Bio-based production of isobutylene from isobutanol by biocatalysis

Recently, the company Global Bioenergies has patented their research on the fermentative production of isobutylene, showing that bio-based isobutylene production is possible. If this compound is produced cost efficiently, its conversion into biofuels, or any other possible products, could become attractive. Therefore, it need to find new efficient process and environmentally friendly methods for isobutylene production. So far, enzymatic dehydration of isobutanol has been considered to be virtually impossible. However, there is an exception that the well-known oleate hydratase has activity for dehydration of isobutanol and other alcohols. The aim of this study was to develop method for high yield of isobutanol conversion to isobutylene by oleate hydratases. To achieve this goal, we conducted examination by molecular aspect as follows; 1) Cloning: The optimized sequences are synthesized by oligonucleotide concatenation and cloned in a pET28a vector. The resulting plasmid, transformed in E. coli ER2566 cells by electroporation. 2) Expression: The cells are grown with shaking (200 rpm) at 37°C in LB media, containing 20 µg/ml kanamycin until reaching an OD at 600 nm comprised between 0.5~0.7 and isopropyl-β-D-thiogalactopyranoside is then added to a final concentration of 0.1mM and protein expression is continued at 16°C overnight. For the result, we obtained optimal reaction conditions for the conversion of isobutylene from isobutanol using 13-LA enzyme were pH 6.5, 30°C, 100rpm, and 3% isobutanol. Under these conditions, 13-LA enzyme produced 0.1 mg/ml of isobutylene for 17h. This result is expected to be helpful for environmentally friendly process of isobutylene production by bioconversion.