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Improving Enzyme Activity and Broadening Selectivity for Biological Desulfurization and Upgrading of Petroleum Feedstocks

Description: The objective of this project was to develop improved biocatalysts for desulfurization and upgrading of petroleum feedstocks. The goal was to improve the activity and broaden the selectivity of desulfurization enzymes using directed evolution as a tool as well as to explore the impact of ring-opening on biological desulfurization
Date: May 12, 2003
Creator: Borole, Abhijeet P.; Hamilton, Choo Y.; Miller, Karen; Davison, Brian; Grossman, Matthew & Shong, Robert
Partner: UNT Libraries Government Documents Department

Extremozymes for bioprocessing

Description: For many years, people have been seeking to employ enzymes, proteins that act as biocatalysts, as environmentally friendly replacements for many currently used industrial processes, as well as for the production of fuels and chemicals from biomass sources such as waste paper and agricultural residues. Current applications of enzymes include enzyme-assisted bleaching of wood pulp, preparation of textiles, design of biosensors, enzyme diagnostic kits, and bioremediation of toxic metals and chlorinated chemicals. However, until fairly recently, most of these enzymes came from mesophilic sources--organisms that grow at moderate temperatures (20--50 C) and pH 5 to 8. Frequently, these enzymes are not robust enough to perform well in industrial processes, have little tolerance for organic solvents or toxic chemicals, lose activity rapidly during catalysis, and cannot be used at high temperatures and alkaline or acidic conditions. But there are microorganisms that are known to grow under extreme conditions such as high temperature (thermophiles), low temperature (psychrophiles), acidic pH (acidophiles), and alkaline pH (alkalinophiles). The enzymes, extremozymes, from such microorganisms are active under the extreme conditions of temperature and pH at which the extremophiles grow. Use of extremozymes extends the potential temperature range for efficient enzymatic reactions to between 40 C and 100 C.
Date: November 1, 1998
Creator: Evans, B.R.; Zhou, J.; Poole, T.L.; Bunick, G.J.; Palumbo, A.V. & Woodward, J.
Partner: UNT Libraries Government Documents Department

Controlled production of cellulases in plants for biomass conversion. Annual report, March 11, 1997--March 14, 1998

Description: The goal of this project is to facilitate conversion of plant biomass to usable energy by developing transgenic plants that express genes for microbial cellulases, which can be activated after harvest of the plants. In particular, the feasibility of targeting an endoglucanase and a cellobiohydrolase to the plant apoplast (cell wall milieu) is to be determined. To avoid detrimental effects of cellulose expression in plants, enzymes with high temperature optima were chosen; the genes for these enzymes are from thermophilic organisms that can use cellulose as a sole energy source. During the past year (year 2 of the grant), efforts have been focused on testing expression of endoglucanase E{sub 1}, from Acidothermus cellulolyticus, in the apoplast of both tobacco suspension cells and Arabidopsis thaliana plants. Using the plasmids constructed during the first year, transgenic cells and plants that contain the gene for the E{sub 1} catalytic domain fused to a signal peptide sequence were obtained. This gene was constructed so that the fusion protein will be secreted into the apoplast. The enzyme is made in large quantities and is secreted into the apoplast. More importantly, it is enzymatically active when placed under optimal reaction conditions (high temperature). Moreover, the plant cells and intact plants exhibit no obvious problems with growth and development under laboratory conditions. Work has also continued to improve binary vectors for Agrobacterium-mediated transformation, to determine activity of E{sub 1} at various temperatures, and to investigate the activity of the 35S Cauliflower Mosaic Virus promoter in E. coli. 9 figs.
Date: June 1, 1998
Creator: Danna, K.J.
Partner: UNT Libraries Government Documents Department

Biochemistry of dissimilatory sulfur oxidation. Progress report, July 1992--June 1994

