Abstract
Twenty five environmental isolates enriched for their ability to grow onN-acetylphenylalanine as sole carbon source were investigated for their hydrolytic action on (+)γ-lactam (2-azabicyclo[2.2.1]hept-5-en-3-one). Strain CMC 3060, a mucoidal Gram-negative rod identified as a strain ofPseudomonas fluorescens, produced high levels of (+)lactamase, and was subsequently found to produce two distinct intracellular enantiomer-selective, γ-lactamases, one for each isomer. The (+)lactamase was produced constitutively whereas the (-lactamase was produced 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 highly selective for the (+)lactam and showed no activity against a wide range 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 monomer of molecular weight 61000 Da.
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References
Bevan J, A Sutherland, I Brackenridge., R Wisdom and J Littlechild. 1995. Purification and characterisation of two different γ-lactamase enzymes with opposite stereospecificity. FEBS Lett (submitted).
Collins AN, GN Sheldrake and J Crosby, eds. 1992. Chirality in Industry. Wiley & Sons, London, UK.
Johnson WS and AJ Lindsey. 1939. An improved universal buffer. Analyst 64: 490–492.
Kilbanov AM. 1983. Immobilized enzymes and cells as practical catalysts. Science 219: 722–727.
Marquez VE and MI Lim. 1986. Carbocyclic nucleosides. Med Res Rev 6: 1–40.
McCague R and C Evans. 1993. Chemical Specialities USA 93 Symposium, Pittsburgh. Spring Innovations Ltd, Stockport, UK.
Poppe L and L Novak. 1992. Selective Biocatalysis. A Synthetic Approach. VCH Publishers, New York, NY, USA.
Santaniello E, P Ferraboschi, P Grisenti and A Manzocchi. 1992. The biocatalytic approach to the preparation of enantiomerically pure chiral building blocks. Chem Rev 92: 1071–1140.
Taylor ST, AG, Sutherland, C Lee, R Wisdom, S Thomas, SM Roberts and CT Evans. 1990. Chemoenzymatic synthesis of (−)-carbovir utilising a whole cell catalysed resolution of 2-azabicyclo[2.2.1]hept-5-en-3-one. J Chem Soc Chem Commun 16: 1120–1121.
Taylor ST, R McCague, R Wisdom, C Lee, K Dickson, G Ruecroft, F O'Brien, J Littlechild, J Bevan, SM Roberts and CT Evans. 1993. Development of the biocatalytic resolution of 2-azabicyclo[2.2.1]hept-5-en-3-one as an entry to single-enantiomer carbocyclic nucelosides. Tetrahedron: Asymmetry 4:(6): 1117–1128.
Vince R and M Hua. 1990. Synthesis and anti-HIV activity of carbocyclic 2′,3′-didehydr-2′,3′-dideoxy 2,6-disubstituted purine nucleosides. J Med Chem 33: 17–21.
White EL, WB Parker, LJ Macy, SC Shaddix, G McCaleb, JA Secrist, RE Vince and WM Shannon. 1989. Comparison of the effect of carbovir, AZT, and dideoxynucleoside triphosphates on the activity of human immunodeficiency virus reverse transcriptase and selected human immunodeficiency virus reverse transcriptase and selected human polymerases. Biochem Biophys Res Commun 161(2): 393–398.
Williamson C, CM Exall, MF Jones, PL Mo, IL Myers, IL Paternoster and R Storer. 1989. Poster presentation at SCI Medicinal Chemistry Symposium, Cambridge, UK.
Wong C-H and GM Whitesides. 1994. Enzymes in Synthetic Organic Chemistry. Tetrahedron Organic Chemistry Series. Vol 12. Pergamon Press, Elsevier Science, Oxford, UK.
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Brabban, A.D., Littlechild, J. & Wisdom, R. Stereospecificγ-lactamase activity in aPseudomonas fluorescens species. Journal of Industrial Microbiology 16, 8–14 (1996). https://doi.org/10.1007/BF01569915
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DOI: https://doi.org/10.1007/BF01569915