There is an ongoing search for green and sustainable alternatives to fossil fuels. Cell culture biofuel production is one such alternative but is hampered by issues of cell toxicity and product yield. A solution to these issues is to purify the required pathway enzymes and design an in vitro biofuel production scheme. Critical to this system is that the enzymes act in a coordinated fashion and retain high activity during production. As the enzymes are no longer within the cell, optimization of reaction conditions is critical. Utilizing previous results from our lab concerning immobilization of enzymes to increase their stability for biofuel production, two more stable/active enzymes that can convert 2-ketoisovaleric acid to isobutanol, (2-ketoisovalerate decarboxylase (KIVD) and alcohol dehydrogenase (ADH)), were immobilized on epoxy functionalized methacrylate resin provided by Resindion (Rome, Italy). A third enzyme, (formate dehydrogenase (FDH)), was also immobilized to facilitate cofactor recycling and reduce product inhibition. We utilized in vivo produced reactant to test the free enzyme reaction scheme and then using those results, as well as stability measurements, we optimized our in vitro reaction scheme. Future work will involve examining nanofiltration to carry out in situ removal of the biofuel product and examining electrochemical production of NADH as a substitute for the recycling enzyme, FDH.

Co-ordinated enzyme catalysis: Designing a cell-free system

2019 Spring National Meeting

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