IMPROVEMENT OF THE CATALYTIC PROPERTIES OF PENICILLIN-G ACYLASE FROM ESCHERICHIA-COLI ATCC-11105 BY SELECTION OF A NEW SUBSTRATE, SPECIFICITY

Cloned penicillin G acylase (PGA) from Escherichia coli ATCC 11105 was mutagenized in vivo using N-methyl-N'-nitro-N-nitrosoguanidine. Mutan ts of PGA were selected by their ability to allow growth of the host s train E. coli M8820 with the new substrates phenylacetyl-beta-alanyl-L -proline (PhAc-beta Ala-Pro) phthalyl-L-leucine (Pht-Leu) or phthalylg lycyl-L-proline (Pht-Gly-Pro) as sole source of proline and leucine re spectively. PGA mutants were purified and immobilized onto spherical m ethacrylate (G-gel). The immobilized form of mutant PGA selected with (PhAc-beta Ala-Pro) hydrolyzed 95% of 9 mmol penicillin G 30% faster t han wild-type PGA using the same specific activities. The specific act ivity of the soluble enzyme was 2.7-fold, and inhibition by phenylacet ic acid was halved. Immobilized PGA mutant selected with Pht-Gly-Pro h ydrolyzed penicillin G 20% faster than wild-type PGA. The K-m of the s oluble enzyme was increased 1.7-fold. Furthermore, the latter two muta nts were also 3.6-fold more stable at 45 degrees C than wild-type PGA. The specific activity of the mutant selected with Pht-Leu was 6.3-fol d lower, and inhibition by phenylacetic acid was increased 13-fold.