Experiments facilitating the engineering of the PETNR active site to accommodate a range of non natural enone substrates with substituents localised on the α and β carbons of the unsaturated bond are described. In order to facilitate the high throughput purification of PETNR libraries poly histidine (PETNRHis) and biotin (PETNRBio) tagged PETNR variants were generated. High throughput protocols were developed for the automated generation, purification and screening of libraries in a 96 well format. Protocols were optimised and trialled using blocks consisting of PETNRHis WT only and characterised in terms of intra block variation. A range of single site saturation mutagenic libraries were generated at positions in the active site consisting of T26, Y68, W102, H181, H184, Y186, Q241 and Y351. Sequencing results indicated randomised libraries with the occasional instance of bias evident. Expression and purification in a 96 well format was monitored by SDS PAGE and protein quantitation. Library activity was quantified and demonstrated to retain varying degrees of activity with the model substrate 2-cyclohexenone. Following this verification of the experimental protocol libraries were screened against a range of substrates analogous to substrates demonstrated to be active with PETNRWT but incorporating substituents at the α and β carbons. 'Hits' generated from these screening reactions were studied further by the determination of the specific activity and quantitation of substrate/product from biotransformation reactions. From these screening experiments totalling 3,600 individual reactions, 35 were identified as potential hits, of these 8 proved to be genuinely improved variants. Substituents at the β carbon were demonstrated to compromise the activity of the WT enzyme most severely. Positions 68, 102, and 351 were demonstrated to play an important role in the accommodation of substituents at the α carbon whilst residues 26 and 351 are important for the β carbon. The best variants demonstrated up to 9 fold improvements in poor substrates which represented rates in excess of those observed for model substrates.

The Old Yellow Enzyme (OYE) family of enzymes has been shown to reduce industrially important chemicals and has been used to study quantum tunnelling during enzymatic hydrogen transfer. Though extensively studied, only mesophilic homologues have been studied within the enzyme family. This thesis discusses the characterisation of Thermophilic Old Yellow Enzyme (TOYE), from Thermoanaerobacter pseudethanolicus, and provides the first published crystal structure of a thermophilic OYE-family member. In addition to increased thermostability compared to mesophilic homologues, thermophilic enzymes are important for use in industrial as often they are more stable towards organic solvents used in industry than their mesophilic homologues while catalysing the same reactions. This makes thermophiles and hyperthermophiles interesting targets for investigating the importance of enzyme dynamics during catalysis. They have also been used to study the linking of protein motion to quantum tunnelling during hydrogen transfer in other enzyme systems. In the work for this thesis, the basic characteristics of TOYE were examined. Thermal stability up to 70 °C was shown by CD and fluorescence studies and the preference towards reductive coenzyme was analysed by stopped-flow studies. Structural studies were conducted using X-ray crystallography, electron microscopy and sedimentation velocity studies. The crystal structure revealed a tetrameric enzyme with a relatively large active site. Evidence for higher oligomeric states was also obtained. The potential use of TOYE as a biocatalyst was explored by steady-state reaction, biotransformation and organic solvent resistance studies. The temperature dependences of kinetic isotope effects were used to examine the presence of tunnelling and importance active site geometry during catalysis and compared to previously described enzymes. These studies introduce a new and unique OYE-family member, allowing for more in-depth analysis of TOYE.

The research carried out within this thesis was aimed at the development and implementation of a biocatalytic route towards Ticagrelor, a platelet-aggregation inhibitor. A bio-retrosynthetic consideration of the target compound yielded different possible strategies, which were analysed in terms of enantioselectivity and efficiency. The ultimate goal was to generate a biocatalyst specifically tailored to the starting material to yield the target compound in high optical purity and conversion. Different approaches to the chemoenzymatic generation of the cyclopropyl subunit (cf figure) in enantiomerically pure form were proposed and tested. The lipase from Thermomyces lanuginosus proved to be the most selective and active enzyme tested and was used as a model enzyme, initially yielding an E of 76 at a conversion of 50% after 48h. Through both reaction engineering and rational protein design approaches the time to attain 50% conversion could be reduced to 24 h while the enantioselectivity of the process increased to 100. Moreover, in a rational protein design approach different residues in the lid of the lipase were identified through analysis of the resolved crystal structures and subsequently mutated in order to investigate the influence of these residues on the overall performance of the lipase towards the target biotransformation. Mutations on Asn88 resulted in the inactivation of the enzyme while an Asp57Asn mutation resulted in a more active enzyme. Ultimately, this research has contributed to making the synthetic route towards Ticagrelor more environmentally sustainable, diminishing the need for the use of toxic, unsustainable and sterically demanding auxiliaries, as well as the amount of waste produced. The principles of green chemistry have been applied to the case studied. The synthetic route towards a key fragment of Ticagrelor has been significantly shortened using a biotransformation with an enzyme that can be recycled and employed in catalytic quantities.

