Browsing by Subject "enzyme"
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Item Analyzing the Limits and Extent of Alpha-Amylase Catalyzed Removal of Starch-Based Filter Cake(2012-02-14) Dharwadkar, Pavan S.The ability of starch to impart functions including fluid-loss control, cuttings transport, and rheological characteristics to water-based drilling fluids has led to its widespread use in the oil industry. The filter cake deposited by these drilling fluids often employs sized solid particles and starch to inhibit fluid loss into the formation. This inherently causes damage to the formation by impairing the permeability and must be removed before production. An alpha-amylase enzyme treatment was found to provide an effective approach to degrading starch in filter cake. In this work, an alpha-amylase enzyme treatment was analyzed by determining the extent of degradation of starch in filter cake using the iodine test, identifying degradants using high performance liquid chromatography, spectrophotometrically monitoring the concentration of enzyme, and measuring the cleanup efficiency of the enzyme treatment using a static filter press apparatus. The alpha-amylase enzyme used in this study was found to have a molecular weight under 30,000. The activity of the alpha-amylase enzyme was found to be sensitive to pH and temperature. The alpha-amylase enzyme was found to denature at temperatures above 165 degrees F and reversibly deactivate at pH below 4. Optimal conditions for alpha-amylase activity were found to be 150 degrees F and pH 6.5. The enzyme treatment works by hydrolyzing the interior glycosidic bonds of amylose and amylopectin residues of starch, creating soluble poly- and oligosaccharides and glucose. The enzyme treatment did not dissolve the calcium carbonate sized solids and a 5 wt. % hydrochloric acid postflush was necessary. The cleanup efficiency of the enzyme at pH 6.5 and room temperature treatment in conjunction with the postflush in a static test was 73% at 10% v/v concentration. Degradants resulting from alpha-amylase were identified chromatographically. Enzyme concentration remained steady prior to and after treatment.Item Discovery of Deaminase Activities in COG1816(2013-04-24) Goble, Alissa MImproved sequencing technologies have created an explosion of sequence information that is analyzed and proteins are annotated automatically. Annotations are made based on similarity scores to previously annotated sequences, so one misannotation is propagated throughout databases and the number of misannotated proteins grows with the number of sequenced genomes. A systematic approach to correctly identify the function of proteins in the amidohydrolase superfamily is described in this work using Clusters of Orthologous Groups of proteins as defined by NCBI. The focus of this work is COG1816, which contains proteins annotated, often incorrectly, as adenosine deaminase enzymes. Sequence similarity networks were used to evaluate the relationship between proteins. Proteins previously annotated as adenosine deaminases: Pa0148 (Pseudomonas aeruginosa PAO1), AAur_1117 (Arthrobacter aurescens TC1), Sgx9403e and Sgx9403g, were purified and their substrate profiles revealed that adenine and not adenosine was a substrate for these enzymes. All of these proteins will deaminate adenine with values of kcat/Km that exceed 105 M-1s-1. A small group of enzymes similar to Pa0148 was discovered to catalyze the hydrolysis of N-6-substituted adenine derivatives, several of which are cytokinins, a common type of plant hormone. Patl2390, from Pseudoalteromonas atlantica T6c, was shown to hydrolytically deaminate N-6-isopentenyladenine to hypoxanthine and isopentenylamine with a kcat/Km of 1.2 x 107 M^-1 s^-1. This enzyme does not catalyze the deamination of adenine or adenosine. Two small groups of proteins from COG1816 were found to have 6-aminodeoxyfutalosine as their true substrate. This function is shared with 2 small groups of proteins closely related to guanine and cytosine deaminase from COG0402. The deamination of 6-aminofutalosine is part of the alternative menaquinone biosynthetic pathway that involves the formation of futalosine. 6-Aminofutalosine is deaminated with a catalytic effeciency of 105 M-1s-1 or greater, Km?s of 0.9 to 6.0 ?M and kcat?s of 1.2 to 8.6 s-1. Another group of proteins was shown to deaminate cyclic- 3?, 5? -adenosine monophosphate (cAMP) to produce cyclic-3?, 5?-inosine monophosphate, but will not deaminate adenosine, adenine or adenosine monophosphate. This protein was cloned from a human pathogen, Leptospira interrogans. Deamination may function in regulating the signaling activities of cAMP.Item Evaluation of Exogenous Enzymes Targeting Non-starch Polysaccharides in Reduced Energy Diets on Broiler Growth Performance and Processing Parameters(2013-12-04) Klein, Joseph ThomasMultiple experiments were conducted to investigate the inclusion of a cocktail NSPase and ?