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dc.contributor.advisorBenedik, Michael J.
dc.creatorWang, Lan
dc.date.accessioned2010-01-15T00:08:10Z
dc.date.accessioned2010-01-16T00:55:19Z
dc.date.available2010-01-15T00:08:10Z
dc.date.available2010-01-16T00:55:19Z
dc.date.created2008-05
dc.date.issued2009-05-15
dc.identifier.urihttp://hdl.handle.net/1969.1/ETD-TAMU-2765
dc.description.abstractThis study generated two cyanide dihydratase (CynD) mutants of Bacillus pumilus C1 with improved activity at higher pH by random mutagenesis. The purpose of this study was to create enzyme variants better suited to degrade cyanide under the harsh conditions of industrial applications. We employed error-prone PCR to construct a library of CynD mutants. A high throughput screening system was developed to screen the library for improved activity. Two mutants were identified that could degrade cyanide at pH10 whereas the wild-type enzyme was inactive at pH9 or higher. The mutants each had three amino acid substitutions compared to the wild-type enzyme. The mutants were also more stable than the wild-type enzyme at 42oC. E327G was identified as one of the key amino acids that are responsible for the improved activity. The goal of the second project was to convert substrate specificity of the Bacillus sp. OxB-1 nitrilase to that of a cyanidase by mutagenesis or construction of hybrid genes. The OxB-1 nitrilase of Bacillus sp. shows a high level of identity with the cyanide dihydratases from B. pumilus C1 and P. stutzeri AK61 but utilizes different substrate. This provides a valuable resource to study the substrate specificity determinants of cyanide degrading enzymes. One deletion mutant and four hybrid proteins were constructed based on the alignment information. The constructed proteins were all unable to degrade cyanide.en
dc.format.mediumelectronicen
dc.format.mimetypeapplication/pdf
dc.language.isoen_US
dc.subjectprotein engineeringen
dc.subjectcyanide remediationen
dc.subjectbioremediationen
dc.subjectcyanide dihydrataseen
dc.subjectnitrilaseen
dc.subjectmicrobiologyen
dc.subjectgene cloningen
dc.subjectgenetic engineeren
dc.titleEngineering pH tolerant mutants of a cyanide dihydratase of Bacillus pumilus C1 and identifying constraints on substrate specificity in nitrilasesen
dc.typeBooken
dc.typeThesisen
thesis.degree.departmentBiologyen
thesis.degree.disciplineMicrobiologyen
thesis.degree.grantorTexas A&M Universityen
thesis.degree.nameMaster of Scienceen
thesis.degree.levelMastersen
dc.contributor.committeeMemberGolden, Susan S.
dc.contributor.committeeMemberWild, James R.
dc.type.genreElectronic Thesisen
dc.type.materialtexten
dc.format.digitalOriginborn digitalen


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