The development of cost-effective and sustainable catalytic methods for the production of enantiomerically pure chiral amines is a key challenge facing the pharmaceutical and fine chemical industries. There is an increasing demand for broadly applicable synthetic methods which deliver the desired amine product in high yield and enantiomeric excess (e.e.). Previously we have described the development of variants of monoamine oxidase from Aspergillus niger (MAO-N) which are able to mediate the complete conversion of racemic amines to the corresponding enantiomerically pure products in a single step. In this thesis we report a panel of MAO-N variants (D5, D9 and D11) developed in our laboratory, which are able to mediate the deracemisation of primary, secondary and tertiary amines with broad structural features. In particular, we have synthesized and subjected to deracemisation a broad range of tetrahydroisoquinolines and tetrahydro-β-carbolines checking enantioselectivity and enantiopreference of our biocatalysts. A relation between lipophilicity of the substituents and enantiopreference of the enzyme has been identified. We have also engineered a new MAO-N variant (D11) with a greatly increased substrate scope and enhanced tolerance for bulky substrates. Application of this engineered biocatalyst is highlighted by the asymmetric synthesis of the generic drugs Solifenacin and Levocetirizine as well as a number of important classes of biologically active alkaloid natural products. We also report a novel MAO-N mediated asymmetric oxidative Pictet-Spengler approach to the synthesis of (R)-harmicine.Another challenge facing the chemist in the new millennium is the development of cleaner and more efficient chemical processes. To this aim the combination of two or more catalytic systems to complete a series of cascade reactions is considered particularly appealing. We have reported a concurrent redox cascade for the deracemisation of pyrrolidines and tetrahydroisoquinolines using our monoamine oxidase-N with a biotinylated Ir-complex within streptavidin (SAV). To achieve the final goal it is necessary to shield the metal inside a host to avoid the mutual inactivation of the two catalysts. We have also described the combination of MAO-N with berberine bridge enzyme (BBE) for the synthesis of berbines (tetrahydroprotoberberines), which represent a sub-class of tetrahydro-isoquinoline alkaloids found in various plants. This bi-enzymatic cascade allows the synthesis of these structures achieving a theoretical 100% yield instead of the 50% given by the kinetic resolution using BBE itself.

As the demand for cost-efficient and environmentally friendly processes increases in the chemical industry, impact of biocatalysis, which is the utilization of enzymes and whole microorganisms for production of fine chemicals, has become more predominant. From pharmaceuticals to cosmetics, biocatalysts are widely used in various sectors, and their significance have dramatically intensified with the introduction of initial protein engineering techniques in 1980s. As the field of protein engineering has evolved over the last few decades, its integration with other disciplines such as process engineering and synthetic biology is now more critical for establishing non-natural pathways and reactions to produce broader range of chemicals. While developing an interdisciplinary approach, few strategies have emerged to be more prevalent: (i) better integration of biocatalysts with (nano)devices, and (ii) use of protein based scaffolds for creating novel synthetic multienzyme cascades. Throughout this doctoral thesis, applicability of these ideas with oxidoreductases was investigated. Oxidoreductases are a class of under-utilized enzymes that catalyze the electron transfer between different metabolites, while at the same time use cofactors (NAD(P)(H), molecular oxygen, etc.) as the electron supplier. In Chapter 2, the electron transfer mechanism of a monooxygenase, cytochrome P450 27B1 (CYP27B1), was mimicked for electrochemical sensing of a vitamin D form (25(OH)D) in solution. Natural electron transfer pathway of this enzyme uses NADPH and two electron transfer proteins for conversion of 25(OH)D to its product. Inspired by this mechanism, this enzyme was mixed with an artificial redox mediator and immobilized on an electrode surface. As a result of rigorous experiments, CYP27B1-modified electrode was found to detect 25(OH)D in its physiological range. This is a significant result as it opens a new way for development of a vitamin D biosensor that can diminish the amount of required cost and time for testing. In the next chapter of the thesis, effects of changing the size of cofactor on catalysis of dehydrogenases were studied in detail. Natural cofactors of two different redox enzymes were chemically modified with PEG, and kinetic experiments were conducted in order to better understand the relation between transport phenomena and biocatalysis. It was found that when the size of the cofactor was increased, two enzymes were affected differently; while efficiency of one enzyme was not altered significantly, that of the other dropped dramatically. Through comprehensive analysis, dominant