A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, fulfilment of the requirements for the degree of Master of Science. Johannesburg 2017. / Pichia pastoris is an established platform for the production of industrial enzymes. This nonfermentative methylotrophic yeast has many attractive features for the production of heterologous protein both in the laboratory and in industry. The PichiaPinkTM multi-copy secreted expression system was selected for the heterologous production of the fluorinase from Streptomyces cattleya. Fluorinase enzymes are useful for the production of fluorinated compounds which are applied in the agrochemical and pharmaceutical industries. The gene was cloned into the pPinkα-HC vector and used to transform four host srains by electroporation. Protein production was induced with 0.5% methanol and expression and activity was analysed by SDS-PAGE and a HPLC activity assay. Construction of the pPinkαHC-fLA expression plasmid and transformation of the host strains proved succesful. The PichiaPinkTM integrants showed genetic instability as the expression cassette showed signs of gene excision, thereby reducing the gene copy number. The wild-type strain1 efficiently secreted the foreign protein into the culture media, but the α-MF secretion signal was not processed correctly and secretion failed for the three protease knockout strains. However, the enzyme in both the secreted and intracellular protein fraction showed activity. Secretion methods need to be optimised and intracellular expression should be explored. The fluorinase enzyme was successfully cloned and expressed in four PichiaPinkTM strains and a biologically active protein was produced. / XL2017

by Nancy Wang. / Bibliography: leaves 126-144 / Thesis (M.Ph.)--Chinese University of Hong Kong, 1984

Thesis (Ph. D.)--School of Chemistry and Biochemistry, Georgia Institute of Technology, 2004. Directed by James C. Powers. / Vita. Includes bibliographical references (leaves 132-151).

Not available / text

Microbial nitrile hydratases are important industrial enzymes that catalyse the conversion of nitriles to the corresponding amides. A thermostable, cobalt-type Bacillus sp. RAPc8 microbial nitrile hydratase was cloned and expressed in E.coli. In this study the primary aim was to determine the molecular structure of Bacillus sp. RAPc8 microbial nitrile hydratase.

<p>The nitrilases are an important class of industrial enzymes that are found in all phyla. These enzymes are expressed widely in prokaryotes and eukaryotes. Nitrilases convert nitriles to corresponding acids and ammonia. They are used in industry as biocatalysts because of their specificity and enantioselectivity. These enzymes belong to the nitrilase superfamily in which members share a common &alpha / &beta / &beta / &alpha / structural fold and a unique cys, glu,lys catalytic triad with divergent N- and C-terminals.</p> <p>There are four atomic structures of distant homologues in the superfamily, namely 1ems, 1erz, 1f89 and 1j31. All structures have two-fold symmetry which conserves the &alpha / &beta / &beta / &alpha / -&alpha / &beta / &beta / &alpha / fold across the dimer interface known as the A surface. The construction of a 3D model based on the solved structures revealed the enzyme has two significant insertions in its sequence relative to the solved structures, which possibly correspond to the C surface. In addition there are intermolecular interactions in a region of a conserved helix, called the D surface. These surfaces contribute additional interactions responsible for spiral formation and are absent in the atomic resolution homologues.</p> <p>The recombinant enzyme from R.rhodochrous J1 was expressed in E. coli BL21 cells and eluted by gel filtration chromatography as an active 480 kDa oligomer and an inactive 80 kDa dimer in the absence of benzonitrile. This contradicts previous observations, which reported the native enzyme exists as an inactive dimer and elutes as a decamer in the presence benzonitrile. Reducing SDS-PAGE showed a subunit atomic mass of ~40 kDa. EM and image analysis revealed single particles of various shapes and sizes, including c-shaped particles, which could not form spirals due to steric hindrances in its C terminal.</p> <p>Chromatographic re-elution of an active fraction of 1-month old J1 nitrilase enabled us to identify an active form with a mass greater than 1.5 MDa. Reducing SDS-PAGE, N-terminal sequencing and mass spectroscopy showed the molecular weight was ~36.5 kDa as result of specific proteolysis in its C terminal. EM revealed the enzyme forms regular long fibres. Micrographs (109) were recorded on film using a JEOL 1200EXII operating at 120 kV at 50K magnification. Two independent 3D reconstructions were generated using the IHRSR algorithm executed in SPIDER. These converged to the same structure and the resolution using the FSC 0.5 criterion was 1.7 nm.</p> <p>The helix structure has a diameter of 13nm with ~5 dimers per turn in a pitch of 77.23 &Aring / . Homology modeling and subsequent fitting into the EM map has revealed the helix is built primarily from dimers, which interact via the C and D surfaces. The residues, which potentially interact across the D surface, have been identified and these confer stability to the helix. The conservation of the insertions and the possibility of salt bridge formation on the D surface suggest that spiral formation is common among microbial nitrilases. Furthermore, the presence of the C terminal domain in J1 nitrilase creates a steric hindrance that prevents spiral formation. When this is lost &ndash / either by specific proteolysis or autolysis - an active helix is formed.</p>

