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<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

The enzymatic production of R-phenylacetylcarbinol (R-PAC), with either whole cells or partially purified pyruvate decarboxylase (PDC) as the biocatalyst, requires high PDC activity and an inexpensive source of pyruvate for an economical feasible biotransformation process. Microbial pyruvate produced by a vitamin auxotrophic strain of Candida glabrata was selected as a potential substrate for biotransformation. With an optimal thiamine concentration of 60 ??g/l, a pyruvic acid concentration of 43 g/l and yield of 0.42 g/g glucose consumed were obtained. Using microbially-produced unpurified pyruvate resulted in similar PAC concentrations to those with commercial pure substrate confirming its potential for enzymatic PAC production. To obtain high activity yeast PDC, Candida utilis was cultivated in a controlled bioreactor. Optimal conditions for PDC production were identified as: fermentative cell growth at initial pH at 6.0 followed by pH downshift to 3.0. Average specific PDC carboligase activity of 392 ?? 20 U/g DCW was achieved representing a 2.7-fold increase when compared to a constant pH process. A mechanism was proposed in which the cells adapted to the pH decrease by increasing PDC activity to convert the accumulated internal pyruvic acid via acetaldehyde to ethanol thereby reducing intracellular acidification. The effect of pH shift on specific PDC activity of Saccharomyces cerevisiae achieved a comparable increase of specific PDC carboligase activity to 335 U/g DCW. The effect of pyruvic acid at pH 3.0 on induction of PDC activity was confirmed by cultivation at pH 3 with added pyruvic acid. Using microarray techniques, genome-wide transcriptional analyses of the effect of pH shift on S. cerevisiae revealed a transient increased expression of PDC1 after pH shift, which corresponded to the increase in specific PDC activity (although the latter was sustained for a longer period). The results showed significant gene responses to the pH shift with approximately 39 % of the yeast genome involved. The induced transcriptional responses to the pH shift were distinctive and showed only limited resemblance to gene responses reported for other environmental stress conditions, namely increased temperature, oxidative conditions, reduced pH (succinic acid), alkaline pH and increased osmolarity.

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<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>

The enzymatic production of R-phenylacetylcarbinol (R-PAC), with either whole cells or partially purified pyruvate decarboxylase (PDC) as the biocatalyst, requires high PDC activity and an inexpensive source of pyruvate for an economical feasible biotransformation process. Microbial pyruvate produced by a vitamin auxotrophic strain of Candida glabrata was selected as a potential substrate for biotransformation. With an optimal thiamine concentration of 60 ??g/l, a pyruvic acid concentration of 43 g/l and yield of 0.42 g/g glucose consumed were obtained. Using microbially-produced unpurified pyruvate resulted in similar PAC concentrations to those with commercial pure substrate confirming its potential for enzymatic PAC production. To obtain high activity yeast PDC, Candida utilis was cultivated in a controlled bioreactor. Optimal conditions for PDC production were identified as: fermentative cell growth at initial pH at 6.0 followed by pH downshift to 3.0. Average specific PDC carboligase activity of 392 ?? 20 U/g DCW was achieved representing a 2.7-fold increase when compared to a constant pH process. A mechanism was proposed in which the cells adapted to the pH decrease by increasing PDC activity to convert the accumulated internal pyruvic acid via acetaldehyde to ethanol thereby reducing intracellular acidification. The effect of pH shift on specific PDC activity of Saccharomyces cerevisiae achieved a comparable increase of specific PDC carboligase activity to 335 U/g DCW. The effect of pyruvic acid at pH 3.0 on induction of PDC activity was confirmed by cultivation at pH 3 with added pyruvic acid. Using microarray techniques, genome-wide transcriptional analyses of the effect of pH shift on S. cerevisiae revealed a transient increased expression of PDC1 after pH shift, which corresponded to the increase in specific PDC activity (although the latter was sustained for a longer period). The results showed significant gene responses to the pH shift with approximately 39 % of the yeast genome involved. The induced transcriptional responses to the pH shift were distinctive and showed only limited resemblance to gene responses reported for other environmental stress conditions, namely increased temperature, oxidative conditions, reduced pH (succinic acid), alkaline pH and increased osmolarity.

(R)-phenylacetylcarbinol or R-PAC is a chiral precursor for the synthesis of pharmaceuticals ephedrine and pseudoephedrine. PAC is produced through biotransformation of pyruvate and benzaldehyde catalyzed by pyruvate decarboxylase (PDC) enzyme. The present research project aims at characterizing a two-phase aqueous/organic process for enzymatic PAC production. In a comparative study of several selected yeast PDCs, the highest PAC formation was achieved in systems with relatively high benzaldehyde concentrations when using C. utilis PDC. C. tropicalis PDC was associated with the lowest by-product acetoin formation although it also produced lower PAC concentrations. C. utilis PDC was therefore selected as the biocatalyst for the development of the two-phase PAC production. From an enzyme stability study it was established that PDC deactivation rates in the twophase aqueous/octanol-benzaldehyde system were affected by: (1) soluble octanol and benzaldehyde in the aqueous phase, (2) agitation rate, (3) aqueous/organic interfacial area, and (4) initial enzyme concentration. PDC deactivation was less severe in the slowly stirred phase-separated system (low interfacial area) compared to the rapidly stirred emulsion system (high interfacial area), however the latter system was presumably associated with a faster rate of organic-aqueous benzaldehyde transfer. To find a balance between maintaining enzyme stability while enhancing PAC productivity, a two-phase system was designed to reduce the interfacial contact by decreasing the organic to aqueous phase volume ratio. Lowering the ratio from 1:1 to 0.43:1 resulted in increased overall PAC production at 4??C and 20??C (2.5 M MOPS, partially purified PDC) with a higher concentration at the higher temperature. The PAC was highly concentrated in the organic phase with 212 g/L at 0.43:1 in comparison to 111 g/L at 1:1 ratio at 20??C. The potential of further two-phase process simplification was evaluated by reducing the expensive MOPS concentration to 20 mM (pH controlled at 7.0) and employment of whole cell PDC. It was found that 20??C was the optimum temperature for PAC production in such a system, however under these conditions lowering the phase ratio resulted in decreased overall PAC production. Two-phase PAC production was relatively low in 20 mM MOPS compared to biotransformations in 2.5 M MOPS. Addition of 2.5 M dipropylene glycol (DPG) into the aqueous phase with 20 mM MOPS at 0.25:1 ratio and 20??C improved the production with organic phase containing 95 g/L PAC. Although the productivity was lower, the system may have the benefit of a reduction in production cost.