Abstract
Penicillin acylase ofE. coli NCIM 2400 has been purified to homogeneity using a combination of hydrophobic interaction chromatography and DEAE-cellulose treatment. A variety of substituted matrices were synthesized using D- or DL-phenylglycine, norleucine, ampicillin, or amoxycillin as ligands, all of which retained penicillin acylase at high concentrations of ammonium sulfate or sodium sulfate. The enzyme could be eluted nonbiospecifically by buffer of lower ionic strength with over 95% recovery of the activity. Ammonium chloride, ammonium nitrate, sodium chloride, sodium nitrate, and potassium chloride were ineffective in either adsorption or elution of the enzyme on these columns. Further purification of this partially pure enzyme with DEAE-cellulose at pH 7.0–7.2 yielded an enzyme preparation of very high purity according to electrophoretic and ultracentrifugal analyses, its specific activity being as high as 37 U/mg protein. The purifiedf enzyme has a molecular weight of 67,000 a sedimentation coefficient of 4.0S, and resolves into two forms upon isoelectric focusing. Overall recoveries ranged between 75 and 85%. Ease of operation, high recoveries, high purity of the enzyme and prolonged reuse of the conjugates make the process economically feasible and possibly of great commercial importance.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Vandamme, E. J., and Voets, J. P. (1974),Adv. Appl. Microbiol. 17, 311.
Lagerloff, E., Nathorst-Westfelt, L., Ekstrom, B., and Sjoberg, B. (1976),Methods Enzymol. 44, 759.
Savidge, T. A., and Cole, M. (1975),Methods Enzymol. 43, 705.
Kutzbach, K., and Rauenbusch, E. (1974),Hoppe-Seyler’s Z. Physiol. Chem. 355, 45.
Shimizu, M., Okachi, R., Kimura, K., and Nara, I. (1975),Agric. Biol. Chem. 39, 1665.
Vandamme, E. J., and Voets, J. P. (1975),Experientia 31, 140.
Schneider, W. J., and Roehr, M. (1976),Biochim. Biophys. Ada 452, 177.
Carleysmith, S. W., Dunnill, P., and Lilly, M. D. (1980),Biotechnol. Bioeng. 22, 735.
Robak, M., and Szewczuk, A. (1981),Acta Biochem. Pol. 28, 275.
Veronese, F., Franchi, D., Boccu, E., Guerrato, A., and Orsolini, P. (1981),Farmaco. Ed. Sd. 36, 663.
Vojtisek, V., Vlasak, J., Barta, M., and Culik, K. (1981), Czech Patent No. 183170.
Moon, Y. L., and Byun, S. M. (1981),Korean Biochem. J. 14, 73.
Svec, F., Kalal, J., Barta, M., Vojtisek, V., and Culik, K. (1982), Czech Patent No. 190556.
Mahajan, P. B., and Borkar, P. S. (1982),Hindustan Antibiotics Bull. 24, 38.
Porath, J., and Axen, R. (1976),Methods Enzymol. 44, 19.
Mahajan, P. B., and Borkar, P. S. (1982), Indian Patent Application Nos. Bom 45/82 and Bom 99/82.
Spies, J. R. (1957),Methods Enzymol. 3, 467.
Smith, J. W. G., DeGrey, G. E., and Patel, V. J. (1967),Analyst 92, 247.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951),J. Biol. Chem. 193, 265.
Gabriel, O. (1971),Methods Enzymol. 22, 565.
Weber, K., and Osburn, M. (1969),J. Biol. Chem. 244, 4406.
Vesterberg, O. (1972),Biochim. Biophys. Acta 257, 11.
Bomstein, J., and Evans, W. G. (1965),Anal. Chem. 37, 567.
Klyosov, A. A., Svedas, V. K., and Galaev, I. Yu. (1977),Bioorg. Khim. 800.
Hjerten, S. (1981),Meth. Biochem. Anal. 27, 89.
Chiang, C., and Bennett, R. E. (1967),J. Bacteriol. 93, 302.
Deshpande, B. S., Mahajan, P. B., and Borkar, P. S. (1982), “Penicillin Acylase ofKluyvera citrophilia,” abstract presented at the 52nd Annual General Meeting of the Society of Biological Chemists of India at Chandigarh in November 1982.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Mahajan, P.B., Borkar, P.S. Novel approaches to the purification of penicillin acylase. Appl Biochem Biotechnol 9, 421–437 (1984). https://doi.org/10.1007/BF02798397
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02798397