Cephalosporin C amidase was covalently attached using a protein loading of 7.0-200 mg protein/g dry carrier on four epoxy-activated Sepabeads differing in particle size and pore diameter. Initial-rate kinetic analysis showed that for Sepabeads with small pore diameters (30-40 nm), the apparent K(M) of the amidase for hydrolysis of cephalosporin C at 37 degrees C and pH 8.0 increased approximately 3-fold in response to increased particle size (approximately 120-400 microm) and increased amount of immobilized enzyme (7.0-70 mg protein/g dry carrier) while maximum specific activity (3.2 U/mg protein; 25% of free amidase) was affected only by particle size. In contrast, for Sepabeads with wide pores (150-250 nm), the K(M) was independent of the enzyme loading. Internal effectiveness factors calculated from observable Thiele modulus reflected the dependence of K(M) on geometrical parameters of the particles. A new method for determination of the overall intraparticle pH was developed based on luminescence lifetime measurements in the frequency domain. Sepabeads were doubly labeled using a lipophilic variant of the pH-sensitive dye fluorescein, and Ru(II) tris(4,7-diphenyl-1,10-phenantroline) whose phosphorescence properties are independent of pH. Luminescent lifetime measurements of doubly labeled particle suspensions showed superior signal-to-noise ratio compared to fluorescence intensity-based measurements using singly labeled particles. The difference at apparent steady state (DeltapH) between bulk (external pH) and intraparticle pH (internal pH) was as large as approximately 0.6 units. The DeltapH was dependent on substrate concentration, particle size, and pore diameter. Therefore, these results characterize the role of carrier characteristics and reaction parameters in the formation of concentration gradients for substrate and acidic product during hydrolysis of cephalosporin C by immobilized amidase. The strong pH dependence of the immobilized amidase underscores the importance of considering intraparticle pH gradients in the design of an efficient carrier-bound biocatalyst.

Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a long-known flavoenzyme whose most important biocatalytic application is currently the industrial production of 7-amino-cephalosporanic acid (7-ACA) from cephalosporin C. Lacking mechanistic foundation, rational stabilization of TvDAO for improved process performance remains a problem. We report on results of thermal denaturation studies at 50 degrees C in which two purified TvDAO forms were compared: the native enzyme, and a site-specifically oxidized protein variant that had the side chain of cysteine108 converted into a sulfinic acid and lost 75% of original specific activity. Although inactivation time courses for both enzymes are fairly well described by simple single-exponential decays, the underlying denaturation mechanisms are shown by experiments and modeling to be complex. One main path leading to inactivation is FAD release, a process whose net rate is determined by the reverse association rate constant (k), which is 25-fold lower in the oxidized form of TvDAO. Cofactor dissociation is kinetically coupled to aggregation and can be blocked completely by the addition of free FAD. Aggregation is markedly attenuated in the less stable Cys108-SO(2)H-containing enzyme, suggesting that it is a step accompanying but not causing the inactivation. A second parallel path, characterized by a k-value of 0.26/h that is not dependent on protein concentration and identical for both enzymes, likely reflects thermal unfolding reactions. A third, however, slow process is the conversion of the native enzyme into the oxidized form (k < 0.03/h). The results fully explain the different stabilities of native and oxidized TvDAO and provide an inactivation mechanism-based tool for the stabilization of the soluble oxidase.

The amination of racemic a-chiral aldehydes, 2-phenylpropanal derivatives, was investigated employing w-transaminases. By medium and substrate engineering the optical purity of the resulting b-chiral chiral amine could be enhanced to reach optical purities up to 99% ee. Using enantiocomplementary w-transaminases allowed us to access the (R)-as well as the (S)-enantiomer in most cases. It is important to note that the stereopreference of the w-transaminases found for a-chiral aldehydes did not correlate with the stereopreference previously observed for the amination of methyl ketones. In one case the stereopreference switched even upon exchanging a methyl substituent to a methoxy group.

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