Improving the enzymatic synthesis of semi-synthetic beta-lactam antibiotics via reaction engineering and data-driven protein engineering

Penicillin G acylase;Beta-lactam antibiotics

Deaguero, Andria Lynn ; Chemical Engineering

University:Georgia Institute of Technology

Department:Chemical Engineering

关键词: Penicillin G acylase;    Beta-lactam antibiotics;   

Others  :  https://smartech.gatech.edu/bitstream/1853/42727/1/deaguero_andria_l_201112_phd.pdf

美国|英语

来源: SMARTech Repository

【 摘 要 】

Semi-synthetic β-lactam antibiotics are the most prescribed class of antibiotics in the world.Chemical coupling of a β-lactam moiety with an acyl side chain has dominated the industrial production of semi-synthetic β-lactam antibiotics since their discovery in the early 1960s.Enzymatic coupling of a β-lactam moiety with an acyl side chain can be accomplished in a process that is much more environmentally benign but also results in a much lower yield.The goal of the research presented in this dissertation is to improve the enzymatic synthesis of β-lactam antibiotics via reaction engineering, medium engineering and data-drive protein engineering.Reaction engineering was employed to demonstrate that the hydrolysis of penicillin G to produce the β-lactam nucleus 6-aminopenicillanic acid (6-APA), and the synthesis of ampicillin from 6-APA and (R)-phenylglycine methyl ester ((R)-PGME), can be combined in a cascade conversion.In this work, penicillin G acylase (PGA) was utilized to catalyze the hydrolysis step, and PGA and α-amino ester hydrolase (AEH) were both studied to catalyze the synthesis step.Two different reaction configurations and various relative enzyme loadings were studied.Both configurations present a promising alternative to the current two-pot set-up which requires intermittent isolation of the intermediate, 6-APA.Medium engineering is primarily of interest in β-lactam antibiotic synthesis as a means to suppress the undesired primary and secondary hydrolysis reactions.The synthesis of ampicillin from 6-APA and (R)-PGME in the presence of ethylene glycol was chosen for study after a review of the literature.It was discovered that the transesterification product of (R)-PGME and ethylene glycol, (R)-phenylglycine hydroxyethyl ester, is transiently formed during the synthesis reactions.This never reported side reaction has the ability to positively affect yield by re-directing a portion of the consumption of (R)-PGME to an intermediate that could be used to synthesize ampicillin, rather than to an unusable hydrolysis product.Protein engineering was utilized to alter the selectivity of wild-type PGA with respect to the substituent on the alpha carbon of its substrates.Four residues were identified that had altered selectivity toward the desired product, (R)-ampicillin.Furthermore, the (R)-selective variants improved the yield from pure (R)-PGME up to 2-fold and significantly decreased the amount of secondary hydrolysis present in the reactions.Overall, we have expanded the applicability of PGA and AEH for the synthesis of semi-synthetic β-lactam antibiotics.We have shown the two enzymes can be combined in a novel one-pot cascade, which has the potential to eliminate an isolation step in the current manufacturing process.Furthermore, we have shown that the previously reported ex-situ mixed donor synthesis of ampicillin for PGA can also occur in-situ in the presence of a suitable side chain acyl donor and co-solvent.Finally, we have made significant progress towards obtaining a selective PGA that is capable of synthesizing diastereomerically pure semi-synthetic β-lactam antibiotics from racemic substrates.