This thesis deals with studies on the hydrophobicity of proteins and enzymes and is divided in four chapters summarized separately below. (1) Traditional methods of coagulating milk for the manufacture of cheese suffer from two major drawbacks, namely, the high cost of the enzyme and the fact that they are batch systems. An obvious solution to both problems would be the use of immobilized enzymes to coagulate milk. Hydrophobic adsorption offers certain potential advantages over other techniques of enzyme immobilization. The objective of this part of the thesis was to immobilize the milk clotting enzymes chymosin and pepsin on various hydrophobic carriers and to assess their suitability for continuous coagulation of skimmilk. All enzyme-carrier preparations exhibited high initial activity on exposure to milk. However, the deactivation rates were very high. The main reason for this rapid deactivation appeared to be the loss of enzyme from the carriers, since soluble activity was detected in all enzyme preparations. The enzyme loss was due to the physical desorption of enzyme from the carriers as well as to the relatively rapid leakage of the ligand from the carrier (phenoxyacetyl cellulose). The best enzyme preparation was obtained with phenoxyacetyl cellulose. However, a study indicated that the continuous coagulation of skimmilk with proteases immobilized on the hydrophobic supports used in this study was not economically feasible. (2) The fat binding capacity (FBC) of food proteins is an essential functional property. However, fat binding as determined by existing methods has been mainly attributed to physical entrapment of the oil rather than to the binding with proteins. A simple turbidimetric method, thus, was developed for determining the FBC of various proteins. The turbidity was dependent on wavelength, blending time and volume of oil. The regression equation for predicting FBC was: FBC (%) = 30.271 + 1.381 S[sub=o] - 0.014 S[sub=o] x s where S[sub=o] and s are surface hydrophobicity and solubility index, respectively. A highly significant correlation (R² = 0.802, P < 0.01) was found between S[sub=o], S[sub=o] x s, and FBC of 11 food proteins tested. Advantages of the method developed include the small amount of sample required and the fact that the measured values would reflect the true fat binding capacity of proteins by minimizing the fat-entrapping effects. (3) The objectives of this part of the thesis were to determine the effects of heating on the emulsifying properties of selected food proteins, and, to assess the value of Sq as a predictor of these properties. The results obtained indicated that the emulsifying properties of the proteins studied were differently affected by heating, and that heat-denaturation was not always accompanied by loss of functionality, but, on the contrary, resulted in great improvement. The emulsifying properties could well be predicted solely on the basis of S[sub=o] level but not on the basis of solubility level, which indicated that S[sub=o] is a very important property determining protein functionality. However, the emulsion activity, emulsion stability and fat binding of the proteins studied could be well explained and more accurately predicted by S[sub=o] and solubility together. (4) The objectives of this part were to evaluate the thermal properties (thickening, coagulation and gelation) of selected food proteins and to assess the value of hydrophobicity as their predictor. The results obtained indicated that the average (S) and not the surface hydrophobicity was important for these properties. The thermal properties studied could not be explained by either the average hydrophobicity or sulfhydryls alone. Instead, they could well be predicted using average hydrophobicity and sulfhydryls together. / Land and Food Systems, Faculty of / Graduate

Bibliography: pages 161-176. / Vibrio alginolyticus is a gram-negative aerobic bacterium that produces several extracellular serine proteases and a collagenase during the stationary growth phase. The aim of this study was to investigate alkaline serine protease production by this organism, and to attempt the cloning and expression of a V.alginolyticus protease gene in Escherichia coli.

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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>