Studies on the isolation of penicillin acylase from Escherichia coli by aqueous two‐phase partitioning

A method of enzyme release and aqueous two‐phase extraction is described for the separation of penicillin acylase from Escherichia coli cells. Butyl acetate, 12% (v/v), treatment combined with freeze–thawing gives up to 70% enzyme release. For polyethylene glycol (PEG) + phosphate two‐phase extraction systems the enzyme purity and yield were rather low. Modified PEG, including PEG–ampicillin, PEG–aniline, PEG–phosphate, and PEG–trimethylamine, were synthesized and used in aqueous two‐phase systems; PEG–trimethylamine is the most satisfactory. A system containing 12% (w/w) PEG4000, 8% (w/w) of which is PEG–trimethylamine, with 0.7 M potasium phosphate at pH 7.2, resulted in the enzyme selective partition being greatly enhanced by charge directed effects. Possible mechanisms for the separation process are discussed. © 1992 John Wiley & Sons, Inc.