Recovery and partial purification of penicillin G acylase from E. coli homogenate and B. megaterium culture medium using an expanded bed adsorption column

• Published in: Biochemical engineering journal Vol. 44; no. 2; pp. 111 - 118
• Main Authors: Pinotti, L.M., Fonseca, L.P., Prazeres, D.M.F., Rodrigues, D.S., Nucci, E.R., Giordano, R.L.C.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15-05-2009; Elsevier
• Subjects: Adsorption; B. megaterium; Bacillus megaterium; Biological and medical sciences; Biotechnology; E. coli; Enzymes; Escherichia coli; Fundamental and applied biological sciences. Psychology; Process integration; Purification; Enzymes; Purification; Adsorption; B. megaterium; E. coli; Process integration; Culture medium; Penicillin amidase; Enzyme; Hydrolases; Operating process
• ISSN: 1369-703X; 1873-295X
• DOI: 10.1016/j.bej.2008.11.006

The adsorption of penicillin G acylase (PGA) from
B. megaterium and from
Escherichia coli on a cationic resin, Streamline SP XL, was studied using both packed and expanded beds. Stability assays showed that penicillin acylases from the two sources presented high irreversible deactivation at pH 4.0 and 4.5, but remained stable at pH 4.8. Adsorption experiments performed in a packed bed (PB), in the pH range 4.8–5.8, showed highest adsorption yields at pH 4.8, for both enzymes. Using small expanded bed adsorption (EBA) columns, PGA was directly recovered and partially purified from
E. coli crude extracts,
E. coli homogenates, and from
B. megaterium centrifuged broth in a single unit operation. Global recovery yields of 91.0, 55.0 and 7.4% and purification factors of 4.5-, 7.5- and 12.7-fold were achieved, respectively. The elution yields of penicillin acylase obtained with these cationic EBA processes when working with
E. coli homogenate and
B. megaterium centrifuged medium were of 100 and 52%, respectively. The comparison of adsorption capacities of
E. coli penicillin acylase from crude extracts onto Streamline SP XL showed similar results for packed-bed and for expanded-bed modes. However, PGA adsorption yields for
E. coli (homogenate) and
B. megaterium (centrifuged medium) were substantially lower than the values obtained for
E. coli crude extract, due to the competition of cell debris and other components present in the
B. megaterium medium.