impact of PEGylation was determined to be due to the differences in the interactions of PEGylated cofactors and enzymes. This study showed that protein engineering methods can be utilized to gain insights into better understanding of the relationship between mass transfer and catalysis in engineered bioprocesses and biocatalytic cascades. In Chapter 4, PEGylated cofactors were used to create artificial multienzyme complexes. In this study, SpyCatcher-SpyTag scaffold was utilized for wiring two redox enzymes and by tethering with PEGylated cofactors, a new biocatalyst with self-contained redox chemistry was obtained. Detailed kinetic analysis showed that this new multienzyme cascade was able to catalyze a reaction that was thermodynamically downhill but kinetically very slow in the absence of any enzyme. This also proved that attached cofactor acts as a ‘swing-arm’, carrying electrons from one enzyme to another; similar to the unique mechanism of pyruvate dehydrogenase complex. Generality of this methodology was investigated by constructing an immobilized three-enzyme-containing biocatalyst, which was hypothesized to catalyze an industrially important reaction under very mild conditions. This work is a significant contribution to the field, and a good demonstration of use of protein engineering for process engineering applications. Chapter 5 concludes this thesis with a study that investigates the practicability of a collagen mimetic peptide as a novel way of constructing multiprotein cascades. Collagen mimetic peptides are composed of three individual strands that might (homotrimer) or might not (heterotrimer) have identical sequences, and in this work, we have utilized a recently designed hydroxyproline-free sequences of a heterotrimer collagen mimetic peptide. Individual strands were attached to different proteins by genetic fusion, and optimum experimental conditions for self-assembly of a multiprotein complex were investigated. Initial results suggested formation of such a complex, but further experiments are required to finalize the confirmation. This new collagen-based platform studied in this chapter is a crucial step towards development of cofactorless multienzyme cascades. Finally, this doctoral thesis demonstrates the prominence of protein engineering in biocatalysis applications by utilizing various strategies together with the electron transfer mechanisms of oxidoreductases. By expanding and building upon these methodologies, it is possible to obtain more improved biosensors and functional artificial multienzyme cascades with industrial applications. Hence, this study is a promising example to exhibit the impact of interdisciplinary approach on industrial biotechnology.

Finding alternatives to petroleum-based energy sources is of interest since it could reduce the emissions of net carbon dioxide to the atmosphere by increasing the usage of renewable energy sources. To do so improvements are needed in the renewable energy production sector. Biogas production is of interest since the anaerobic digestion process can degrade many different biomolecules and is, contrary to e.g. bioethanol and biodiesel, not dependent on specific molecules. Thus, many wastes such as slaughterhouse waste, sludge from waste water treatment and lignocellulose residual material etc. can be used as substrates for biogas production. However, there are limitations in the degradation process depending on the composition of the selected substrate. To overcome these limitations such as inhibition of different microorganisms, or recalcitrant substrate, different methods can be used to increase the biogas production.  In this study different substrates were selected and analyzed/treated for remedies of early stage rate limiting problems of the anaerobic digestion process. Different analyzes and techniques were selected depending on the limitations correlated to the main problematic component of the specific substrate.  Improvements could be reached for the degradation of slaughterhouse waste by augmentation with the clay mineral zeolite. Addition of different enzymes to the substrate environment of different waste water treatment plant sludges resulted in limited life time of the selected enzymes. However, certain enzymes proved to be promising candidates with an effect of increased biogas production rate and yield for the time that the enzyme remained active. In an additional experiment, cellulolytic enzymes, naturally produced by a biogas producing microbial community, were induced, collected and added to a biogas experiment of ensiled forage ley, by which it was shown that these cellulases led to an increase in biogas production rate and yield. Thus, the studies demonstrate different techniques for improving the anaerobic digestion process of different types of substrates. / <p>Handledare saknas</p>

