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Purification and characterization of the hydroxylaminobenzoate lyase from pseudomonas pickettii YH105, cloned in escherichia coli
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Title
Purification and characterization of the hydroxylaminobenzoate lyase from pseudomonas pickettii YH105, cloned in escherichia coli
Name
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Hunter
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Farley Allen
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Farley Allen Hunter
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author
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Theodore
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Advisory Committee
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Theodore Chase
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chair
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Antoine
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Alan
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Advisory Committee
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Alan Antoine
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internal member
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Zylstra
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Gerben
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Advisory Committee
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Gerben Zylstra
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Rutgers University
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degree grantor
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Graduate School - New Brunswick
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school
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Text
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theses
OriginInfo
DateCreated
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2008
DateOther
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2008-01
Language
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English
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electronic
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application/pdf
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text/xml
Extent
ix, 34 pages
Abstract
The hydroxylaminolyase enzyme of Pseudomonas pickettii YH105 (now Ralstonia pickettii) removed the hydroxylamino group from p-hydroxylaminobenzoate with the concomitant formation of protocatechuate. In prior work, Brian A. Koller transferred the YH105 gene coding for hydroxyaminolyase into Escherichia coli now identified as BAK100. Although transferred, the hydroxyaminolyase enzyme was not characterized with respect to protein size and activity.
Utilizing samples of BAK100, the bacteria was grown on Luria-Bertani broth, induced with isopropyl β-D-1-thiogalactopyranoside (IPTG), separated by centrifugation, washed and weighed. After suspension in morpholinopropane sulfonic acid (MOPS) buffer, the enzyme was released from the crude cells by processing through a French press. Following centrifugation to remove non-target cell fragments, protamine sulfate treatment was used to remove RNA. The resulting solution was concentrated and non-target protein removed utilizing ammonium sulfate treatment prior to gradient DEAE and Sepharose 6L-6B gel filtration. Other purification techniques evaluated and discarded included dialysis, hydroxylapatite treatment, polyethylene glycol precipitation, and pH precipitation. Overall, the protein was purified relative to the crude cell extract, increasing by a factor of 2.7. The purification steps resulted in a substantial loss of activity. The overall yield was only 5%.
Several SDS electrophoresis gels all showed multiple prominent bands preventing the determination of enzyme size. Kinetic studies utilizing HPLC to quantifying protocatechuate formation provided a Km of 0.079 mM and Vmax of 0.16 µmol/min·mg for protamine sulfate treated crude cell extract.
Substrates other than p-hydroxylaminobenzoate were tested to evaluate enzyme specificity including m-hydroxylaminobenzoate, 3-methyl-4-hydroxylaminobenzoate and
p-hydroxylamino phenyl acetic acid. Only 3-methyl-4-hydroxylaminobenzoate showed slight conversion, suggesting a high level of enzyme specificity.
Utilizing samples of BAK100, the bacteria was grown on Luria-Bertani broth, induced with isopropyl β-D-1-thiogalactopyranoside (IPTG), separated by centrifugation, washed and weighed. After suspension in morpholinopropane sulfonic acid (MOPS) buffer, the enzyme was released from the crude cells by processing through a French press. Following centrifugation to remove non-target cell fragments, protamine sulfate treatment was used to remove RNA. The resulting solution was concentrated and non-target protein removed utilizing ammonium sulfate treatment prior to gradient DEAE and Sepharose 6L-6B gel filtration. Other purification techniques evaluated and discarded included dialysis, hydroxylapatite treatment, polyethylene glycol precipitation, and pH precipitation. Overall, the protein was purified relative to the crude cell extract, increasing by a factor of 2.7. The purification steps resulted in a substantial loss of activity. The overall yield was only 5%.
Several SDS electrophoresis gels all showed multiple prominent bands preventing the determination of enzyme size. Kinetic studies utilizing HPLC to quantifying protocatechuate formation provided a Km of 0.079 mM and Vmax of 0.16 µmol/min·mg for protamine sulfate treated crude cell extract.
Substrates other than p-hydroxylaminobenzoate were tested to evaluate enzyme specificity including m-hydroxylaminobenzoate, 3-methyl-4-hydroxylaminobenzoate and
p-hydroxylamino phenyl acetic acid. Only 3-methyl-4-hydroxylaminobenzoate showed slight conversion, suggesting a high level of enzyme specificity.
Note
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M.S.
Note
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Includes bibliographical references (p. 33-34).
Subject
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Topic
Microbiology and Molecular Genetics
Subject
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Topic
Biodegradation
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Topic
Hydrogenation
Subject
(ID = SUBJ4);
(authority = ETD-LCSH)
Topic
Nitro compounds
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(ID = SUBJ5);
(authority = ETD-LCSH)
Topic
Amino compounds
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(ID = SUBJ6);
(authority = ETD-LCSH)
Topic
Aromatic compounds
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Graduate School - New Brunswick Electronic Theses and Dissertations
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rucore19991600001
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http://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.17139
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ETD_652
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doi:10.7282/T3G44QNJ
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ETD graduate
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The author owns the copyright to this work.
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Copyright protected
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Open
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Name
Farley Hunter
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Rutgers University. Graduate School - New Brunswick
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Non-exclusive ETD license
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Author Agreement License
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I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
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