Ampicillin Synthesis Using a Two‐Enzyme Cascade with Both α‐Amino Ester Hydrolase and Penicillin G Acylase

Abstract

The current enzymatic production of semisynthetic β-lactam antibiotics requires isolation and purification of the intermediate 6-aminopenicillanic acid which adds cost and complexity to the manufacturing process. In this work, we took advantage of the unique substrate specificity of aamino ester hydrolases to perform a purely aqueous one-pot production of ampicillin from penicillin G and D-phenylglycine methyl ester, catalyzed by α-amino ester hydrolase and penicillin G acylase. The synthesis was performed in both a one-pot, one-step synthesis resulting in a maximum conversion of 39%, and a one-pot, two-step process resulting in a maximum conversion of 47%. The two-enzyme cascade reported in this paper is a promising alternative to the current enzymatic two-step, two-pot manufacturing process for semisynthetic β-lactam antibiotics which requires intermittent isolation of 6-aminopenicillanic acid.

Nonribosomal peptide synthetases and their biotechnological potential in Penicillium rubens

“…In the following step (either enzymatic or chemical), 6-APA is acylated using “non-natural” donor substrates to produce the antibiotic with the desired moiety. This has led to the production of common broad-spectrum antibiotics such as ampicillin and amoxicillin (Blum et al., 2010 ; Deng et al., 2016 ; Moody & Boesten, 2006 ; Wu et al., 2010 ). During the years, many more SSPAs have been developed, each presenting their own advantages or disadvantages in terms of antibiotic activity spectrum and pharmacokinetics.…”

“…Furthermore, the use of organic solvents, catalysts and other compounds that can produce by-products is often required. To render the process less costly and more environmentally-friendly, in vitro one-pot biocatalytic cascades that use penicillin acylases (and mutated variants) have been developed (Blum et al., 2010 ; Deng et al., 2016 ; Gabor & Janssen, 2004 ; Jager et al., 2008 ; Wu et al., 2010 ; Zhang et al., 2010 ).…”

Multidisciplinary approaches for enzyme biocatalysis in pharmaceuticals: protein engineering, computational biology, and nanoarchitectonics

“…For a detailed overview of various enzyme biocatalysis applications, please refer to Table 1. 51,52,58,59,61,62,65,66,[71][72][73][74][77][78][79][80][81][82][83][84][85][86][87][88][89][90][91][92][93]…”

“…For a detailed overview of various enzyme biocatalysis applications, please refer to Table 1. 51,52,58,59,61,62,65,66,71–74,77–93…”

Strategies to Improve the Biosynthesis of β-Lactam Antibiotics by Penicillin G Acylase: Progress and Prospects

“…Among them, both reactions 5 and 6 are performed at neutral pH (pH 7.0). In the case of reaction 5, penicillin G is hydrolyzed by PGA to obtain 6-APA, then 6-APA is reacted with D-PGME, using α-amino ester hydrolase (AEH) as a catalyst to form ampicillin at the final yield of 47% ( Blum et al, 2010 ), whereas in reaction 6, nitrile hydratase is first used to hydrolyze d -phenylglycine nitrile (D-PG-CN) into D-PGA, then D-PGA is reacted with 7-ADCA under PGA catalysis to synthesize cephalexin. After complexation with 1,5-dihydroxynaphthalene, a final yield of 79% is obtained ( Wegman et al, 2002 ).…”