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The use of stereoselective aminohydrolases (acylases) in kinetic resolutions is a commonly employed industrial route to both L- and D- α-amino acids from Nacetylated- DL-starting materials. However, a flaw in this process is the need for repeated racemisation steps of the non-desired enantiomer to achieve yields >50%. A solution to this drawback would be a dynamic kinetic resolution driven by an in situ racemisation step that would allow the yield to approach 100% A cheap and “green” catalyst for this racemisation would be an enzyme such as N-acyl amino acid racemase (NAAAR) from the actinobacteria Amycolatopsis sp. Ts-1-60. This enzyme requires no organic cofactor, is stable at high temperatures (~60°C) and importantly, shows no racemase activity toward free amino acids. Unfortunately, the activity of NAAAR with N-acetyl substrates is low and reported to suffer from inhibition above substrate concentrations of 50 mM, prohibiting its use in NAAAR/acylase coupled dynamic kinetic resolutions under industrial conditions. In an attempt to remove these limitations, directed evolution has been applied to the Amycolatopsis Ts-1-60 NAAAR to engineer a variant enzyme with increased activity towards N-acetyl substrates. An improved variant NAAAR may allow for a commercial NAAAR/acylase coupled dynamic resolution process. Directed evolution has proven to be a highly versatile and successful tool for protein engineering. However, the ability to screen for improved variants is often technically difficult or time consuming and in the case of NAAAR this is especially true as the substrate and product are simply enantiomers of an N-acetyl amino acid. To overcome this, an enantioselective genetic selection system has been employed to allow the screening of mutagenic NAAAR libraries. After several rounds of selection, a NAAAR variant (NAAAR G291D F323Y) has been isolated with increased racemase activity towards a range of synthetically useful N-acetyl substrates. This enzyme has been over-expressed, purified and its characteristics compared to the wild-type and other variants discovered during the evolution process. NAAAR G291D F323Y has been crystallised to 2.71 Å allowing a molecular basis for the increase in catalytic activity to be proposed. Coupling of this enzyme with a stereoselective acylase has been used to produce enantiopure amino acids in >99% yield, twice the inherent 50% maximum yield of acylase based resolutions. Early results suggest this NAAAR variant has the potential to be employed on a multi kilogram scale for the economical production of enantiopure L- and D- α-amino acids.

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Two novel lectins were purified from the <I>Moraceae</I> plant <I>Morus nigra</I> or Black Mulberry. These lectins were named <I>Morus nigra</I> agglutinin I (MNA I) and <I>Morus nigra</I> agglutinin II (MNA II). Data obtained from carbohydrate inhibition studies indicated that MNA I specifically bound N-acetylgalactosamine and D-galactose, and in contrast, MNA II selectivity bound <I>α</I>-methyl-D-mannoside and D-mannose. MNA I is composed of two glycosylated α-chains and two <I>β</I>-chains, both of which share a high degree of amino acid sequence identity with the N-terminal regions of the <I>α</I>- and <I>β</I>-chains of the <I>Moraceae</I> lectins, Jacalin and <I>Macluar pomifera</I> agglutinin (MPA). MNA II consists of three dominant subunits, each of which has the same N-terminal amino acid sequence. This sequence shares a degree of N-terminal amino acid sequence identity with the <I>Moraceae</I> D-mannose binding lectin KM+ and the <I>β</I>-chains of Jacalin, MPA and MNA I. Glycosylation of the MNA II subunits was not detected. The binding characteristics of <I>Moraceae</I> lectins to the intestinal brush border were assessed using both <I>in vitro</I> and <I>in vivo</I> approaches. Initially <I>in vitro</I> studies using Brush Border Vesicle preparations were conducted. Thereafter <I>in situ</I> experiments using rat intestinal ligated loops were carried out. The <I>in vitro</I> and <I>in situ</I> binding studies showed that MNA I had a low binding capacity for the rat intestinal brush border, however Western blot studies illustrated that MNA I selectively bound to two rat intestinal brush boarder proteins. Initial <I>in vitro</I> data indicated that MNA II also had a low binding capacity for the rat intestinal brush border. However, additional <I>in vitro</I> and <I>in situ</I> studies showed MNA II bound extensively to the jejunal villi. Furthermore, considerable MNA II uptake into the jejunal enterocytes and lamina propria was detected.

Amino acids are the basic building blocks of proteins. The determinationof their structures plays an important role in correctly describing the functionsof the proteins. This thesis is devoted to theoretical studies on the potentialenergy surface of amino acids, in particular the infrared and soft X-ray spectralfingerprints of their most stable conformers.The stable structures of amino acids can be explored by different methods.We have used a full space systematic search strategy to determine the potentialenergy surface of deprotonated arginine and revealed several new conformers.With that, the calculated thermodynamic parameters are finally in good agreementwith their experimental counterparts. We have also proposed a molecularfragment based step-by-step strategy to search for the most stable conformers oflarge biomolecules. The high efficiency and good accuracy of this strategy havebeen firmly illustrated by the modeling of several polypeptides.Infrared (IR) spectroscopy has become one of the most applied techniques tocharacterize the structures of gas-phase amino acids. A direct comparison betweenexperimental and calculated infrared spectra provides an efficient way to describethe conformation exchanges of the amino acids. It is found that the conformersof an amino acid are not always necessary to reach the thermal equilibrium undercertain experimental conditions. The local minima could be responsible for theappearance of the measured spectra. This important point has been highlightedby the calculations of deprotonated tyrosine and cysteine, as well as the arginine.The near-edge X-ray absorption fine structure (NEXAFS) spectra and X-rayphotoelectron spectra (XPS) have also been simulated for neutral, deprotonatedand protonated arginine. The influences of intra-, and intermolecular hydrogenbonds on the electronic structure of the arginine have been carefully examined. Itis suggested that the XPS is capable of distinguishing the canonical and zwitterinicisomers of arginine, and works much better than any other tools available. / <p>QC 20140522</p>