Redirecting the inactivation pathway of penicillin amidase and increasing amoxicillin production via a thermophilic molecular chaperone

Abstract

We have previously shown that a single-subunit thermosome from Methanocaldococcus jannaschii (rTHS) can stabilize enzymes in semi-aqueous media (Bergeron et al., 2008b). In the present study, rTHS was used to stabilize penicillin amidase (PGA) in methanol-water mixtures. Including methanol in the reaction medium for amoxicillin synthesis can suppress unwanted hydrolysis reactions but inactivate PGA. Inactivation and reactivation pathways proposed for PGA illustrate the predictability of enzyme stabilization by rTHS in co-solvents. Calcium was necessary for reversible dissociation of the two PGA subunits in methanol-water and the presence of calcium resulted in an enhancement of chaperone-assisted stabilization. rTHS also acted as a stabilizer in the enzymatic synthesis of the beta-lactam antibiotic amoxicillin. rTHS stabilized PGA, increasing its half-life in 35% methanol by fivefold at 37 degrees C. Stabilization by rTHS was enhanced but did not require the presence of ATP. Including rTHS in fed-batch reactions performed in methanol-water resulted in nearly 4 times more amoxicillin than when the reaction was run without rTHS, and over threefold higher selectivity towards amoxicillin synthesis compared to aqueous conditions without rTHS. The thermosome and other thermophilic chaperones may thus be generally useful for stabilizing enzymes in their soluble form and expanding the range of conditions suitable for biocatalysis.

Self‐renaturing enzymes: Design of an enzyme‐chaperone chimera as a new approach to enzyme stabilization

“…The filtrate of each centrifuged sample was frozen at À208C for no more than 4 days until HPLC analysis was performed. HPLC analysis was carried out on a Waters HPLC setup (Waters Corp., Milford, MA) as described previously (Bergeron et al, 2009) with an Altima HP C18 AQ column (Alltech, Deerfield, IL).…”

“…Previously, rTHS was used to stabilize PGA in 30% MeOH, resulting in an increase of amoxicillin produced (Bergeron et al, 2009). For this study, substrates 6-aminopenicillanic acid (6-APA) and p-hydroxy-phenyl glycine methyl ester (POHPGME) were reacted in the presence of 30% v/v MeOH, 378C for 96 h (60 reactor average-residence times) and amoxicillin production was recorded.…”

Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli

“…The use of cytoplasmic or periplasmic chaperones in the folding assistance of PACs overproduced in heterologous hosts has been widely reported [22-26]. As for Tth PAC, TF and GroEL/ES proved to be the best choices at improving Eco PGA production [22].…”

Expression levels of chaperones influence biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and Pseudomonas putida Baeyer–Villiger monooxygenase

“…Molecular chaperones are involved in proper folding of cellular proteins and maintenance of preexisting proteins in their native states (Kim et al, ; Kolaj et al, ). Thereby, molecular chaperones have been widely used not only to increase the functional expression level of heterologous proteins/enzymes in microbial cells but also to improve their structural stability under reaction conditions, particularly in the presence of organic solvents and chemicals (Bergeron et al, ; Bergeron et al, ; Bergeron et al, ; Kolaj et al, ; Lee et al, ). For example, a single subunit recombinant thermosome (r‐THS) of Methanocaldococcus jannaschii , which was originally assembled into a homooligomer comprised of 16 identical subunits in wild‐type strains (Bergeron et al, ), was successfully used to stabilize penicillin amidase in 35% methanol (Bergeron et al, ).…”