Grantee Research Project Results
Final Report: Development of Novel Proteins to Enhance Cellulose Deconstruction for Ethanol Production
EPA Contract Number: EPD08033Title: Development of Novel Proteins to Enhance Cellulose Deconstruction for Ethanol Production
Investigators: Sella Kapu, Nuwan U.
Small Business: Expansyn Technologies Inc.
EPA Contact: Richards, April
Phase: I
Project Period: March 1, 2008 through August 31, 2008
Project Amount: $69,668
RFA: Small Business Innovation Research (SBIR) - Phase I (2008) RFA Text | Recipients Lists
Research Category: SBIR - Emission Reductions and Biofuels , Small Business Innovation Research (SBIR)
Description:
This Environmental Protection Agency Small Business Innovative Research (SBIR) Phase 1 project explored the feasibility of using a novel group of plant proteins, homologs of expansin domain 2 (HED2), as an additive to improve the performance of cellulase in the digestion of cellulosic biomass. Our objectives for Phase I were two-fold: (1) produce gram amounts of ZM3 (a HED2 protein from maize pollen) in Escherichia coli, and (2) test the recombinant protein for cellulase synergism using real-world forms of biomass.
Summary/Accomplishments (Outputs/Outcomes):
Protein coding sequence of ZM3 was cloned into a commercially available expression vector and E. coli strain BL21 transformed with the construct. Expression of the recombinant protein was optimized with respect to several parameters such as temperature and duration of induction. Based on optimization experiments, it was possible to reach a recombinant ZM3 yield of more than 50 percent of total soluble protein. To produce gram levels of recombinant ZM3, E. coli was grown in a 16 L culture and protein expression induced under optimal conditions. This approach resulted in the collection of more than 600 g of E. coli cell paste carrying recombinant ZM3. Purification of recombinant ZM3 required only a single ion exchange chromatography step resulting in a protein yield of approximately 400 mg/L of culture. Cellulase synergistic activity of the recombinant ZM3 was verified using filter paper as the substrate. Examination of structural features of recombinant ZM3 in comparison with native ZM3 by circular dichroism and NMR spectroscopy suggested proper folding of ZM3 expressed in E. coli.
The ability of recombinant ZM3 to enhance cellulase performance was tested using different types of biomass and different cellulase preparations. Depending on dosage, recombinant ZM3 demonstrated 20-80 percent cellulase synergistic effect when dilute sulfuric acid pretreated corn stover (PCS) was used as the substrate. Under the cellulase digestion conditions used, PCS conversion was approximately 35-45 percent. Interestingly, the synergistic effect of recombinant ZM3 against raw corn stover was only 3-5 percent suggesting that the phenomenon is dependent upon the type of biomass. ETI currently is continuing cellulase hydrolysis experiments to optimize reaction conditions with the aim of minimizing the required ZM3 dose while maximizing biomass conversion and maintaining acceptable levels of synergy (at least 10%). Considering the observed difference between PCS and raw corn stover, the type of biomass is considered an important parameter for optimization.
Conclusions:
The results of Phase I demonstrate the technical feasibility of; (1) producing gram amounts of synergistically active recombinant ZM3 in E. coli, and (2) using ZM3 as an additive to enhance cellulase performance in the hydrolysis of biomass. Furthermore, Phase I experimental results show that expansins have a synergistic effect in improving cellulase activity and, therefore, may enable a significant reduction in cellulase cost. Significant strides have been made by enzyme manufacturers to reduce cellulase costs to an estimated $0.40/gallon cellulosic ethanol produced; however, it is recognized that further improvements in overall efficiency are required to reach the commercial target enzyme cost of less than $0.10/gallon. The development of new technology, including accessory proteins such as ZM3, will play a critical role in the ongoing quest to enhance cellulase performance in the conversion of cellulosic biomass to simple sugars for production of biofuels.
Supplemental Keywords:
small business, SBIR, EPA, cellulosic biomass, alternative fuels, ethanol, alternative energy, energy feedstock, plant protein, enzymes, deconstruction of cellulosic biomass, hydrolysis of crystalline cellulose, greenhouse gas emissions, engine emissions reduction, vehicle emissions reduction, sustainable industry/business, scientific discipline, RFA, technology for sustainable environment, sustainable environment, environmental chemistry, biomass, automotive industry, alternative energy source, emission controls, cellulase, cost, bioethanol, HED2,, RFA, Scientific Discipline, Sustainable Industry/Business, Environmental Chemistry, Sustainable Environment, Technology for Sustainable Environment, automotive industry, biomass, emission controls, alternative fuel, ethanol, alternative energy sourceThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.