-mannanase, separately and in combination, in reduced energy diets on broiler growth performance and processing yield. Each experiment contained a positive control (PC), negative control (NC) diet (-132 kcal/kg AME), and the inclusion of enzymes in the NC to evaluate enzyme effectiveness. The reduction in energy negatively impacted performance and processing parameters in all experiments. The inclusion of NSPase negated the negative effects of energy reduction in experiment 1. Experiment 2 evaluated increased pelleting temperature on NSPase activity. Body weight (BW) was increased (P<0.05) with the inclusion of NSPase pelleted at 80, 85, and 90 C throughout the experiment compared to NC; however, the treatment pelleted at 80 C outperformed the other NSPase pelleted treatments. The inclusion of NSPase pelleted at 80 C reduced (P<0.05) feed conversion ratio (FCR) compared to the NC throughout the experiment. At the conclusion of the trial, NSPase inclusion pelleted at 85 and 90 C yielded FCR similar to PC. The experimental design of experiment 3 and 4 included five dietary treatments including a PC, NC, NC supplemented with ?-mannanase, NSPase, and ?-mannanase/NSPase. Performance parameters were evaluated on d 14, 28, 42, and 47 and a subset of broilers were processed on day 48. Increases (P<0.05) in BW were observed with the inclusion of NSPase and ?-mannanase/ NSPase on day 14 and with all treatment groups on day 28. An additive effect was observed with reduced FCR through day 28 with the combination of ?-mannanase/ NSPase. In experiment 4, performance was evaluated on days 14, 27, 35, and 41 and carcass yields determined on day 42. Increases in day 14 BW were observed with the inclusion of the NSPase alone and ?-mannanase/NSPase to reach a similar weight as the PC. Inclusion of ?-mannanase/NSPase increased (P<0.05) BW compared to the NC. Inclusion of the NSPase reduced (P<0.05) cumulative FCR through 41 days of age. Inclusion of ?-mannanase/NSPase resulted in reduced (P<0.05) FCR in the finisher phase and cumulatively throughout the trial to levels of the PC. The combination of ?-mannanase/NSPase did increase (P<0.05) WOG weight similar to observations in BW. These data confirm that enzyme supplementation in low energy diets improve performance and indicate that additive effects of a combination of enzymes could potentially be a cost saving strategy for producers.Item Functional Exploration and Characterization of the Deaminases of Cog0402(2014-02-24) Hitchcock, Daniel StephenHigh throughput sequencing technology and availability of this information has changed the way enzyme families can be studied. Sequence information from large public databases such as GenBank and UniProtKB can easily retrieved for the purpose of identifying unique enzymatic activities. The strategy adopted for this study is to identify characterized enzymes and the sequence features which give rise to their substrate specificity. Homologues of these enzymes are retrieved, and any active site variations can be readily identified. Cluster of Orthologous Groups (cog) 0402 is a family of enzymes which comprise a portion of the amidohydrolase superfamily. This group catalyzes a deamination reaction, releasing free ammonia and replacing it with a tautomerized oxygen. Cog0402 is most well known for guanine and cytosine deaminase, however other functions exist. One such function was that of S-adenosylhomocysteine deaminase, which was related to a large group of uncharacterized enzymes. These enzymes were predicted by us to deaminate 5?-modified adenosines. The enzymes were physically characterized these predictions were confirmed and a 5?-deoxyadenosine deaminase was discovered in addition to an 8-oxoadenine deaminase. During this study it was noted that background isoguanine deaminase activity was found at appreciable rates in E. coli. This activity was purified and identified using nanoLC-MS/MS and found to be caused by E. coli cytosine deaminase. E. coli cytosine deaminase itself is found in a cluster of uncharacterized enzymes with a single amino acid difference in the active site. Representative enzymes were purified and a 5-methylcytosine deaminase was discovered. This enzyme is capable of rescuing thymine auxotrophs in the presence of 5-methylcytosine, and will confer sensitivity to 5-fluorocytosine. Finally, an enzyme distantly related to cytosine deaminase was purified and found to be a unique pterin deaminase. It was most efficient for oxidized pterin rings and would accept a variety of substituents on the C6 