Bacterial P450-catalyzed polyketide hydroxylation on a microfluidic platform.
The incorporation of a multicomponent, cofactor-dependant P450 into a microfluidic biochip is demonstrated. The PikC hydroxylase Streptomyces venezuelae was incorporated into a PDMS-based microfluidic channel. The enzyme was immobilized to Ni-NTA agarose beads via in situ attachment following the addition of the beads to the microchannel. The enzyme loading was approximately 6 microg per mg of beads resulting in a microchannel loading of 10.7 mg/mL. This high enzyme loading enabled the rapid hydroxylation of the macrolide YC-17 to methymycin and neomethymycin in about equal amounts with a conversion of >90% at a flow rate of 70 nL/min. This high reactivity allowed rapid hydroxylation reactions to be performed with short residence times, which is critical for complex enzymes with limited inherent stability.
Srinivasan
Aravind
A
Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
Bach
Horacio
H
Sherman
David H
DH
Dordick
Jonathan S
JS
D019149:Bioreactors; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D004789:Enzyme Activation; D004800:Enzymes, Immobilized; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006900:Hydroxylation; D018942:Macrolides / Q000138:chemical synthesis* / Q000737:chemistry*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods*; D013302:Streptomyces / Q000201:enzymology*
D004800:Enzymes, Immobilized; D018942:Macrolides; C116391:YC-17 macrolide; C008517:methymycin; C008594:neomethymycin; D003577:Cytochrome P-450 Enzyme System