Not available / text

Submitted in fulfilment of the academic requirements for the degree of Master of Science in School of Molecular and Cell biology University of the Witwatersrand Johannesburg, South Africa / Esterases are hydrolytic enzymes that have many industrial applications. They are used in food, pharmaceutical, pulp and paper, cosmetics, biofuels and many other industries. This gives research of these enzymes major importance. Esterase genes received from CSIR Biosciences were cloned in E. coli DH5α cells. The plasmids carrying these genes were pET20b(+) for genes named Est1, Est2, Est3, Est4, Est5, Est6, Est7, Est8, Est9, Est10, Est12, Est13, Est14 and pET28a(+) for Est11. These plasmids were extracted from the cloning host and transformed into the expression host which was E. coli BL21. The cells were then induced for expression and the presence of the protein bands representing the products of expression were confirmed by running the crude enzyme extract on SDS-Page. The enzyme extracts were tested for activity using pNp-acetate. All 14 esterases were active and they were characterised in terms of pH optima, temperature optima and kinetics. The enzymes showed a pH range of 6.0 to 9.0 and temperature range of 30°C to 50°C. The enzymes were investigated for substrate specificity and they showed a greater preference for short acyl chain substrates over long acyl chain substrates. Further testing was done for activity of the enzymes using α-naphthylbutyrate and naphthol AS-D chloroacetate alongside lipases. A total of 87 enzymes were tested using these colorimetric assays and 36 of the enzymes were found to be active including all 14 esterases. These 36 enzymes were tested for use in enzymatic resolution of three different chemical compounds available as racemic mixtures. No success was observed for two of the compounds but one of them showed some enantioselectivity. This research will be furthered on at large scale to allow continued synthesis of potential HIV-1 protease inhibitors.

<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>

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

<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>

No description available.

No description available.