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 0.7M 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.