Summary
δ-(l-α-Aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase activity has been partially-purified from cell-free extracts of Streptomyces clavuligerus by ammonium sulfate precipitation. The salt precipitated enzyme was immobilized on an anion exchange resin and synthesis of ACV was observed by exposing the immobilized enzyme preparation to a reaction mixture containing l-α-aminoadipic acid, l-valine and l-cysteine in the presence of appropriate cofactors. Reaction mixtures containing l-α-aminobutyric acid(aB) in place of l-valine synthesized the ACV analog ACaB. Immobilized ACV synthetase can be reused, and after six cycles of reaction, 28.9% of original activity remains.
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Bahadur GA, Baldwin JE, Usher JJ, Abraham EP, Jayatilake GS, White RL (1981) Cell-free biosynthesis of penicillins. Conversion of peptides into new β-lactam antibiotics. J Am Chem Soc 103:7650–7651
Baldwin JE, Adlington RM, Coates JB, Crabbe JC, Crouch NP, Keeping JW, Knight GC, Schofield CJ, Ting H-H, Vallejo CA, Thorniley, M, Abraham EP (1987) Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities. Biochem J 245:831–841
Banko G, Wolfe S, Demain AL (1986) Cell-free synthesis of δ-(l-α-aminoadipyl)-l-cysteine, the first intermediate of penicillin and cephalosporin biosynthesis. Biochem Biophys Res Commun 137:528–535
Banko G, Demain AL, Wolfe S (1987) δ-(l-α-Aminoadipyl)-l-cysteinyl-d-valine synthetase (ACV synthetase): a multifunctional enzyme with broad substrate specificity for the synthesis of penicillin and cephalosporin precursors. J Am Chem Soc. 109:2858–2860
Castro JM, Liras P, Laiz L, Cortes J, Martin JF (1988) Purification and characterization of the isopenicillin N synthase of Streptomyces lactamdurans. Biochem J 134:133–141
Cortes J, Martin JF, Castro JM, Laiz L, Liras P (1987) Purification and characterization of a 2-oxoglutarate-linked ATP-independent deacetoxycephalosporin C synthase of Streptomyces lactamdurans. J Gen Microbiol 133:3165–3174
Dotzlaf JE, Yeh W-K (1987) Copurification and characterization of deacetoxycephalosporin C synthetase/hydroxylase from Cephalosporium acremonium. J Bacteriol 16:1611–1618
Hollander IJ, Shen Y-Q, Heim J, Demain AL, Wolfe S (1984) A pure enzyme catalyzing penicillin biosynthesis. Science 224:610–612
Jensen SE, Westlake DWS, Wolfe S (1984) Production of penicillins and cephalosporins in an immobilized enzyme reactor. Appl Microbiol Biotechnol 20:155–160
Jensen SE, Leskiw BK, Vining LC, Aharonowitz Y, Westlake DWS, Wolfe S (1986) Purification of isopenicillin N synthetase from Streptomyces clavuligerus. Can J Microbiol 32:953–958
Jensen SE, Westlake DWS, Wolfe S (1988) Production of the penicillin precursor δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV) by cell free extracts from Streptomyces clavuligerus. FEMS Microbiol Lett 49:213–218
Koshaka M, Demain AL (1976) Conversion of penicillin N to cephalosporin(s) by cell-free extracts of Cephalosporium acremonium. Biochem Biophys Res Commun 70:464–473
Wolfe S, Demain AL, Jensen SE, Westlake DWS (1984) Enzymatic approach to the syntheses of unnatural beta-lactams. Science 226:1386–1392
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Jensen, S.E., Wolfe, S. & Westlake, D.W.S. Synthesis of the penicillin precursor, δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV), by an immobilized enzyme preparation. Appl Microbiol Biotechnol 30, 111–114 (1989). https://doi.org/10.1007/BF00263995
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DOI: https://doi.org/10.1007/BF00263995