Economic bioethanol production is critically dependent upon the ability to convert both the hexose (C6) and pentose (C5) sugars resulting from cellulose and hemicellulose components of biomass. C5 sugars are not readily fermentable by native organisms. Genetically Modified Organisms (GMOs) are designed to ferment xylose, but their stability, ethanol yield, environmental impact, and survival under conditions of industrial fermentation are unproven. As an alternative, we developed a novel scheme for efficient fermentation of both C5 and C6 sugars using native yeasts. Rather than the traditional approach of genetically modifying yeast to ferment xylose, we pursue the unique approach of transforming xylose into a form that native yeast can successfully ferment. The projected economics of our process are very favorable in comparison to the costs associated with engineering, licensing and propogating GMOs. This novel fermentation technology is readily accessible to rural farming economies for implementation in cellulosic ethanol production facilities. OBJECTIVES: Show feasibility of developing a cost-effective biocatalysts capable of increasing product yield in the biological conversion of lignocellulosic biomass as well as enabling the long-term goal of simultaneous saccharification and fermentation (SSF) that currently limits the economic efficiency of biofuels production. The high level goals are: 1. Optimization of the use of Urease: To achieve optimal isomerization in the co-immobilized pellet system, the urease loading on the Xylose Isomerase (XI) particles and urea concentration in the bulk solution need to be optimized. We will address the optimization necessary to achieve the target pH difference by varying the urease coating thickness, urea concentrations and the medium temperature in a systematic manner. 2. Crude Urease Production: The production cost of coimmobilized enzyme pellets has a strong bearing on the ethanol production cost in our technology. The XI particles needed in our technology are commercially available Sweetzyme (from Novozyme) or GenSweet (from Genencor) pellets used in the high fructose corn syrup (HFCS) industry and, as such, are extremely inexpensive (~$0.02/gal EtOH). The very small quantities of urease needed to modify these pellets could be immobilized from a crude extract obtained at a fraction of the cost of the purified forms available from enzyme manufacturers. Accordingly, our final task for Phase I is to isolate crude urease extracts from the two sources: jack beans and pigeon peas and assess the possibility of coating the commercial XI particles with these crude urease extracts to achieve functional bi-layered pellets. APPROACH: The following illustrates the methods used in our preliminary analysis and are indicative of the methods and analysis that will be continued in Phase I research. General scientific methods Chemicals: Novo Sweetzyme(Sigma Aldrich G4166, 350 U/g or more activity based on isomerization of glucose to fructose), which is immobilized glucose isomerase produced from Streptomyces murinus, will be used for the isomerization of xylose. The glucose isomerase has optimal activity for glucose/fructose isomerization at pH 7.5 and 60 deg C (as per the manufacturer). The Sweetzyme pellets are dry, brown, cylinder-shaped granules with a diameter of approximately 1-3 mm. Jack bean urease (Sigma U4002, 70,400 U/g) will be used for generating the co-immobilized enzyme pellets used in the isomerization studies. Urease has optimal activity at pH 7.0 and 25 deg C (as per manufacturer). Both enzymes were stored at 4 deg C. Additional chemicals, including xylose, urea, borax, magnesium chloride, cobalt chloride, sodium citrate, and Tris will all be purchased from Sigma Aldrich (St. Louis, MO). Immobilization of urease on Sweetzyme pellets: For co-immobilization of urease on the Sweetzyme pellets, 500ml of 1g/l or 2g/l urease solution and 2g of Sweetzyme pellets are added to a 1 liter beaker. The beaker is left on the benchtop at room temperature for 24 or 48hrs. The pellets were separated from the solution by decanting and gravity filtration and dried on a paper towel at room temperature for 24hrs or until dry. Co-immobilized pellets are stored at 4 deg C until use. Activity of immobilized urease was measured at pH 7.5 and 25 deg C using a standard assay procedure that measures the rate of ammonia liberation. The urease activities obtained with our immobilization procedure are in the range of 550-577 U/g pellets, where a Unit liberates 1 micromol of ammonia per minute under the assay conditions. The effectiveness will be evaluated by the yield of ethanol during fermentation as well as the economics of co-immobilization
