Purification Of An Enantioselective(-) γ-Lactamase And Study On Enzyme Assays

Biotrasformations have been widly used in synthetic chemistry. Many advantages make biocatalysis an effective method to produce enantiomerically pure2-azabicyclo [2.2.1] hept-5-en-3-one(y-lactam). Sixty-nine environmental isolates enriched for their ability to utilize N-acetylphenylalanine as sole carbon source were investigated for their hydrolytic activity on y-lactamase. We found L29-9, a strain of Microbacterium hydrocarbonoxydans, screened by our lab produces high levels of both (+) and (-) γ-lactamase.By use of hydrophobic interaction chromatography, followed by ion exchange chromatography and size exclusion chromatography, SDS-PAGE showed that the molecular weight of the lactamase was about31kDa. The (-) γ-lactamase was purified3.3-fold with a high activity of61.3U mg-1. The conversion percentage and ee of (-) Y-lactamase were64.8%and93.1%, respectively. Km=2.262X10-3mol/L. The highest activity was found at the optimal temperature 30℃and pH8.0, respectively. Only11.7%of the activity was left when it was incubated for15min at60℃, and the ee of the products was2.3%. No increase or decrease of activity was found with the addition of metal ions.The result of N-terminal amino acid sequence analysis is GCITVGNENS. From a search of the protein sequence database (NCBI, EMBL), no similarity with other reported amidases could be found.