Project Number: 5325-41000-046-00
Project Type: Appropriated

Start Date: Mar 24, 2005
End Date: Mar 23, 2010

Objective:
Objective 1: Develop novel starch-degrading enzymes and enzyme-based systems that can convert starch from corn, wheat, and barley to glucose more efficiently. The aim is to improve activity at low pH, and increase catalytic conversion to lower the chemical and energy cost of liquefaction and saccharification. Objective 2: Develop novel enzymes and enzyme-based systems for solubilization and saccharification of hemicellulose components of corn fibers, crop residues, straws and grasses to simple sugars. Special focus will be to engineer high activity xylanases with decreased end-product inhibition and that can be produced in microbes, which make active cellulases. Objective 3: Utilize and evaluate the evolved enzymes and enzyme systems for the hydrolysis of corn, wheat, and barley starch (Objective 1) and solubilization and saccharification of hemicellulose components of corn fibers, crop residues, straws, and grasses (Objective 2) in laboratory and pilot scale operations. The improved enzymes will be evaluated on industrial substrates. Large-scale process development will be conducted in collaboration with corn ethanol companies. Objective 4: Develop and evaluate ethanol-based separation systems for the biorefining of wheat to produce conventional and new fractions as platforms for both food and non-food applications. Characterize the functional material properties of the refined fractions produced in these new systems for their use in both food and non-food applications. Objective 5: Develop and evaluate improved biorefining methods utilizing barrier or membrane-based separation technologies to manage the recovery of ethanol or other biobased chemicals for use as a platform chemical, a biofuel, and a processing fluid.

Approach:
New biocatalysts are created through directed evolution, in which libraries of enzyme variants are created by the mutation of encoding genes. The libraries are screened for target properties, and the candidate molecules selected can further undergo cycles of mutation, amplification, and selection until the desired enzyme variant is obtained. To create novel starch-degrading enzymes toward improved performance, the following steps are involved: (1) isolate, clone and express amylase genes; (2) mutate the gene sequences to create libraries; (3) screen the libraries for desired functions; (4) Purify the evolved enzymes for biochemical studies; (5) define the structure-function relationship; and (6) construct novel hybrid enzymes. To develop enzymes for the solubilization of hemicelluloses, the following approach is used: (1) screen by molecular techniques for a full set of genes encoding xylanolytic enzymes that are individually or collectively highly active in xylan degradation; (2) clone and express the genes in suitable expression systems; (3) conduct directed evolution by computational and experimental enzyme mutation; (4) purify the evolved enzymes for biochemical studies; (5) test the enzymes by combinatorial approaches; and (6) produce the enzymes in microbial systems which also produce active cellulases. As the best-fit enzymes are evolved and selected, the enzyme will be purified to homogeneity for the determination of kinetic properties. A variety of microscopic techniques will be applied to elucidate the action of the individual enzymes developed in this research. Several formats of bench scale enzyme reactions will be evaluated. These include liquefaction and saccharification as a distinct process, liquefaction and saccharification integrated with fermentation, and differentiated liquefaction and saccharification at high solids concentrations. To develop new separation systems, determine and account for (a) unique wheat gluten protein properties in the hydrated and developed state that enhance separation from starch and (b) unique solvent properties of ethanol that enable subfractionation of proteins, recovery of minor components (pentosan), and production of a dry product. Evaluate processing strategies such as countercurrent, batch, and crosscurrent extraction. Collaborate with ARS cost engineers and process-design firms where appropriate. To develop ethanol enrichment and recovery methods develop unique barrier materials for separation operations based pm pervaporation, perstraction, etc. Evaluate new materials for improved permeability and selectivity for ethanol recovery and concentration from fermentation broth (5-10% ethanol). Replacing 5325-41000-041-00D (February 2005). Combining 5325-41000-047-00D (7/08)