Description: The aims of the current proposal were to initiate the systematic identification and quantification of the relevant sulfur-transformation enzymes encoded and expressed by different species of the thiobacilli. The experimental plan had two major goals: (1) to purify known sulfur-transformation enzymes to electrophoretic homogeneity; and (2) To perform immunochemical analyses of protein expression using cell-free extracts and polyclonal antibodies directed against each protein purified in goal number one.
Date: October 1, 1997
Creator: Blake, R. II
Partner: UNT Libraries Government Documents Department

Structure-function correlation for ras p21 and the molecular origin of cancer

Description: In the past five years the authors followed different routes in correlating the structure and function of p21{sup ras} on an atomic level. The main project focused on understanding the GTPase mechanism catalyzed by p21{sup ras} and other GTP-binding proteins. The progress on this front is summarized. The starting point was the crystal structure of p21{sup ras} that was solved by the Kim group and the Wittinghofer group and paved the way for any attempt of understanding the hydrolysis mechanism in this protein. The crystallographic analysis has identified a water molecule (Wat175) in a position that makes it likely to be able to act as the nucleophile in the hydrolysis reaction. This water is directly located between the {gamma}-phosphate and the side chain of Gln61 in one of its possible orientations. This arrangement and the fact that mutations of Gln61 decrease the GPTase reaction rate led to the suggestion that this residue plays an important role in catalysis by acting as the general base for the nucleophilic water molecule and that it is assisted by Glu63.
Date: December 1, 1997
Partner: UNT Libraries Government Documents Department

Immobilization, stabilization and patterning techniques for enzyme based sensor systems.

Description: Sandia National Laboratories has recently opened the Chemical and Radiation Detection Laboratory (CRDL) in Livermore CA to address the detection needs of a variety of government agencies (e.g., Department of Energy, Environmental Protection Agency, Department of Agriculture) as well as provide a fertile environment for the cooperative development of new industrial technologies. This laboratory consolidates a variety of existing chemical and radiation detection efforts and enables Sandia to expand into the novel area of biochemically based sensors. One aspect of this biosensor effort is further development and optimization of enzyme modified field effect transistors (EnFETs). Recent work has focused upon covalent attachment of enzymes to silicon dioxide and silicon nitride surfaces for EnFET fabrication. They are also investigating methods to pattern immobilized proteins; a critical component for development of array-based sensor systems. Novel enzyme stabilization procedures are key to patterning immobilized enzyme layers while maintaining enzyme activity. Results related to maximized enzyme loading, optimized enzyme activity and fluorescent imaging of patterned surfaces will be presented.
Date: January 1, 1997
Creator: Flounders, A.W.; Carichner, S.C.; Singh, A.K.; Volponi, J.V.; Schoeniger, J.S. & Wally, K.
Partner: UNT Libraries Government Documents Department

Regional Neonatal Associates for cooperative study of platelet-activating factor (PAF). Summary report

Description: Lipid inflammatory mediators are thought to play an important role in the pathogenesis of the respiratory distress syndrome, including neonatal lung injury and bronchopulmonary dysplasia (BPD). One such mediator is platelet-activating factor (PAF), a potent bioactive phospholipid that induces adverse airway, vascular, and microcirculatory responses. To study the role of PAF in neonatal lung disease, we used an {sup 125}I-radioimmunoassay to measure PAF in whole blood and tracheal lavage in very low birthweight infants at 1, 3, 5, 9, 21 and 28 days after birth. PAF was found in the pulmonary lavagate and blood of ventilated infants as early as one day after birth. Lavagate levels of PAF increased with acute injury (pneumothorax, pneumonia) but were not associated with BPD. Our results indicate PAF could be associated with the pathogenesis of BPD. We suggest that as a consequence of the pathophysiologic processes associated with BPD, PAF is released by pulmonary cells. Our preliminary data indicate that low birthweight infants also have lower PAF acetylhydrolase levels in cord blood and tracheal lavagate as compared to adults. Therefore, it is possible the increased levels of PAF in the blood of low birthweight infants might be due to persistent transient increases in PAF alveolar levels coupled with lower blood acetylhydrolase activities and could be important in the development of symptoms associated with BPD. Future plans for this project call for completing the enzymatic study of acetylhydrolase activity in pulmonary lavage of the BPD infants.
Date: November 1, 1992
Creator: Snyder, F.
Partner: UNT Libraries Government Documents Department

