Redirecting the inactivation pathway of penicillin amidase and increasing amoxicillin production via a thermophilic molecular chaperone.
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.
Department of Chemical Engineering, University of California, Berkeley, California 94720, USA.
Lisa M
LM
Bergeron
Talar
T
Tokatlian
Lizabeth
L
Gomez
Douglas S
DS
Clark
D000255:Adenosine Triphosphate / Q000378:metabolism; D000658:Amoxicillin / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D019843:Archaeal Proteins / Q000378:metabolism*; D018833:Chaperonins / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D017017:Methanococcus / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism*; D010399:Penicillin Amidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D056405:Thermosomes
D000900:Anti-Bacterial Agents; D019843:Archaeal Proteins; D018832:Molecular Chaperones; D000658:Amoxicillin; D000255:Adenosine Triphosphate; D010399:Penicillin Amidase; D018833:Chaperonins; D056405:Thermosomes