The increasing demand to find a more carbon-neutral energy source has fueled the search for biologically-derived products.  The current method for producing alcohol biofuels, in particular butanol, is through metabolic cellular processes. Limitations on biofuel production using cell culture include low product (alcohol) concentrations (2-5 vol%) due to feedback inhibition, instability of cells, and lack of economic product recovery processes.  Several research groups are using metabolic engineering and systems biology to overproduce butanol and increase tolerance.  To overcome these challenges, we propose an alternate simplified biofuel production approach based on a cell-free immobilized enzyme system.  Optimization of immobilization techniques and conditions leads to stable enzyme kinetics when compared with free enzyme in solution.  We study the activity of two multi-domain enzymes, alcohol dehydrogenase and keto-acid decarboxylase, after immobilization on multiple surface curvatures and chemistries, including silica nanoparticles, mesopourous silica, and methacrylate.  This process is attractive because (i) there are no cells that may experience product feedback inhibition, (ii) enzymes are produced at high titer using standard fermentation sans feedback limitations, (iii) protein engineering is used to modify the two enzymes to high product titer and high temperature, (iv) enzymes are stabilized through immobilization, and (v) the reaction is driven toward product through its continual removal (recovery).      In this work we used amine functionalized methacrylate resin as a support material for our enzymes.  Covalently immobilized alcohol dehydrogenase (ADH) exhibits stable reproducible enzymatic activity over multiple batch cycles.  Keto acid decarboxylase (KdcA) was engineered to be more active and stable using error-prone PCR directed evolution. The complete immobilized ADH and engineered KdcA system produces isobutanol from ketoisovaleric acid.

Optimizing immobilized enzyme performance: In vitro production of isobutanol

247th National Meeting (2014)

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