Understanding and targeting a novel plant viral proteinase/substrate interaction. Final report, July 1, 1989--June 30, 1995

Description: The past 3 years of funding have focused our efforts on trying to understand the molecular basis of a unique substrate interaction displayed by a viral proteinase. We have made good progress and during this funding period we have made four contributions to the scientific literature and have developed the application of the proteinase in the expression and purification of recombinant fusion proteins. A comprehensive review of virus-encoded proteinases, written during the funding period, emphazing the tremendous similarity of viral proteinases with their cellular counterparts and at the same time detail the unique characteristics which permit them to function in a cellular environment. The focus of the research effort was the tobacco etch virus (TEV) 27kDa NIa proteinase.
Date: October 1, 1995
Creator: Dougherty, W.
Partner: UNT Libraries Government Documents Department

Bacterial quorum sensing and nitrogen cycling in rhizosphere soil

Description: Plant photosynthate fuels carbon-limited microbial growth and activity, resulting in increased rhizosphere nitrogen (N)-mineralization. Most soil organic N is macromolecular (chitin, protein, nucleotides); enzymatic depolymerization is likely rate-limiting for plant N accumulation. Analyzing Avena (wild oat) planted in microcosms containing sieved field soil, we observed increased rhizosphere chitinase and protease specific activities, bacterial cell densities, and dissolved organic nitrogen (DON) compared to bulk soil. Low-molecular weight DON (<3000 Da) was undetectable in bulk soil but comprised 15% of rhizosphere DON. Extracellular enzyme production in many bacteria requires quorum sensing (QS), cell-density dependent group behavior. Because proteobacteria are considered major rhizosphere colonizers, we assayed the proteobacterial QS signals acyl-homoserine lactones (AHLs), which were significantly increased in the rhizosphere. To investigate the linkage between soil signaling and N cycling, we characterized 533 bacterial isolates from Avena rhizosphere: 24% had chitinase or protease activity and AHL production; disruption of QS in 7 of 8 eight isolates disrupted enzyme activity. Many {alpha}-Proteobacteria were newly found with QS-controlled extracellular enzyme activity. Enhanced specific activities of N-cycling enzymes accompanied by bacterial density-dependent behaviors in rhizosphere soil gives rise to the hypothesis that QS could be a control point in the complex process of rhizosphere N-mineralization.
Date: October 1, 2008
Creator: DeAngelis, K. M.; Lindow, S. E. & Firestone, M. K.
Partner: UNT Libraries Government Documents Department

Enzyme Activity and Biomolecule Templating at Liquid and Solid Interfaces

Description: There are two main components of this research program. The first involves studies of the adsorption and catalytic activity of proteins at fluid-fluid and fluid-solid interfaces; the second employs biological macromolecules as templates at the solid-liquid interface for controlled crystallization of inorganic materials, to provide materials with specific functionality.
Date: December 1, 2004
Creator: Blanch, Harvey W.
Partner: UNT Libraries Government Documents Department

Control of light saturated photosynthesis: Concentration and activity of ribulose bisphosphate carboxylase. Final report, September 1, 1993--February 28, 1997

Description: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is one of the most abundant enzymes on the planet and is responsible for catalysing the net fixation of CO{sub 2} into organic matter. It is central, therefore, to primary productivity in marine and terrestrial ecosystems. Rubisco is a large enzyme with low substrate affinity and low catalytic efficiency and is considered to limit the rate of light-saturated photosynthesis. This report summarizes research into the molecular basis of the regulation of phytoplankton photosynthesis. It describes experimental and theoretical studies of the role of Rubisco in regulating the photosynthetic rate of phytoplankton. It also describes the integration of a mechanistically based phytoplankton growth model into a description of primary productivity in the sea. This work was conducted as part of the Ocean Margins Program.
Date: May 1, 1997
Creator: Geider, R.J.
Partner: UNT Libraries Government Documents Department