positions. Futhermore, it was thought to catalyze the first step of an undescribed pterin degradation pathway.Item Identification of Structural Changes Associated with Regulation of Tyrosine Hydroxylase(2011-10-21) Wang, ShanzhiTyrosine hydroxylase (TyrH) is the first and rate-limiting enzyme of catecholamine synthetic pathway, and its regulation is critical for controlling catecholamine synthesis. The well recognized regulatory mechanisms are inhibition by catecholamine binding and re-activation upon Ser40 phosphorylation. Catecholamines bind to TyrH tightly, while phosphorylation of TyrH at Ser40 decreases the binding affinity by several hundred-fold. Regulation of TyrH is accompanied by conformational changes of the protein. This study focuses on the identification of the conformational changes of TyrH upon dopamine binding and Ser40 phosphorylation, using hydrogen deuterium exchange mass spectrometry (HDMS) and fluorescence spectroscopy. HDMS identifies three peptides undergoing conformational changes upon dopamine binding, peptide 35-41, 42-71 and 295-299. Peptides 35-41 and 42-71 are on the regulatory domain, while peptide 295-299 is at the active site entrance. Upon dopamine binding, all three peptides are protected from exchange; phosphorylation of TyrH at Ser40 has opposite effects on the exchange kinetics of peptide 295-299, but peptides 35-41 and 42-71 could not be detected by MS after phosphorylation. This suggests that the structural effects of dopamine binding and Ser40 phosphorylation are opposite. The fluorescence spectroscopy of mutant enzymes containing a single tryptophan at position 14, 34 or 74 was performed before and after phosphorylation. F34W/F3W TyrH has a significant decrease in steady-state fluorescence anisotropy, an increase in the bimolecular quenching rate constant kq and dynamic anisotropy upon phosphorylation at Ser40, while F14W/F3W TyrH and F74W/F3W TyrH exhibit much smaller differences. This suggests that phosphorylation of TyrH at Ser40 increases the flexibility of the regulatory domain. The results are consistent with TyrH existing in two conformations, a closed conformation stabilized by dopamine in which the N-terminal regulator domain of TyrH covers the active site entrance and an open conformation stabilized by phosphorylation in which the regulatory domain has moved away from the active site entrance.Item Mechanistic and Functional Characterization of Lactonases of COG3618 in the Amidohydrolase Superfamily(2014-05-06) Hobbs, Merlin EricThe postgenomic era of scientific research has yielded an inundation of gene and protein sequences which are available in public databases. This torrent of sequences, literally in the millions, has altered the strategies, methodologies, and approaches taken toward function discovery and annotation. At the forefront, are interdisciplinary approaches; such as, genomic enzymology. Genomic enzymology bridges aspects of classical enzymology, structural and functional genomics, and comparative genomics. These methods also take advantage of evolutionarily related proteins, which have relatively similar sequence similarity but, yield different functions. The amidohydrolase superfamily (AHS), contains proteins of similar chemistry and topology, which are related to one another through a common ancestral progenitor. The AHS has been organized into smaller groups based only on sequence similarity, which are known as clusters of orthologous groups (COG). There are currently 24 COGs within this superfamily. Cog3618 is comprised of primarily lactonase enzymes. The first identified member of cog3618 was LigI, which catalyzes the reversible hydrolysis of 2-pyrone-4,6-dicarboxylate. The chemical mechanism was elucidated and it was determined that this protein is the first member of the AHS that does not require a metal cofactor for catalysis. The mechanistic characteristics of LigI, along with genomic enzymology, were utilized to predicted and identify the functions of two other COG members. BmulJ_04915 and BVU_0220 were identified as L-fucono-1,5-lactonase and L-galactono-1,5-lactonase, respectively. NMR analysis revealed that these enzymes give preference to 6-member lactones, as opposed to their more stable counterparts. In addition, it is proposed that L-galactono-1,5-lactone participates in a novel L-galactose catabolic pathway, which appears to converge with D-galacturonate degradation through the intermediate of an adjacent dehydrogenase (BVU_0222). BVU_0222 was determined to oxidize L-galactonate to D-tagaturonate, which is a pathway intermediate for the degradation of D-glucuronate.Item Structure Dynamics Guided Enzyme Improvement of ENDO-BETA-1, 4-XYLANASE I(2013-06-06) Uzuner, UgurEnzyme structure dynamics has recently