(384g) Immobilization Optimization of ?-Amino Ester Hydrolase from Xanthomonas Campestris Pv. Campestris for Enzymatic Production of ?-Lactam Antibiotics

Authors: 
Lagerman, C. - Presenter, Georgia Institute of Technology
Salami, H., University of Maryland
Grover, M., Georgia Tech
Rousseau, R., Georgia Institute of Technology
Bommarius, A., Georgia Institute of Technology
Enzymatic production of β-lactam antibiotics has recently been a focus to replace inefficient chemical production. The use of soluble enzyme for β-lactam antibiotic production is limited due to issues with recoverability, recyclability, and enzyme deactivation. Enzyme operational stability can be improved through immobilization, but this often introduces mass transfer limitations to the enzyme within the porous resins, especially if reaction rates are high. Optimizing enzyme activity and stability on a platform that can be separated and reused is a multidimensional problem that must consider diffusion, intrinsic enzyme kinetics, and resin characteristics.

α-amino ester hydrolase (AEH) from Xanthomonas campestris pv. campestris has been explored by our group for β-lactam antibiotic synthesis [1]. We studied AEH immobilization by covalent attachment to porous beads via surface epoxides for β-lactam antibiotic production. Binding efficiency exceeded 98% on ChiralVision Immobeads® and 91% on Resindion Relizyme® at 40 mg AEH/g dry resin; however, efficiency decreased by 10% at 100 mg/g. Effectiveness factors were calculated using a Thiele modulus that incorporates cephalexin hydrolysis kinetics, effective diffusion coefficients, and enzyme loading [2]. Decreasing enzyme loading in Relizyme® beads from 107 mg AEH/g dry resin to 1.9 mg/g increases the effectiveness factor from 0.02 to 0.19. Increasing enzyme loading decreases synthesis selectivity as the produced antibiotic hydrolyzes. The decreased radius of Relizyme® beads (80 μm) compared to that of Immobeads® (200 μm) more than doubles the effectiveness factor. For feasible reactor operation, optimum enzyme loading for beads >100 μm sacrifice effectiveness to ensure a low slurry density capable of being well mixed. Our predictions suggest that resins <10 μm can achieve >99% effectiveness while keeping solid volume under 5%.

References

[1] J.K. Blum, A.S. Bommarius, J. Mol. Catal. B: Enzym. 67 (2010) 21–28.

[2] R.J. Barros, E. Wehtje, F. Garcia & P. Adlercreutz, Biocatal. Biotransfor. 16 (1998) 67-85