Investigation of the determinants of thermal stability of the nitrile hydratase from Geobacillus pallidus RAPc8

dc.contributor.advisorSewell, Bryan Trevoren_ZA
dc.contributor.authorKianja, John Mainaen_ZA
dc.date.accessioned2017-06-01T10:07:39Z
dc.date.available2017-06-01T10:07:39Z
dc.date.issued2016en_ZA
dc.description.abstractThe mechanisms of thermal stability have been a long studied subject for many years with the aim of enhancing thermal stability of protein molecules to enhance their application in industry. The nitrile hydratases group of enzymes catalyse the hydrolysis of nitriles to amides using an exothermic catalytic mechanism. Understanding and applying specific amino acid residue mutations at specific regions in protein structures has been important for engineering of thermal stability into these often tetrameric thermolabile nitrile hydratases currently used in industry globally. At the near atomic level, the interatomic interaction(s) between specific amino acid residues governs the structure and function of nitrile hydratases. This study investigated several possible interactions responsible for conferring thermal stability to several thermostability-enhanced nitrile hydratase composite mutants generated from the wild type Geobacillus pallidus RAPc8 nitrile hydratase (NHase), namely: L103S+Y127N+F36L+D4G, M43K+T150A+S169R and D96E+D167V+M188V each labelled as 9E, 9C and 8C respectively. The composite mutants were previously developed using error-prone PCR of the wild type nitrile hydratase genes coding for the alpha and beta subunits from Geobacillus pallidus RAPc8. These composite mutants presented an opportunity to understand intramolecular thermostabilising mechanisms in this nitrile hydratase. Each individual mutation found in the composite mutants, was separately introduced into the DNA coding for the Geobacillus pallidus RAPc8 NHase by site directed mutagenesis. These individual mutants were over-expressed from E. coli and purified for further study. Using activity assays and protein melting curves, their individual thermal stability contributions were determined and represented as the difference in free energy of thermal unfolding (change in Gibbs free energy) of the single and composite mutants relative to the wild type nitrile hydratase. The measured residual activity following thermal inactivation was used together with the Arrhenius equation and a three parameter non-linear fit to determine the free energy of thermal unfolding. The change in Gibbs free energy resulting from each thermostabilising mechanism coupled to the analysis of their crystal structures was used to suggest the contributing mechanisms. This study found that intersubunit interactions through hydrogen bonds and salt bridges are especially important for contributing towards thermal stability of tetrameric nitrile hydratases. Hydrophobic interaction through the formation of a water shell around hydrophobic side-chains and packing of hydrophobic side-chains was also observed to contribute to thermal stability. These results suggest a path towards rational design and engineering of thermostabilising mutations into nitrile hydratases. Increased thermostability would improve their large scale application in industry by allowing these enzymes to be more active for longer at higher temperatures and decrease the cost of amide production.en_ZA
dc.identifier.apacitationKianja, J. M. (2016). <i>Investigation of the determinants of thermal stability of the nitrile hydratase from Geobacillus pallidus RAPc8</i>. (Thesis). University of Cape Town ,Faculty of Health Sciences ,Division of Medical Biochemistry & Structural Biology. Retrieved from http://hdl.handle.net/11427/24445en_ZA
dc.identifier.chicagocitationKianja, John Maina. <i>"Investigation of the determinants of thermal stability of the nitrile hydratase from Geobacillus pallidus RAPc8."</i> Thesis., University of Cape Town ,Faculty of Health Sciences ,Division of Medical Biochemistry & Structural Biology, 2016. http://hdl.handle.net/11427/24445en_ZA
dc.identifier.citationKianja, J. 2016. Investigation of the determinants of thermal stability of the nitrile hydratase from Geobacillus pallidus RAPc8. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Kianja, John Maina AB - The mechanisms of thermal stability have been a long studied subject for many years with the aim of enhancing thermal stability of protein molecules to enhance their application in industry. The nitrile hydratases group of enzymes catalyse the hydrolysis of nitriles to amides using an exothermic catalytic mechanism. Understanding and applying specific amino acid residue mutations at specific regions in protein structures has been important for engineering of thermal stability into these often tetrameric thermolabile nitrile hydratases currently used in industry globally. At the near atomic level, the interatomic interaction(s) between specific amino acid residues governs the structure and function of nitrile hydratases. This study investigated several possible interactions responsible for conferring thermal stability to several thermostability-enhanced nitrile hydratase composite mutants generated from the wild type Geobacillus pallidus RAPc8 nitrile hydratase (NHase), namely: L103S+Y127N+F36L+D4G, M43K+T150A+S169R and D96E+D167V+M188V each labelled as 9E, 9C and 8C respectively. The composite mutants were previously developed using error-prone PCR of the wild type nitrile hydratase genes coding for the alpha and beta subunits from Geobacillus pallidus RAPc8. These composite mutants presented an opportunity to understand intramolecular thermostabilising mechanisms in this nitrile hydratase. Each individual mutation found in the composite mutants, was separately introduced into the DNA coding for the Geobacillus pallidus RAPc8 NHase by site directed mutagenesis. These individual mutants were over-expressed from E. coli and purified for further study. Using activity assays and protein melting curves, their individual thermal stability contributions were determined and represented as the difference in free energy of thermal unfolding (change in Gibbs free energy) of the single and composite mutants relative to the wild type nitrile hydratase. The measured residual activity following thermal inactivation was used together with the Arrhenius equation and a three parameter non-linear fit to determine the free energy of thermal unfolding. The change in Gibbs free energy resulting from each thermostabilising mechanism coupled to the analysis of their crystal structures was used to suggest the contributing mechanisms. This study found that intersubunit interactions through hydrogen bonds and salt bridges are especially important for contributing towards thermal stability of tetrameric nitrile hydratases. Hydrophobic interaction through the formation of a water shell around hydrophobic side-chains and packing of hydrophobic side-chains was also observed to contribute to thermal stability. These results suggest a path towards rational design and engineering of thermostabilising mutations into nitrile hydratases. Increased thermostability would improve their large scale application in industry by allowing these enzymes to be more active for longer at higher temperatures and decrease the cost of amide production. DA - 2016 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2016 T1 - Investigation of the determinants of thermal stability of the nitrile hydratase from Geobacillus pallidus RAPc8 TI - Investigation of the determinants of thermal stability of the nitrile hydratase from Geobacillus pallidus RAPc8 UR - http://hdl.handle.net/11427/24445 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/24445
dc.identifier.vancouvercitationKianja JM. Investigation of the determinants of thermal stability of the nitrile hydratase from Geobacillus pallidus RAPc8. [Thesis]. University of Cape Town ,Faculty of Health Sciences ,Division of Medical Biochemistry & Structural Biology, 2016 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/24445en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDivision of Medical Biochemistry and Structural Biology
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherMedical Biochemistryen_ZA
dc.titleInvestigation of the determinants of thermal stability of the nitrile hydratase from Geobacillus pallidus RAPc8en_ZA
dc.typeMaster Thesis
dc.type.qualificationlevelMasters
dc.type.qualificationnameMSc (Med)en_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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