Utilization of biocatalysts in cellulose waste minimization

Description: Cellulose, a polymer of glucose, is the principal component of biomass and, therefore, a major source of waste that is either buried or burned. Examples of biomass waste include agricultural crop residues, forestry products, and municipal wastes. Recycling of this waste is important for energy conservation as well as waste minimization and there is some probability that in the future biomass could become a major energy source and replace fossil fuels that are currently used for fuels and chemicals production. It has been estimated that in the United States, between 100-450 million dry tons of agricultural waste are produced annually, approximately 6 million dry tons of animal waste, and of the 190 million tons of municipal solid waste (MSW) generated annually, approximately two-thirds is cellulosic in nature and over one-third is paper waste. Interestingly, more than 70% of MSW is landfilled or burned, however landfill space is becoming increasingly scarce. On a smaller scale, important cellulosic products such as cellulose acetate also present waste problems; an estimated 43 thousand tons of cellulose ester waste are generated annually in the United States. Biocatalysts could be used in cellulose waste minimization and this chapter describes their characteristics and potential in bioconversion and bioremediation processes.
Date: September 1, 1996
Creator: Woodward, J. & Evans, B.R.
Partner: UNT Libraries Government Documents Department

Tissue-specific changes of glutamine synthetase activity in oats after rhizosphere infestation by Pseudomonas syringae pv. tabaci. Final report

Description: Oats (Avena sativa L. lodi) tolerant of rhizosphere infestation by Pseudomonas syringae pv. tabaci when challenged by the pathogen experience tissue-specific alterations of ammonia assimilatory capabilities. Altered ammonia assimilatory potentials between root and leaf tissue result from selective inactivation of glutamine synthetase (GS) by the toxin Tabtoxinine-B-lactam (TBL). Root GS is sensitive and leaf GSs are resistant to TBL inactivation. With prolonged challenge by the pathogen root GS activity decreases but leaf GS specific activity increase. Higher leaf GS activity is due to decreased rates of degradation rather than increased GS synthesis. Higher leaf GS activity and elevated levels of GS polypeptide appear to result from a limited interaction between GS and TBL leading to the accumulation of a less active but more stable GS holoenzyme. Tolerant challenged oats besides surviving rhizosphere infestation, experience enhanced growth. A strong correlation exists between leaf GS activity and whole plant fresh weight, suggesting that tissue-specific changes in ammonia assimilatory capability provides the plant a more efficient mechanism for uptake and utilization of nitrogen.
Date: May 15, 1996
Creator: Knight, T.J.; Temple, S. & Sengupta-Gopalan, C.
Partner: UNT Libraries Government Documents Department

Mechanisms of lignin biosynthesis during xylogenesis in Zinnia elegans. Final report, July 1, 1992--June 30, 1996

Description: This project initially focused on identifying and characterizing three components of the extracellular lignification reaction: peroxidases, hydrogen peroxide production, and oxygen dependent oxidases. Zinnia elegans was utilized for the model organism. Laccase activity was found to be more tightly correlated with lignification than peroxidase activity.
Date: May 1, 1997
Creator: Eriksson, K.E.L. & Dean, J.F.D.
Partner: UNT Libraries Government Documents Department

Rational enhancement of enzyme performance in organic solvents. Final technical report, 1992--1996

Description: This research focused on the following: the dependence of enzymatic activity of several model hydrolases in nonaqueous solvents; control of substrate selectivity of the protease subtilisin Carlsberg by the solvent; control of catalytic activity and enantioselectivity of this enzyme in organic solvents by immobilization support; lipase-catalyzed acylation of sugars in anhydrous hydrophobic media; the possibility of accelerating enzymatic processes in organic solvents by certain cosolvents; whether lipase catalysis in organic solvents can be enhanced by introducing interfaces in the in the reaction medium; the structure of proteins suspended in organic solvents; improving enzymatic enantioselectivity in organic solvents; analyzing the plunge in enzymatic activity upon replacing water with organic solvents; and the structural basis for the phenomenon of molecular memory of imprinted proteins in organic solvents.
Date: December 31, 1996
Creator: Klibanov, A.M.
Partner: UNT Libraries Government Documents Department