<p>The nitrilases are an important class of industrial enzymes that are found in all phyla. These enzymes are expressed widely in prokaryotes and eukaryotes. Nitrilases convert nitriles to corresponding acids and ammonia. They are used in industry as biocatalysts because of their specificity and enantioselectivity. These enzymes belong to the nitrilase superfamily in which members share a common &alpha / &beta / &beta / &alpha / structural fold and a unique cys, glu,lys catalytic triad with divergent N- and C-terminals.<br /> <br /> There are four atomic structures of distant homologues in the superfamily, namely 1ems, 1erz, 1f89 and 1j31. All structures have two-fold symmetry which conserves the &alpha / &beta / &beta / &alpha / -&alpha / &beta / &beta / &alpha / fold across the dimer interface known as the A surface. The construction of a 3D model based on the solved structures revealed the enzyme has two significant insertions in its sequence relative to the solved structures, which possibly correspond to the C surface. In addition there are intermolecular interactions in a region of a conserved helix, called the D surface. These surfaces contribute additional interactions responsible for spiral formation and are absent in the atomic resolution homologues.<br /> <br /> The recombinant enzyme from R.rhodochrous J1 was expressed in E. coli BL21 cells and eluted by gel filtration chromatography as an active 480 kDa oligomer and an inactive 80 kDa dimer in the absence of benzonitrile. This contradicts previous observations, which reported the native enzyme exists as an inactive dimer and elutes as a decamer in the presence benzonitrile. Reducing SDS-PAGE showed a subunit atomic mass of ~40 kDa. EM and image analysis revealed single particles of various shapes and sizes, including c-shaped particles, which could not form spirals due to steric hindrances in its C terminal.</p> <p>Chromatographic re-elution of an active fraction of 1-month old J1 nitrilase enabled us to identify an active form with a mass greater than 1.5 MDa. Reducing SDS-PAGE, N-terminal sequencing and mass spectroscopy showed the molecular weight was ~36.5 kDa as result of specific proteolysis in its C terminal. EM revealed the enzyme forms regular long fibres. Micrographs (109) were recorded on film using a JEOL 1200EXII operating at 120 kV at 50K magnification. Two independent 3D reconstructions were generated using the IHRSR algorithm executed in SPIDER. These converged to the same structure and the resolution using the FSC 0.5 criterion was 1.7 nm.<br /> <br /> The helix structure has a diameter of 13nm with ~5 dimers per turn in a pitch of 77.23 &Aring / . Homology modeling and subsequent fitting into the EM map has revealed the helix is built primarily from dimers, which interact via the C and D surfaces. The residues, which potentially interact across the D surface, have been identified and these confer stability to the helix. The conservation of the insertions and the possibility of salt bridge formation on the D surface suggest that spiral formation is common among microbial nitrilases. Furthermore, the presence of the C terminal domain in J1 nitrilase creates a steric hindrance that prevents spiral formation. When this is lost &ndash / either by specific proteolysis or autolysis - an active helix is formed.</p>

Thesis (Ph.D.)--McMaster University, 2003. / Advisor: Gerard D. Wright. Also available via World Wide Web.

