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.
Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
Aravind
A
Srinivasan
Horacio
H
Bach
David H
DH
Sherman
Jonathan S
JS
Dordick
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