Intraparticle concentration gradients for substrate and acidic product in immobilized cephalosporin C amidase and their dependencies on carrier characteristics and reaction parameters

Abstract

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.

Advanced characterization of immobilized enzymes as heterogeneous biocatalysts

“…Screening of suitable immobilization procedures is also a very interesting application of internal sensing whereby optimization of carrier geometries is of particular relevance for biocatalyst design. Immobilization conditions can be optimized in a target-oriented manner once conditions of the heterogeneous environment are well known [167,170]. Results of characterization of immobilized enzymes with internal sensing methods support the idea that determination of kinetic and mass-transfer parameters for heterogeneous biocatalysts is strongly supported when the available evidence is not just from the external environment [178].…”

“…Self-referenced measurements and fluorescence lifetime [191] determinations exhibit superior analytical performance in agitated systems. Dual lifetime referencing (DLR), in particular [170,171,192], offers high versatility, independent on catalyst concentration, reactor configuration and scale of operation [170,171].…”

Nanofibrous membranes for single-step immobilization of hyperthermophilic enzymes

“…In contrast, using acetone-based crosslinking, there was little increase in activity with increased enzyme loading (constant electrospinning time of 4 h). Even increasing the enzyme loading by 4-fold (from 0.05% to 0.2%) resulted in a less than 2-fold increase in apparent activity; therefore, at increased enzyme loadings the diffusion of the substrate becomes increasingly rate limiting as in previous reports [61]. This result suggests that when using acetone-based crosslinking the diffusion of the substrate to and product from the immobilized enzyme within the fiber contributes to the lower apparent activity of the enzyme upon immobilization.…”

“…As we mentioned previously, the "electrospinning window" or time over which to collect fibers (time to δ4851) increased with higher enzyme loadings. As a result, the electrospinning time and mat thickness increased [63,64]; therefore, the decrease in activity could be due to increased mass thickness (indicating inter-fiber mass transfer limitations) and/or increased exposure time to GA and HCl which may deactivate the enzyme [48,61,65]. Using the acetone based crosslinking we varied the electrospinning time while holding the enzyme loading and crosslinking time constant and determined that the apparent specific activity of the immobilized enzyme was inversely proportional to electrospinning time (data not shown).…”

Biosilica and bioinspired silica studied by solid-state NMR

“…In operando characterization of the catalyst (i.e. : in realistic conditions of applications of the biocatalyst) have been carried out by applying diverse analytical methods directly within the solid support: examples include optochemical sensing [68][69][70][71] as well as spectroscopic imaging and electrochemical sensing (see table 5 in reference [51]). These methods yield direct handle on reagents' local concentration, gradients and diffusion properties with both temporal and spatial information.…”