Nitrile biocatalysts are of use in the chemical and pharmaceutical industries for the synthesis of carboxyamides and carboxylic acids. In particular, the application of biocatalysts in the synthesis of single enantiomer compounds is of increasing interest, but requires novel substrate specific highly stereoselective biocatalysts. Addition to the limited toolbox of known nitrile biocatalysts requires definitive characterisation of the biocatalysts through accurate determination of the substrate profiles and quantification of activity. The accurate quantification of stereoisomers chiral mixtures to determine biocatalyst stereoselectivity remains a significant challenge due to the difficulty in separating stereoisomers by physical methods. The known nitrile metabolising organism, Rhodococcus rhodochrous ATCC BAA-870, was grown in a defined medium and harvested, providing whole cell biocatalyst. Additional biomass was disrupted to provide a cell free enzyme extract, which was put through an enzyme purification protocol to provide a solution with specific activity of 351 U.mg⁻¹. A portion of the enzyme was self immobilised using the SphereZyme™ technique. The nitrile hydratase SphereZymes™ (1.2 U.mg⁻¹ initial activity) that were prepared had pH and temperature optima of 6 and 30°C respectively, and could be recovered by repeated washing. The particles retained activity in the presence of the organic solvents isooctane and n-hexadecane saturated with 50 mM phosphate buffer (pH 7.5). An initial analytical system was devised for quantification of the nitrile hydratase activity using the non-chiral substrate benzonitrile. An improved reversed phase high performance liquid chromatography method was developed to separate and quantify benzamide, benzoic acid and benzonitrile. The mobile phase consisting of 0.1% trifluoroacetic acid in H₂O and acetonitrile (70:30, %v/v), at a flow rate of 0.5 ml.ml⁻¹, 25°C, resolved all three analytes in 3.5 minutes on a Waters X-Terra MS C18 3.5μm column. UV detection was carried out at 210 nm. Analytical methods to determine activity and enantioselectivity of the whole cell biocatalyst were subsequently developed for both β-amino nitriles and β-hydroxy nitrile substrates and hydrolysis products.

Submitted in partial fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Durban Institute of Technology, 2000 / M

Melting of elemental solids can be identified and appreciated as a particularly simple example of discontinuous phase transitions involving condensed phases. Motivated, on the one hand, by the need to improve the microstructural quality of laser-crystallized columnar-grained polycrystalline Si films for manufacturing advanced AMOLED displays and, on the other hand, to investigate the fundamental details associated with phase transformations transpiring in condensed systems, this thesis examines the initiation and evolution of melting in polycrystalline thin films. Distilling the essence of the classical nucleation theory and extending its description to address more general cases of phase initiation and evolution, a general thermodynamic method based on capillarity effect is developed and applied to determine the shape of solid/liquid interfaces that are in mechanical equilibrium. We first explicitly identify and build our analysis based on how the shape of solid/liquid interfaces must comply with the contact angle conditions at the junctions and also the property of constant mean curvature. Bi-crystal and tri-crystal models are presented to capture the microstructural features such as junctions and vertices of interfaces in polycrystalline thin films. At each of the potential melt initiating sites, the parameter space of contact angles is divided into domains depending on the shape of the solid/liquid interface that can be established in mechanical equilibrium. Melting initiation mechanisms are subsequently determined based on the permissible shape for each domain. This analysis is further extended to the edges and corners of embedded cubic crystals (with nonidentical contact angles at different faces). Secondly, in order to facilitate the thermodynamic analysis of the melting initiation and interface propagation, we extend our curvature-evolution-centric method to identify and develop what we consider as the central function for discontinuous phase transitions. Specifically, starting with a local governing condition, identifies and builds on two curvatures: ρ^E (𝑉) and ρ* (𝑇). ρ^E (𝑉) captures the evolution of the mean curvature of the solid/liquid interface as a function of liquid volume for the case in which the mechanical equilibrium condition is satisfied, whereas ρ* (𝑇) incorporates the temperature effect on the difference between the volumetric free energy of solid and liquid phases using the corresponding equilibrium mean curvature. We define and identify the interface driving stress function ƒ(𝑉,𝑇)=∂𝐺/∂𝑉=σ(ρ^E (𝑉)-ρ* (𝑇)) of the phase transition as being an important fundamental quantity, which can be directly derived by taking the difference of the two curvature terms. In contrast to the conventional analysis that requires integration of volumetric and interfacial free energy terms over various geometric domains to derive the total free energy as a function of volume for a given temperature, this formation completely disentangles geometry from the thermodynamic aspects of the phase transition and allows them to be treated separately. In addition to providing essentially all relevant thermodynamic information about the phase initiation and evolution, the above method readily permits the use of powerful general-purpose numerical tools to calculate the potentially complex geometry of the solid/liquid and other interfaces and obtain ρ^E (𝑉) directly as the output. Plotting the ρ^E (𝑉) function together with the temperature-dependent iso-curvature line, ρ* (𝑇), unveils the critical thermodynamic information regarding the melting transition at the temperature, such as whether equilibrium points exist, the number of equilibrium points, their stability, and their corresponding volumes. The change of free energy as a function of liquid volume can be derived through integration of the interface driving stress function. The velocity of the solid/liquid interface is simply proportional to the interface driving stress function. The application of this method is demonstrated in both shape-preserving (which we term as isomorphic) and shape-changing (which we term as non-isomorphic) examples. The analysis and findings presented in this thesis are relevant and useful for understanding discontinuous phase transitions, in general, and can be particularly so for small, confined, and embedded systems that are increasingly being utilized in modern technologies.

