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

Belfort, Georges¹  Grimaldi, Joseph J.¹ 

[1] Rensselaer Polytechnic Inst., Troy, NY (United States). Dept. of Chemical and Biological Engineering

关键词: biofuel;    cell-free;    enzymes;    immobilized;    isobutanol;    pervaporation;    brush membranes.;   

DOI  :  10.2172/1168672
RP-ID  :  DOE-RPI--6520-1
PID  :  OSTI ID: 1168672

学科分类：燃料技术

美国|英语

来源: SciTech Connect

【 摘 要 】

Limitations on biofuel production using cell culture (Escherichia coli, Clostridium, Saccharomyces cerevisiae, brown microalgae, blue-green algae and others) include low product (alcohol) concentrations (??��0.2 vol%) due to feedback inhibition, instability of cells, and lack of economical product recovery processes. To overcome these challenges, an alternate simplified biofuel production scheme was tested based on a cell-free immobilized enzyme system. Using this cell free system, we were able to obtain about 2.6 times higher concentrations of iso-butanol using our non-optimized system as compared with live cell systems. This process involved two steps: (i) converts acid to aldehyde using keto-acid decarboxylase (KdcA), and (ii) produces alcohol from aldehyde using alcohol dehydrogenase (ADH) with a cofactor (NADH) conversion from inexpensive formate using a third enzyme, formate dehydrogenase (FDH). To increase stability and conversion efficiency with easy separations, the first two enzymes were immobilized onto methacrylate resin. Fusion proteins of labile KdcA (fKdcA) were expressed to stabilize the covalently immobilized KdcA. Covalently immobilized ADH exhibited long-term stability and efficient conversion of aldehyde to alcohol over multiple batch cycles without fusions. High conversion rates and low protein leaching were achieved by covalent immobilization of enzymes on methacrylate resin. The complete reaction scheme was demonstrated by immobilizing both ADH and fKdcA and using FDH free in solution. The new system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.5 % (v/v). Further increases in titer will require continuous removal of the isobutanol using our novel brush membrane system that exhibits a 1.5 fold increase in the separation factor of isobutanol from water versus that obtained for commercial silicone rubber membranes. These bio-inspired brush membranes are based on the presence of glycocalyx filaments coating the luminal surface of our vasculature and represent a new class of synthetic membranes. They thus meet the requirements/scope of the Bimolecular Materials program, Materials Science and Engineering Div., Office of Science, US DOE.