Production of immobilized penicillin acylase using aqueous polymer systems for enzyme purification and in situ immobilization

• Published in: Enzyme and microbial technology Vol. 28; no. 2; pp. 218 - 224
• Main Authors: Guan, Y.H, Lilley, T.H, Brook, A.H
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Inc 01-02-2001; Elsevier Science
• Subjects: Biological and medical sciences; Biotechnology; Enzyme engineering; Escherichia coli; Fundamental and applied biological sciences. Psychology; Immobilization of enzymes and other molecules; Immobilization techniques; Improved methods for extraction and purification of enzymes; Methods. Procedures. Technologies; penicillin acylase; Purification; Enzyme; Escherichia coli; In situ; EC 3.5.1.11; Immobilization; Ethylene oxide polymer; Separation method; Penicillin amidase; Biphasic system; Bacteria; Hydrolases; Aqueous solution; Immobilized enzyme; Enterobacteriaceae
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/S0141-0229(00)00305-7

A novel approach for the isolation and purification of penicillin acylase (PA), which couples aqueous two-phase partitioning and enzyme immobilization has been investigated.
A PA yield of 90% was achieved by treating
E. coli cells with 4% butyl acetate, freeze-thawing step, and pressure homogenization. PA purification (93% recovery) was achieved by (1) removing cell debris
via precipitation with polyethylene glycol (PEG 2000); (2) aqueous two-phase partitioning using a PEG 2000 + phosphate system (87% recovery).
An in situ enzyme immobilization approach, using oxirane acrylic or aldehyde-agarose beads dispersed in the PEG-rich phase, was explored for the conversion of penicillin G to 6-aminopenicillanic acid. An appropriate immobilization reaction time was found. The catalytic performance of the enzyme, when immobilized, was found not to be affected by recycling of the phase-forming components.