Optimizing Immobilized Enzyme Performance in Cell-Free Environments to Produce Liquid Fuels

2012 AIChE Annual Meeting: Cleaner Energy, Stronger Economy, Better Living

Pittsburg, PA

Fuels and Petrochemicals Division

20002 Alternative Fuels I (Oral Talk)

October 28^th - November 2^nd, 2012

Optimizing immobilized enzyme performance in

cell-free environments to produce liquid fuels

Joseph J. Grimaldi, Cynthia H. Collins and Georges Belfort

Howard P Isermann Department of Chemical and Biological Engineering, and

Center of Biotechnology and Interdisciplinary Studies

Rensselaer Polytechnic Institute, Troy, NY 12180-3590

Active research to produce energy via biofuel from cells, or through the bioconversion of sugars to liquid fuels, offers exciting new alternates to fossil fuel.  We focus on the bioconversion of ketoisovaleric acid, a derivative of sugar, to iso-butanol using a two-enzyme system.  While these enzymatic routes offer great promise and excellent selectivity for the production of biofuels, enzymes exhibit slow kinetics, low volume capacity in solution, and product feedback inhibition.  These limitations must be overcome so that biofuels can be produced economically.  We utilize a novel approach to address these limitations.  Here, enzymes are first synthesized via recombinant DNA technology and then immobilized on solid substrates.  This cell-free enzyme system will be coupled with membrane separations to continuously remove the desired iso-butanol and reduce feed-back inhibition.  A model immobilized enzyme, beta-galactosidase, with a simple color change for assessing reactivity, was first studied to determine the optimal substrate geometry (flat, convex, or concave).  Preliminary data indicates that crowding and orientation of the immobilized enzymes have a large effect on enzyme kinetics. Serial reactions of immobilized keto-acid decarboxylase and alcohol dehydrogenase to produce butanol ex-vivo was then evaluated in solution and bound to substrates.  Finally, The effect of iso-butanol on the kinetics of the enzymes, both in solution and immobilized, was evaluated.