No abstract

Penicillin acylase from Alcaligenes faecalis has a very high affinity for both natural (benzylpenicillin, K m -0.0042 mM) and colorimetric (6-nitro-3-phenylacetamidobenzoic acid, K m = 0.0045 mM) substrates as well as the product of their hydrolysis, phenylacetic acid (A\ = 0.016 mM). The enzyme is partially inhibited at high benzylpenicillin concentrations but the triple SES complex formed still retains 43% of the maximal catalytic activity; the affinity of benzylpenicillin for the second substrate molecule binding site is much lower (As' = 54 mM) than for the first one. Phenylmethylsulfonyl fluoride was shown to be a very effective irreversible inhibitor, completely inactivating the penicillin acylase from A. faecalis in a few minutes at micromolar concentrations; this compound was used for enzyme active site titration. The absolute values of the determined kinetic parameters for enzymatic hydrolysis of 6-nitro-3-phenylacetamidobenzoic acid (k cat = 95 s _1 and k cat IK m = 2.1 X10 7 M _1 s _1 ) and benzylpenicillin (A cat = 54 s _1 and k cat IK m -1.3 X10 -7 M 1 s 1 ) by penicillin acylase from A. faecalis were shown to be highest of all the enzymes of this family that have so far been studied.

A concurrent bienzymatic cascade for the synthesis of optically pure (S)-4-methoxymandelonitrile benzoate ((S)-3) starting from 4-anisaldehyde (1) has been developed. The cascade involves an enantioselective Manihot esculenta hydroxynitrile lyase-catalyzed hydrocyanation of 1, and the subsequent benzoylation of the resulting cyanohydrin (S)-2 catalyzed by Candida antarctica lipase A in organic solvent. To accomplish this new direct synthesis of the protected enantiopure cyanohydrin, both enzymes were immobilized and each biocatalytic step was studied separately in search for a window of compatibility. In addition, potential cross-interactions between the two reactions were identified. Optimization of the cascade resulted in 81% conversion of the aldehyde to the corresponding benzoyl cyanohydrin with 98% enantiomeric excess.

The bio-based 2,5-furandicarboxaldehyde (diformylfuran, DFF) proved as effective as glutaraldehyde in the covalent immobilization of enzymes on amino-functionalized carriers. The binding is attributed to stable imine bond formation.