 February 1996)