Despite decades of research robotic manipulation systems outside of highly-structured industrial applications are still far from ubiquitous. Perhaps particularly curious is the fact that there appears to be a large divide between the theoretical grasp modeling literature and the practical manipulation community. Specifically, it appears that the most successful approaches to tasks such as pick-and-place or grasping in clutter are those that have opted for simple grippers or even suction systems instead of dexterous multi-fingered platforms. We argue that the reason for the success of these simple manipulation systemsis what we call passive stability: passive phenomena due to nonbackdrivable joints or underactuation allow for robust grasping without complex sensor feedback or controller design. While these effects are being leveraged to great effect, it appears the practical manipulation community lacks the tools to analyze them. In fact, we argue that the traditional grasp modeling theory assumes a complexity that most robotic hands do not possess and is therefore of limited applicability to the robotic hands commonly used today. We discuss these limitations of the existing grasp modeling literature and setout to develop our own tools for the analysis of passive effects in robotic grasping. We show that problems of this kind are difficult to solve due to the non-convexity of the Maximum Dissipation Principle (MDP), which is part of the Coulomb friction law. We show that for planar grasps the MDP can be decomposed into a number of piecewise convex problems, which can be solved for efficiently. Despite decades of research robotic manipulation systems outside of highlystructured industrial applications are still far from ubiquitous. Perhaps particularly curious is the fact that there appears to be a large divide between the theoretical grasp modeling literature and the practical manipulation community. Specifically, it appears that the most successful approaches to tasks such as pick-and-place or grasping in clutter are those that have opted for simple grippers or even suction systems instead of dexterous multi-fingered platforms. We argue that the reason for the success of these simple manipulation systemsis what we call passive stability: passive phenomena due to nonbackdrivable joints or underactuation allow for robust grasping without complex sensor feedback or controller design. While these effects are being leveraged to great effect, it appears the practical manipulation community lacks the tools to analyze them. In fact, we argue that the traditional grasp modeling theory assumes a complexity that most robotic hands do not possess and is therefore of limited applicability to the robotic hands commonly used today. We discuss these limitations of the existing grasp modeling literature and setout to develop our own tools for the analysis of passive effects in robotic grasping. We show that problems of this kind are difficult to solve due to the non-convexity of the Maximum Dissipation Principle (MDP), which is part of the Coulomb friction law. We show that for planar grasps the MDP can be decomposed into a number of piecewise convex problems, which can be solved for efficiently. We show that the number of these piecewise convex problems is quadratic in the number of contacts and develop a polynomial time algorithm for their enumeration. Thus, we present the first polynomial runtime algorithm for the determination of passive stability of planar grasps. For the spacial case we present the first grasp model that captures passive effects due to nonbackdrivable actuators and underactuation. Formulating the grasp model as a Mixed Integer Program we illustrate that a consequence of omitting the maximum dissipation principle from this formulation is the introduction of solutions that violate energy conservation laws and are thus unphysical. We propose a physically motivated iterative scheme to mitigate this effect and thus provide the first algorithm that allows for the determination of passive stability for spacial grasps with both fully actuated and underactuated robotic hands. We verify the accuracy of our predictions with experimental data and illustrate practical applications of our algorithm. We build upon this work and describe a convex relaxation of the Coulombfriction law and a successive hierarchical tightening approach that allows us to find solutions to the exact problem including the maximum dissipation principle. It is the first grasp stability method that allows for the efficient solution of the passive stability problem to arbitrary accuracy. The generality of our grasp model allows us to solve a wide variety of problems such as the computation of optimal actuator commands. This makes our framework a valuable tool for practical manipulation applications. Our work is relevant beyond robotic manipulation as it applies to the stability of any assembly of rigid bodies with frictional contacts, unilateral constraints and externally applied wrenches. Finally, we argue that with the advent of data-driven methods as well as theemergence of a new generation of highly sensorized hands there are opportunities for the application of the traditional grasp modeling theory to fields such as robotic in-hand manipulation through model-free reinforcement learning. We present a method that applies traditional grasp models to maintain quasi-static stability throughout a nominally model-free reinforcement learning task. We suggest that such methods can potentially reduce the sample complexity of reinforcement learning for in-hand manipulation.We show that the number of these piecewise convex problems is quadratic in the number of contacts and develop a polynomial time algorithm for their enumeration. Thus, we present the first polynomial runtime algorithm for the determination of passive stability of planar grasps.

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