STEREOSPECIFIC GAMMA-LACTAMASE ACTIVITY IN A PSEUDOMONAS-FLUORESCENS SPECIES

Twenty five environmental isolates enriched for their ability to grow on N-acetylphenylalanine as sole carbon source were investigated for t heir hydrolytic action on (+)gamma-lactam (2-azabicyclo[2.2.1]hept-5-e n-3-one). Strain CMC 3060, a mucoidal Gram-negative rod identified as a strain of Pseudomonas fluorescens, produced high levels of (+)lactam ase, and was subsequently found to produce two distinct intracellular enantiomer-selective gamma-lactamases, one for each isomer, The (+)lac tamase was produced constitutively whereas the (-)lactamase was produc ed only in the presence of the substrate, The (+)lactamase was stable when stored as a frozen cell paste but unstable as a protein solution, losing activity during purification and storage, This enzyme was high ly selective for the (+)lactam and showed no activity against a wide r ange of similar compounds, By use of rapid purification techniques and the inclusion of protease inhibitors and protein stabilisers, the (+) lactamase was purified to homogeneity by FPLC and found to be a monome r of molecular weight 61000 Da.