(357av) Development of an improved ?-amino ester hydrolase for the continuous reactive crystallization of ?-lactam antibiotics

β-lactam antibiotics are continually the most prescribed antibiotics in the world. Semi-synthetic β-lactam antibiotics can be produced enzymatically as an environmentally conscious and cost-effective alternative to traditional chemical synthesis. The enzymatic reaction by penicillin G acylase (PGA) couples an activated acyl side chain with a β-lactam nucleus to produce the desired β-lactam antibiotic. In situ crystallization during the synthesis of beta-lactam antibiotics improves yields by protecting the antibiotic, an intermediate in the reaction pathway, from enzymatic degradation. This enzymatic reactive crystallization process can be further optimized in a continuous process to enable process intensification, simplify process control, and improve the overall performance of the process¹. In this work, we demonstrate a pilot-scale continuous, enzymatic reactive crystallization of amoxicillin and cephalexin along with a novel continuous, magnetophoretic solid-solid separator to isolate pure antibiotic API from solid biocatalyst.

In addition to process development around PGA-catalyzed production of β-lactam antibiotics, a small class of enzymes known as α-Amino ester hydrolases (AEHs) were studied as a potential alternative to PGA for the synthesis of cephalexin^2,3. Despite their rapid kinetics, AEHs have had limited use primarily due to their low stability, aggregation, and rapid deactivation. Recently, we identified a complete kinetic mechanism for AEH catalyzed cephalexin synthesis to account for substrate inhibition that further complicates AEH use in large scale continuous processes^4,5. We demonstrate experimentally that AEH suffers from substrate inhibition and present a new kinetic model herein to fully describe the AEH catalyzed synthesis of cephalexin⁴. We demonstrate that AEH has significant potential for high cephalexin productivity, but substrate inhibition and deactivation severely limit reasonable use of AEH which will be addressed through our protein engineering efforts.

AEH deactivation kinetics were identified using temperature ramping in a continuous activity assay by monitoring β-lactam hydrolysis during temperature ramping. Coupled with differential scanning fluorimetry (DSF) and analytical ultracentrifugation (AUC) experiments, the relationships between AEH deactivation, monomer stability, and oligomericity were better described. Ultimately, the deactivation kinetics coupled with the synthesis kinetics allow for quantification of AEH total turnover numbers (TTN).

To improve AEH stability, computational protein engineering methods have been explored. Mutations for AEH stability improvements were evaluated using energy- and evolutionary-based calculations through available toolboxes such as HotSpot Wizard and FireProt^6,7. In addition, whole variants were constructed using multi-mutation designs from both Pross⁸ and FireProt⁶. With this work, we hope to gain structural insights into AEH denaturation as well as identify new variants with vastly improved thermostability. Overall, additional understanding of and improvement to AEH could make this enzyme a competitive biocatalyst for industrial synthesis of cephalexin.

[1] H Salami, MA McDonald, PR Harris, CE Lagerman, RW Rousseau, MA Grover*, AS Bommarius*, “Continuous reactive crystallization to amoxicillin and cephalexin”, (to be submitted)

[2] T.R.M. Barends, J.J. Polderman-Tijmes, P.A. Jekel, C.M.H. Hensgens, E.J. De Vries, D.B. Janssen, B.W. Dijkstra, The Sequence and Crystal Structure of the α-Amino Acid Ester Hydrolase from Xanthomonas citri Define a New Family of β-Lactam Antibiotic Acylases, Journal of Biological Chemistry 278(25) (2003) 23076-23084. https://doi.org/10.1074/jbc.m302246200.

[3] J.K. Blum, A.S. Bommarius, Amino ester hydrolase from Xanthomonas campestris pv. campestris, ATCC 33913 for enzymatic synthesis of ampicillin, J Mol Catal B Enzym 67(1-2) (2010) 21-28. https://doi.org/10.1016/j.molcatb.2010.06.014.

[4] C.E. Lagerman, M.A. Grover, R.W. Rousseau, A.S. Bommarius, Amino Ester Hydrolase- Catalyzed Synthesis of Cephalexin, Frontiers in Bioengineering and Biotechnology 10 (2022). https://doi.org/10.3389/fbioe.2022.826357.

[5] C.E. Lagerman, M.A. Grover, R.W. Rousseau, A.S. Bommarius, Kinetic model development for α-amino ester hydrolase (AEH)-catalyzed synthesis of β-lactam antibiotics, Chemical Engineering Journal 426 (2021) 131816. https://doi.org/https://doi.org/10.1016/j.cej.2021.131816.

[6] D. Bednar, K. Beerens, E. Sebestova, J. Bendl, S. Khare, R. Chaloupkova, Z. Prokop, J. Brezovsky, D. Baker, J. Damborsky, FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants, PLoS Comput Biol 11(11) (2015) e1004556.

[7] A. Pavelka, E. Chovancova, J. Damborsky, HotSpot Wizard: a web server for identification of hot spots in protein engineering, Nucleic Acids Res 37(Web Server issue) (2009) W376-83. https://doi.org/10.1093/nar/gkp410.

[8] Goldenzweig A, Goldsmith M, Hill SE, Gertman O, Laurino P, Ashani Y, Dym O, Unger T, Albeck S, Prilusky J, Lieberman RL, Aharoni A, Silman I, Sussman JL, Tawfik DS, Fleishman SJ. Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability. Mol Cell. (2016) 63(2):337-346. doi: 10.1016/j.molcel.2016.06.012.

Research Interests

As the use of biocatalysis in the pharmaceutical industry continues to expand, interesting problems have evolved related to accelerating enzyme discovery and optimization as well as fully leveraging the advantages of biocatalysis in process development. I am interested in research relating to all aspects of improvement and development of biocatalytic processes as my dissertation work has demonstrated. More specifically, I am excited for opportunities in either protein engineering projects or biocatalytic process design.