Establishing a biocatalytic process on a carrier-bound immobilized preparation of the used enzyme is desirable for several reasons. A heterogeneous biocatalyst is readily recovered from the liquid bulk, hence is multiply re-usable [1
]. Reactions are readily implemented in continuous or repeated batch modes of operation. Enzymes are often stabilized significantly against inactivation by (multipoint) attachment onto a carrier surface, substantially enhancing their total turnover in the reaction process [2
]. However, the carrier and the immobilization procedures require extra expenditure in biocatalyst preparation; in addition, carrier-bound enzymes are usually (much) less active than their native soluble counterparts. The observed loss of enzyme activity comprises a real
and an apparent
]. In real inactivation, a combination of diverse denaturation processes occur, likely resulting from attachment of the enzyme to the carrier. These processes include conformational distortion, chemical modification and improper orientation of the enzyme on the solid surface [3
]. Unfortunately, causal relationships between enzyme immobilization and denaturation at the molecular level are, to date, not understood enough. Apparent inactivation refers to diffusional limitations and their consequences on the observable enzymatic rate, and arises from two factors. First, if the characteristic times for mass transport into and out of the porous carrier are of the same order of magnitude as the characteristic time for the enzymatic reaction, the overall conversion rate for the immobilized preparation will be lower than that for the native enzyme used in homogeneous biocatalysis [4
]. Second, diffusional limitations may induce concentration gradients between bulk liquid and the interior of the carrier, which may steer the microenvironment of the immobilized enzyme away from optimal for activity, stability or both [5
]. Moreover, the chemical transformation may be itself negatively affected with respect to selectivity or equilibrium position under these conditions [4
]. Knowledge-based selection of a carrier whose geometrical properties (particle size, pore size) support optimum performance of the enzymatic reaction by attenuating the diffusion effects would facilitate development of efficient heterogeneous catalysts for applied biocatalysis.
We consider the case in which the enzymatic transformation proceeds with production or consumption of protons. This situation applies to a diverse range of conversions catalyzed by esterases, lipases, and some amidases of widespread use in the biocatalysis [1
]. In previously published studies, kinetic modeling has been used to delineate spatiotemporal features of pH gradient formations in immobilized enzymes [8
]. Results of these studies suggest that diffusional limitations may result in a substantial difference in the proton concentration between carrier and bulk. However, only a few studies have experimentally determined the internal pH of immobilized enzymes. Kasche, Spiess and coworkers published significant measurements of the pH drop in carrier resulting from enzymatic conversion of penicillin G using the pH-responsive fluorophore fluorescein isothiocyanate (FITC) [7
] and compared their data to predictions from a reaction-diffusion model. In their method, change in FITC luminescence intensity was related to the change in the proton concentration. However, intensity-based measurements may be strongly affected by drifts in the optoelectronic assemblage (light sources and light detectors) and by variations in sensor layer properties (dye concentration or thickness) [11
]. Moreover, large noise in the recorded signals may complicate the analysis of stirred or agitated suspensions.
Combining the pH indicator with a reference dye and measuring emission or excitation at two different wavelengths may overcome some of these drawbacks. Referenced intensity measurements have been performed in two previous studies of heterogeneous biocatalysts. Internal proton concentration was determined by confocal laser scanning microscopy, and the enzyme was fixed on either porous beads [12
] or in a membrane [13
]. The ability to record pH gradients within the carrier material at spatial resolution is a clear advantage of this method. However, light scattering and reflection are not referenced because luminescence is measured at two wavelengths [14
]. Furthermore, the required equipment is expensive and not suitable for the study of particles in suspension.
Lifetime measurements of indicators whose lifetime depends on pH are highly accurate because lifetime is independent of intensity or wavelength interferences [14
]. Spiess et al. [15
] evaluated the diffusion of propionic acid into hydrogels by measuring the pH-dependent lifetime of resorufin using confocal laser scanning microscopy and pulsar excitation. Kuwana et al. [16
] applied a frequency domain proton migration technique to measure the phase shift and thus the lifetime of a pH-sensing fluorophore, carboxy seminaphthofluorescein-1 immobilized on poly(ethylene glycol) microparticles. However, lifetime measurements of fluorescent pH indicators in the nanoseconds range require sophisticated optoelectronic equipment [14
Given the critical limitations of the above-described approaches in the design, selection and optimization of enzyme immobilizates, we here present an alternative method for pH determination in enzyme carriers. The method is based on a procedure known as dual lifetime referencing (DLR) [17
], previously applied in the development of optochemical sensors for pH, oxygen, chloride, copper and ammonia [16
]. DLR utilizes a pair of luminophores, one being the pH indicator and the other the reference standard. The method relies on simultaneous excitation of the indicator and the reference and on measuring the overall phase shift resulting from the ratio of the two intensities [18
]. DLR application requires that several criteria are satisfied: (1) that reference and indicator have large different decay times; (2) that spectral properties of reference are not affected by the sample; (3) that the excitation spectra of indicator and reference overlap (enabling both luminophores to be excited at a single band of wavelength), and (4) that the emission of both luminophores can be detected at a common wavelength or band of wavelength using a single photodetector. The result is a self-referenced measurement that is unaffected by optical system interferences. Specific details of the principle of measurement are described in Huber et al. [17
The pH dependence of the phase shift is utilized for pH determination. Phase-shift data are relatively independent of particle movements and can thus be collected flexibly and at useful signal-to-noise ratio from a wide range of reaction systems including the common stirred tank. Reaction time course monitoring enables measurements to be completed in a time-resolved manner, as required. Moreover, the required instrumentation is inexpensive and easy to handle. The chosen procedural set-up provides information about the average pH inside the immobilizate, but does not elucidate the spatial resolution across characteristic dimensions of the carrier. However, biocatalytic process development with immobilized enzymes typically involves comparative evaluation of different process options. Because of the importance of the time required to complete this development, the applied analysis methods must balance adequate throughput with quality and information content of the acquired results. The DLR approach would seem to present a practically useful compromise, as shown in our recent report [21
]. In the present paper, we describe details of the method and its application to immobilized enzyme characterization. The hydrolysis of cephalosporin C to 7-amino cephalosporanic acid (7-ACA) and D-α-amino adipic acid (DAAA) catalyzed by cephalosporin C amidase covalently tethered on epoxy-activated Sepabeads EC-EP is examined as a model system of industrial relevance (Figure ).
Figure 1 Cephalosporin C hydrolysis catalyzed by CCA. Formation of the DAAA product results in release of the molar equivalent of protons. In the case of immobilized CCA, proton formation may lead to a drop in intraparticle pH relative to the external pH in bulk. (more ...)