January 1, 2010.
The use of lignocellulosic biomass as part of a comprehensive energy portfolio could potentially meet up to 25% of world primary energy needs by 2050, and do so in a domestically sourced, sustainable, and environmentally friendly way1. A major hurdle for large scale adoption of biofuels made from lignocellulosic biomass is inhibitors of microbial growth and metabolism that are present in pre-treated biomass feedstocks. These inhibitors reduce the efficiency of the overall biofuel conversion, and thereby drive up costs. One approach at circumventing this problem is to enhance the native ability of Zymomonas mobilis to convert these toxic inhibitors into less toxic compounds. A major class of inhibitors is the furalaldehydes, primarily furfural (2-furaldehyde) and 5-hydroxymentyl-2-furaldehyde (HMF). Previous work suggests that they can be detoxified by conversion to compounds which are less toxic. The purpose of this investigation was to identify metabolic proteins responsible for furalaldehyde conversion as a first step in enhancing the detoxification rates. An important factor in the purification of proteins is the development of a specific and robust assay. In this case, the disappearance of HMF and furfural from cell lysates which requires NADH or NADPH as cofactors was monitored using a microplate spectrophotometer. Further method optimizations involved identifying growth conditions and lysis conditions to yield maximal activities. Z. mobilis furfural activity was found to be highest in early stationary phase cells, and the addition of furfural did not increase total or specific activity. Lysis was found to be optimal using the Bugbuster HT® lysis reagent (Novagen) with 7500 units/mL of lysozyme. Ammonium sulfate precipitation of proteins is routinely used in protein purifications following cell lysis. Furalaldehyde reduction activity was found to precipitate at greater than 60% ammonium sulfate, and this purification led to an increase in specific activity of 30%. Following precipitation of Z. mobilis lysates by ammonium sulfate, extracts were loaded onto a Fast Protein Liquid Chromatography (FPLC) hydrophobic interaction column (HIC) . Fractions from the FPLC column were then assayed for furfural reduction activity and protein concentration. Active fractions were run on SDS-PAGE in order to identify protein bands that correlated with enzyme activity. Separation by FPLC resulted in modest enrichment of some SDS-PAGE gel bands, but specific and total activity were reduced, when compared to samples taken before column loading. FPLC variables to be tested in the future include using different types of FPLC columns, such as ion exchange or stronger hydrophobic interaction columns and then scaling up to produce large enough quantities for identification by mass spectroscopy (MS). Metabolic pathways identified by these methods will provide targets for genetic manipulation and up regulation.
National Renewable Energy Laboratory (NREL)