Enantiopure epoxides are versatile compounds in the production of single enantiomer drugs, and are of high value as building blocks and intermediates in the preparation of more complex single enantiomer pharmaceuticals and agrochemicals. Epoxide hydrolases, ubiquitous enzymes in nature, can be versatile tools in the biocatalytic production of these single enantiomer epoxides due to their capability of selectively hydrolysing one enantiomer of a wide range of these compounds, and thus rendering an enantiopure epoxide and diol. The value of epoxide hydrolases for the kinetic resolution of epoxide compounds are dependant on factors such as availability, ease of production, long term stability, activity and the displayed enantioselectivity. The first objective of this study was to investigate and optimise the growth media and time for the production of two red yeasts, Rhodotorula glutinis and Rhodospondium toruloides, and their epoxide hydrolysing enzymes. Maximum and minimum epoxide hydrolase (EH) activity for R. glutinis was respectively observed with the YMvit (0,26 mM.min"1) and malt (0,17 mM.min"1) media, while peak biomass production was observed from the YM medium (64,9 mg.mL"1). For R. toruloides, the highest biomass was produced in the YM (130,8 mg.mL"1) medium, with similar epoxide hydrolase activities (average c = 0,75 ± 0,01) displayed for the YM, YMvit and malt grown biocatalysts. With varying the YM medium glucose concentration (0,5 - 2,0 %) the most biomass was produced for R. glutinis with the addition of 1,5 % glucose (60,0 + 0,9 mg.mL"1), with a slight drop in the biomass observed with the addition of 2,0% glucose (56,0 + 1,7 mg.mL"1). No significant differences in epoxide hydrolase activity was observed for the lower glucose additive concentrations (0,5 - 1,5 %), while 2,0 % (m/v) rendered a biocatalyst with almost 20 % higher activity (0,29 mM.min"1). For R. toruloides an increase in the glucose concentration lead to a significantly higher biomass production while the time needed to attain the stationary phase increased progressively from 40 to 96 hours. Almost equal activity was observed for the top three glucose concentrations (average c = 0,82 ± 0,01) at 36 hours growth time, but in all cases a decrease in the EH activity was observed during the stationary phase, with the most pronounced decrease for the 2,0 % (m/v) glucose concentration, that showed a drop in conversion of almost 62 % at 144 hours growth time. The second objective was to synthesise meta and para nitro-, methyl- and methoxystyrene oxides and the successive production of their single enantiopure epoxides through R. glutinis EH mediated kinetic resolution, and the determination of the absolute configuration of the pure residual enantiomers through VCD analysis. R. glutinis selectively hydrolysed the whole range of styrene oxide derivatives, with the highest activity displayed towards the meta substituted derivatives in the order of methyl > methoxy > nitro. m-Methylstyrene oxide reached a % e.e. of >98 within 60 minutes, with an exceptionally high yield of 42,5 %. The absolute configuration of the residual epoxide enantiomers of /n-nitro, m-methyl and m-methoxystyrene oxides were determined to be of the (S)-configuration, indicating that R. glutinis EH preferentially hydrolyses the (R)-epoxides. Thirdly, we attempted to increase the R. glutinis EH activity through the addition of hydroxypropyl-p-cyclodextrin (HPB) and to correlate the rate of chemical and R. glutinis EH mediated enzymatic hydrolysis, and the enzyme's enantioselectivity to the electronic properties of their substituents and the spatial arrangement of the substrates in relation to the EH catalytic triad of the EH active site. An increase in the HPB concentration (0 - 20 % w/v) lead to a substantial increase in both the solubility as well as enzyme activity for p-N02 (para-nitrostyrene oxide) with a significant increase in the solubility of between 2,89 and 6,28 times for the substrate range with the addition of 5 % HPB in comparison to the buffer solution. The acid induced chemical and R. glutinis EH mediated enzymatic reaction rate was correlated to both the Hammett constant as well as the Mulliken charge distributions. The Mulliken charge distribution over the protonated epoxides was correlated to the acid induced chemical hydrolysis rates, while the Mulliken charge distribution over the neutral epoxides could be correlated to the enzymatic reaction rates. An increase in the electron-donating properties of the styrene oxide substituent groups was correlated to an increase in both the chemical as well as the R. glutinis EH mediated hydrolysis reaction rates of the styrene oxide derivatives. Docking of the possible conformers of the (R)- and (S)-enantiomers of these meta and para substituted styrene oxides into the EH binding site of the closely related Aspergillus niger displayed a closer and more preferential fit of the (R)-epoxides which is the faster reacting enantiomerfor both A. niger and R. glutinis EHs. The proven relationship between R. glutinis EH activity and selectivity and the electronic properties of substituent groups, as well as the relationship between spatial arrangement of the epoxide hydrolase binding site and the enantioselectivity of the enzyme, could open up the possibility to correctly predict both the enantioselectivity as well as the activity of R. glutinis EH, and possibly other red yeasts, towards more complex epoxide substrates without the need of time consuming screenings. / Thesis (Ph.D. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2008.

