• Introduction 
• The Challenge
• Our Resources
• Our Leadership
  • Jay D. Keasling
  • Harvey Blanch
  • Ellen Ford
  • Kristin Balder-Froid
  • Paul Adams
  • Pam Ronald
  • Blake Simmons

 

> Back to Scientific and Leadership Staff

---

Current Research

Synergistic Activities

Engineering of enzymes isolated from extremophiles for enhanced biomass saccharification (PI). This is an internally funded three-year LDRD project at Sandia National Laboratories. This project has the objective of modifying the enzyme activity of a cellulase enzyme, endoglucanse, which hydrolyzes the cellulose polymers into cellobiose to make it functional at lower pH (<3) and higher temperatures (between 60 and 80 oC). The development of these enzymes would establish a potential path forward for consolidation of biorefinery process steps, such as combined pretreatment and saccharification. The specific characteristics listed are required for the enzyme to be active for the pretreatment of the cellulosic raw material using dilute acid technology. However, instead of engineering pH and temperature tolerance into the enzyme and the making it highly efficient catalyst, we chose to work with enzymes from extremophilic archaea. The hyperthermophilic archaea, which thrive under extremes of temperature, pH, salinity etc., have received considerable attention because of the high thermostability of their enzymes. The crenarchaeote Sulfolobus solfataricus, which thrives in acidic volcanic hot springs, is a thermoacidophile growing optimally at approx. 80 °C and pH 2– 4. As such, the proteins isolated from this organism are functional at high temperatures and low pH. The genome of S. solfataricus has been sequenced, and three genes (sso1354, sso1949 and sso2534) encoding potentially secreted endo-?-glucanases of glycoside hydrolases (GH) family 12 are found in the genome. We focused our attention on the sso1949 gene which has been shown to be an endo-?-glucanase (EC 3.2.1.4) and exhibits an exceptional activity at extremely low pH and is thermostable, a combination of acid and heat stability which has not yet been reported for any other GHs. Our objective is to engineer this enzyme to be highly efficient catalyst as defined by high turnover and low affinity for binding of the cellulose substrate. We obtained a clone from the lab of Dr. G. Lipps at the Institute of Biochemistry, University of Bayreuth, Bayreuth, Germany and we have established conditions for efficient heterologous expression of the protein, high-throughput purification, assaying, and characterizing the protein. We are now moving forward with engineering this enzyme for improved kinetics through directed evolution and site-directed mutagenesis techniques. Efforts at JBEI within the Deconstruction and Fuels Synthesis Divisions will benefit from the knowledge base generated, as well as the enzymes developed and characterized, by this project.

Polymeric microsystems for high-throughput water analysis and prokaryotic separation and enrichment (PI). Recent national and global events have drawn attention to the need for the rapid and accurate monitoring of water distribution networks. The focus of this project is to develop efficient, disposable, high-throughput microsystem platforms that enable the detection of pathogens in water at low concentrations. To detect particles or pathogens at low concentrations in raw liquid samples, it is vital to develop selective techniques to collect, concentrate, and deliver particles of interest for testing and identification. Dielectrophoresis (DEP) has been shown to be an effective means to manipulate such particles. DEP is the motion of particles driven by conduction effects in a nonuniform electric field. It has been shown that DEP can be used to transport suspended particles utilizing either oscillating (AC) or steady (DC) electric fields . DEP is suitable for differentiating biological particles (e.g., cells, spores, viruses, DNA) because it can collect specific types of particles rapidly and reversibly based on intrinsic properties including size, shape, conductivity and polarizability. Another approach is insulator-based dielectrophoresis (iDEP), which uses insulating obstacles - instead of electrodes - to produce spatial nonuniformities in an electric field that is applied through the suspending liquid. This iDEP technique was first presented by Masuda and subsequently developed further as a means of separating particles by Lee et al. It has been demonstrated that by utilizing insulating glass structures and AC electric fields iDEP can separate DNA molecules, Escherichia coli cells, and red blood cells. Similarly, Zhou et al. and Suehiro et al. used channels filled with insulating glass beads and applied AC electric fields to separate and concentrate yeast cells in water. We expanded these experimental findings to microfluidic glass- based devices using iDEP with a DC electric field applied across an array of insulating posts inside the microfluidic channel. Examples include trapping of polystyrene particles, separating live from dead bacteria, differentiating live species of bacterial prokaryotic cells, and the trapping and concentration of viruses. We have also demonstrated that ridge-based architectures can be used in addition to post-based archictures. This project is one of the core projects on the development of microsystem platforms at Sandia, and will be leveraged by the JBEI Technology Division.