Philosophiae Doctor - PhD / Microbial nitrile hydratases (NHases) are important industrial enzymes that catalyse the conversion of nitriles to the corresponding amides. A thermostable, cobalt-type Bacillus sp. RAPc8 NHase was previously cloned and expressed in E. coli. In this study, the primary aim was to determine the molecular structure of Bacillus sp. RAPc8 NHase. The heterotetrameric enzyme was purified to near homogeneity using heatpurification, hydrophobic interaction chromatography and ion exchange chromatography. Purified NHase was crystallised using the hanging-drop vapourdiffusion method. Crystals produced in the presence of 30% PEG 400, 0.1M MES pH 6.5 and 0.1M magnesium chloride were selected for X-ray diffraction studies. These crystals diffracted well, with diffraction spots visible beyond 2.4Å, with little mosaicity. At 2.5Å, the data were 93% complete. The crystal structure of Bacillus sp. RAPc8 NHase was solved via molecular replacement using the crystal structure of Pseudonocardia thermophila NHase as a search model. The final refined structure had good refinement statistics and geometry. The overall fold was very similar to that of previously determined NHase structures. Bacillus sp. RAPc8 NHase was most similar to Bacillus smithii NHase (0.355År.m.s.d.) and least similar to Rhodococcus sp. R312 NHase (1.191Å r.m.s.d.). One cobalt atom per heterodimer was bound to a typical NHase metal-binding motif, with post-translationally modified cysteine residues among the ligands to the metal. The substrate-binding and catalytic cavity of Bacillus sp. RAPc8 NHase was identified and described in detail. Surface representation of the structure revealed an extended, curved solvent accessible channel with access to bulk solvent from two locations in the heterodimer. The amino-acid residues forming the channel were identified and the geometric dimensions measured. Enzyme inhibition kinetics indicated that benzonitrile was a potent uncompetitive inhibitor of NHase. This information was used to aid the genetic engineering of aromatic substrate specificity into Bacillus sp. RAPc8 NHase. Site-directed mutants of NHase were prepared using the Quickchange mutagenesis procedure. Mutant W76G showed a two to three fold decrease in benzonitrile inhibition compared with the wild-type. Analysis of the substrate channel of this mutant NHase showed an 11% increase in volume and a 20% increase in inner surface area compared to that of the wild-type NHase. Due to the lack of other significant differences between the two structures (an r.m.s.d. of only 0.101Å was observed), this difference was thought to be responsible for the decrease in benzonitrile inhibition. A structure-modelling based approach for assessing the likely structural differences that may result as a result of a specific mutation was suggested and tested. This approach may be of value in future mutagenesis work. / South Africa

It has been observed that thermophilic enzymes are often more sluggish at lower temperatures but comparable active as their mesophilic homologues at their corresponding living temperatures. Although these thermophilic enzymes exhibit high structural stability, the increased stability leads to a decreased flexibility of the thermophilic enzymes in return. To yield further advances in analysis of the interrelationships between flexibility and activity of enzymes, also the molecular basis of enzyme adaptation, we used a pair of thermo-meso acylphosphatase homologues with high level of similarity isolated from hyperthermophilic archeaon Pyrococcus horikoshii (PhAcP) and human (HuAcP) as model to study this issue. Despite the fact that their active-site residues are highly conserved, activity (kcat) of PhAcP is remarkably reduced compared with HuAcP at low temperatures. Based on crystal structure comparison, an extra salt bridge was formed between active site residue and C-terminus of PhAcP. To examine the role of salt bridge plays in catalytic reaction of AcPs, we designed a mutant PhG91A to disrupt the salt bridge in thermophilic PhAcP. In parallel, a salt bridge was re-engineered into mesophilic HuAcP to create HuA99K. Interestingly, the thermophilic variant PhG91A exhibited a more mesophilic-like manner in terms of activity and thermodynamic parameters. On the contrary, mesophilic HuA99K displayed a more thermophilic-like character. This is supplemented by detailed molecular dynamics (MD) simulations, revealing good qualitative agreement with experimental findings. Both theory and experiment results had provided evidences that the presence of a specific salt bridge is directly associated with the temperature adaptation of AcPs by reducing the catalytic site flexibility. / Lam, Yan. / Adviser: K. B. Wong. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3364. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 120-127). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.