A pilot plant scale reactor/separator for ethanol from cellulosics. Quarterly report No. 1 & 2, October 1, 1997--March 30, 1998

Description: The basic objective of this project is to develop and demonstrate a continuous, low energy process for the conversion of cellulosics to ethanol. This process involves a pretreatment step followed by enzymatic release of sugars and the consecutive saccharification/fermentation of cellulose (glucans) followed by hemi-cellulose (glucans) in a multi-stage continuous stirred reactor separator (CSRS). During year 1, pretreatment and bench scale fermentation trials will be performed to demonstrate and develop the process, and during year 2, a 130 L or larger process scale unit will be operated to demonstrate the process using straw or cornstalks. Co-sponsors of this project include the Indiana Biomass Grants Program, Bio-Process Innovation, Xylan Inc as a possible provider of pretreated biomass.
Date: June 1998
Creator: Dale, M. C.
Partner: UNT Libraries Government Documents Department

Large scale solubilization of coal and bioconversion to utilizable energy. Quarterly report, July 1, 1996--September 30 1996

Description: A purification of the Neurospora protein with coal solubilization activity (CSA) using DEAE cellulose chromatography is described. The protein is heavily glycosylated suggesting that it is different than tyrosinase or common phenol oxidases even though it resembles these proteins in enzyme activity and molecular weight.
Date: December 31, 1996
Creator: Mishra, N.C.
Partner: UNT Libraries Government Documents Department

Catalytic mechanism of hydrogenase from Azotobacter vinelandii. Final technical report, August 1, 1994--July 31, 1997

Description: This project is focused on investigations of the catalytic mechanism of the hydrogenase found in the aerobic, N{sub 2}-fixing microorganism Azotobacter vinelandii. This report summarizes the progress during the first two years of the current project and include the anticipated course of the research for the remaining year of the current project. Because the current proposal represents a change in direction, the authors also include a brief progress report of prior DOE-sponsored research dealing with hydrogenases.
Date: October 1, 1997
Creator: Arp, D.J.
Partner: UNT Libraries Government Documents Department

Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing. Final report, June 1, 1988--January 31, 1996

Description: This project has focused on the DNA polymerase of phage T7 for use in DNA sequencing. A complex of T7 DNA polymerase and E. coli thioredoxin form a highly processive DNA polymerase. The exonuclease activity of the enzyme can be reduced by chemical or genetic modifications resulting in an enzyme that has several properties useful in sequencing including high processivity and lack of discrimination against dideoxynucleotides. Manganese ion eliminates all discrimination against ddNTPs allowing sequence determination based on band intensity. A single tyrosine residue in the active site of T7 DNA polymerase is responsible for the efficient incorporation of ddNMPs. Replacement of the phenylalanine at this position in Klenow or Taq DNA polymerase with tyrosine eliminates discrimination against ddNTPs, a property that has advantages for cycle sequencing. Pyrophosphorolysis catalyzed by a polymerase results in the hydrolysis of specific fragments in DNA sequencing reactions, a problem that is eliminated by the addition of pyrophosphatase. The thioredoxin domain of gene 5 protein has been identified and transferred to Klenow DNA polymerase to make it processive. We have crystallized a complex of T7 DNA polymerase/thioredoxin bound to a primer-template in the presence of a dNTP.
Date: August 1, 1996
Creator: Richardson, C.C.
Partner: UNT Libraries Government Documents Department

International symposium on cellular and molecular biology of phosphate and phosphorylated compounds in microorganisms: Proceedings

Description: This report contains the abstracts of papers presented at the conference. Attention is focused on the following topics: regulation of phosphate metabolism in bacteria; structure-function of alkaline phosphatase; regulation of phosphate metabolism in yeast; transport of phosphate and phosphorylated compounds; and phosphate regulation in pathogenesis and secondary metabolism.
Date: December 31, 1993
Partner: UNT Libraries Government Documents Department