Development of an Improved Amino Ester Hydrolase for Continuous Reactive Crystallization Of Beta-Lactam Antibiotics
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Lagerman, Colton Eric
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Abstract
α-amino ester hydrolases (AEH) are a class of enzymes that are capable of synthesizing β-lactam antibiotics but are less studied than the industrially used penicillin G acylase (PGA). While both classes of enzymes synthesize a variety of β-lactam antibiotics, AEHs are promising candidates for enzymatic synthesis of cephalexin due to their rapid kinetics and low pH optimum of activity but suffer from low thermostability and substrate inhibition. The goal of this thesis is to further develop a thermostable AEH optimized for synthesis of β-lactam antibiotics and demonstrate a pilot plant for enzymatic reactive crystallization of both amoxicillin and cephalexin. While the pilot plant was designed using PGA, further development of a stable AEH allows for future testing of AEH in a continuous reactive crystallization process. AEH kinetics were first characterized in batch reactions, and a kinetic model was developed to describe cephalexin synthesis (Chapter 2). The relationships between AEH solution stability, oligomericity, and deactivation were analyzed (Chapter 3). Using the kinetic model developed in Chapter 2, reactor designs were modelled and evaluated for AEH-catalyzed synthesis of cephalexin (Chapter 4). An improved AEH was developed to address low thermostability using computationally guided rational design (Chapter 5). Using PGA, a pilot plant was developed for the enzymatic reactive crystallization of both cephalexin and amoxicillin (Chapter 6), and a novel magnetic separation system was developed to recycle immobilized PGA and isolate pure crystalline API (Chapter 7).
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2023-04-30
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Dissertation