been revealed to be essential for structure-function relationship. Among various structure dynamics analysis platforms, hydrogen deuterium exchange mass spectrometry stands as an efficient and high-throughput way to analyze protein dynamics upon ligand binding, protein folding, and enzyme catalysis. HDX-MS can be used to study the regional dynamics of proteins based on the m/z value or percentage of deuterium incorporation for the digested peptides in the HDX experiments. Various software packages have been developed to analyze HDX-MS data. However, for the accurate, enhanced, and explicit statistical analysis of HDX-MS data statistical analysis of software was developed as HDXanalyzer. The capability of HDX-MS analysis for the identification of enzyme structure dynamics was tested by using model catalysis endoxylanase A (XYN I) from Trichoderma longibrachiatum. The HDX data of XYN I revealed a highly dynamic personality of XYN I through the interaction with two substrates. The dynamic data which certainly restricts the targeted regions for the protein engineering efforts provided useful knowledge about the essential structural modifications for the catalysis of XYN I. The obtained knowledge was then employed for the engineering studies in order to improve the certain characteristics of XYN I protein. The high level stabilization of XYN I protein was gathered and the two highly active and moderately thermostable XYN I recombinants were developed based on the HDX-MS data which further confirmed the efficiency of the current strategy for the rational designs of catalytic proteins. A differential dynamics analysis of the two structurally similar catalysts was also performed through HDX-MS. The functionally and sequentially different but structurally highly similar XYN I and endoglucanase (Eg1A) enzymes revealed distinct structure dynamic characteristics. Compared to XYN I, Eg1A from Aspergillus niger indicated quite restricted structural motions. The data clearly postulated that the intrinsic dynamic modifications of during the enzymatic catalysis may not be the only driving force in all cases. In summary, the integration of the structure dynamics knowledge to the current biochemical and biophysical data of catalysts may provide novel insights to further enzyme improvement applications.Item The chemical mechanisms of flavin-dependent amine oxidases and the plasticity of the two-his one-carboxylate facial triad in tyrosine hydroxylase(2009-05-15) Ralph, Erik C.Despite a number of kinetic and spectroscopic studies, the chemical mechanisms of amine oxidation by flavoenzymes remain widely debated. The mechanisms of by Nmethyltryptophan oxidase (MTOX) and tryptophan 2-monooxygenase (TMO) were probed using a combination of pH and primary deuterium, solvent, and 15N kinetic isotope effects. Slow substrates were chosen for these studies; MTOX was characterized with N-methylglycine and TMO was characterized with L-alanine. Primary deuterium kinetic isotope effects of 7.2 and 5.3 were observed for sarcosine oxidation by MTOX and for alanine oxidation by TMO, respectively, independent of the substrate concentration and pH. Monitoring the reduction of flavin spectroscopically revealed no intermediate flavin species with both enzyme-substrate systems. Furthermore, the magnitudes of the 15N kinetic isotope effects observed with both systems suggest that nitrogen rehybridization and C-H bond cleavage are concerted. These results are consistent with both enzymes utilizing a hydride transfer mechanism for amine oxidation. The role of the iron ligands of tyrosine hydroxylase (TyrH) was also investigated. TyrH contains one iron per monomer, which is held by three conserved amino acid residues, two histidines and a glutamate. As a probe of the plasticity of the metal binding site, each of the metal ligands in TyrH was substituted with glutamine, glutamate, or histidine. The resulting proteins were characterized for metal content, catalytic activity, and dopamine binding. The H336E and H336Q enzymes retain substantial catalytic activity. In contrast, the E376Q enzyme retains about 0.4% of the wild-type catalytic activity, and the E376H enzyme has no significant activity. The H331E enzyme oxidizes tetrahydropterin in a tyrosine-independent manner. The position of the charge-transfer absorbance band for the H336E and H336Q enzyme-inhibitor complexes is shifted relative to that of the wild-type enzyme, consistent with the change in the metal ligand. In contrast, the E376H and E376Q enzymes catalyze dopamine oxidation. These results provide a reference point for further structural studies of TyrH and the other aromatic amino acid hydroxylases, and for similar studies of other enzymes containing this ironbinding motif.