Skip Navigation

Student Research Projects

Purification of a heat-stable alcohol dehydrogenase from Thermoanaerobacter pseudethanolicus

Student Nicolletta Cuthbert '14
Faculty Mentor(s) Dr. Joyce Easter
Department Chemistry
Course Summer Internship

Abstract

Alternative energy sources are establishing their importance as we continue to deplete the non-renewable reserves we have relied upon for so long. Alternative strategies, such as the conversion of lignocellulosic material to ethanol, have been developed; however, their efficiency is currently lacking. In order to use lignocellulosic material such as wood and grasses to produce biofuels, a pretreatment step is generally required to improve enzymatic saccharification. However, the ability of microorganisms to produce ethanol during the fermentation of pretreated lignocellulosic feedstocks is hindered, by the production of inhibitory compounds that are formed during pretreatment. In order to increase the production of ethanol, it is required to mitigate the toxicity of these inhibitory compounds. With the knowledge that certain anaerobic thermophiles are able to grow in the presence of these inhibitory compounds, we looked at how an enzyme discovered from Thermoanaerobacter psuedethanolicus 39E can detoxify certain compounds that are created during pretreatment. While cell-free extracts have demonstrated activity to pretreatment inhibitors such as furfural, we aimed to overexpress a Fe-dependent alcohol dehydrogenase enzyme in E.coli and purify it using a 6X histidine affinity tag translationally fused to the protein. Overexpression was achieved using a vector encoded T7-lac promoter induced by adding isopropyl-?-D-1-thiogalactopyranoside (IPTG). Induction experiments were conducted to determine the optimal overexpression parameters. Once verified, the culture volume was increased to 1 liter and a purification protocol was employed using nickel-based purification columns to selectively bind the tagged protein of interest. Optimal binding and washing conditions were established resulting in an isolated protein of the correct molecular mass. The purified alcohol dehydrogenase was able to oxidize butyraldehyde to butanol using NADPH in the process as expected, even under aerobic conditions. With the pure enzyme we were able to examine basic enzyme properties in more detail including temperature stability, pH range, and substrate specificity. The enzyme exhibited activity up to 70oC and also converted toxic aldehydes such as furfural to less toxic alcohols. With this knowledge, it is possible that this enzyme could be expressed in cellulolytic biofuel producing thermophiles to reduce pretreatment derived toxicity in the future.

 

Website Comments/Feedback

©2014 Virginia Wesleyan College