Enantiopure epoxides are versatile compounds in the production of single enantiomer drugs, and are of high value as building blocks and intermediates in the preparation of more complex single enantiomer pharmaceuticals and agrochemicals. Epoxide hydrolases, ubiquitous enzymes in nature, can be versatile tools in the biocatalytic production of these single enantiomer epoxides due to their capability of selectively hydrolysing one enantiomer of a wide range of these compounds, and thus rendering an enantiopure epoxide and diol. The value of epoxide hydrolases for the kinetic resolution of epoxide compounds are dependant on factors such as availability, ease of production, long term stability, activity and the displayed enantioselectivity. The first objective of this study was to investigate and optimise the growth media and time for the production of two red yeasts, Rhodotorula glutinis and Rhodospondium toruloides, and their epoxide hydrolysing enzymes. Maximum and minimum epoxide hydrolase (EH) activity for R. glutinis was respectively observed with the YMvit (0,26 mM.min"1) and malt (0,17 mM.min"1) media, while peak biomass production was observed from the YM medium (64,9 mg.mL"1). For R. toruloides, the highest biomass was produced in the YM (130,8 mg.mL"1) medium, with similar epoxide hydrolase activities (average c = 0,75 ± 0,01) displayed for the YM, YMvit and malt grown biocatalysts. With varying the YM medium glucose concentration (0,5 - 2,0 %) the most biomass was produced for R. glutinis with the addition of 1,5 % glucose (60,0 + 0,9 mg.mL"1), with a slight drop in the biomass observed with the addition of 2,0% glucose (56,0 + 1,7 mg.mL"1). No significant differences in epoxide hydrolase activity was observed for the lower glucose additive concentrations (0,5 - 1,5 %), while 2,0 % (m/v) rendered a biocatalyst with almost 20 % higher activity (0,29 mM.min"1). For R. toruloides an increase in the glucose concentration lead to a significantly higher biomass production while the time needed to attain the stationary phase increased progressively from 40 to 96 hours. Almost equal activity was observed for the top three glucose concentrations (average c = 0,82 ± 0,01) at 36 hours growth time, but in all cases a decrease in the EH activity was observed during the stationary phase, with the most pronounced decrease for the 2,0 % (m/v) glucose concentration, that showed a drop in conversion of almost 62 % at 144 hours growth time. The second objective was to synthesise meta and para nitro-, methyl- and methoxystyrene oxides and the successive production of their single enantiopure epoxides through R. glutinis EH mediated kinetic resolution, and the determination of the absolute configuration of the pure residual enantiomers through VCD analysis. R. glutinis selectively hydrolysed the whole range of styrene oxide derivatives, with the highest activity displayed towards the meta substituted derivatives in the order of methyl > methoxy > nitro. m-Methylstyrene oxide reached a % e.e. of >98 within 60 minutes, with an exceptionally high yield of 42,5 %. The absolute configuration of the residual epoxide enantiomers of /n-nitro, m-methyl and m-methoxystyrene oxides were determined to be of the (S)-configuration, indicating that R. glutinis EH preferentially hydrolyses the (R)-epoxides. Thirdly, we attempted to increase the R. glutinis EH activity through the addition of hydroxypropyl-p-cyclodextrin (HPB) and to correlate the rate of chemical and R. glutinis EH mediated enzymatic hydrolysis, and the enzyme's enantioselectivity to the electronic properties of their substituents and the spatial arrangement of the substrates in relation to the EH catalytic triad of the EH active site. An increase in the HPB concentration (0 - 20 % w/v) lead to a substantial increase in both the solubility as well as enzyme activity for p-N02 (para-nitrostyrene oxide) with a significant increase in the solubility of between 2,89 and 6,28 times for the substrate range with the addition of 5 % HPB in comparison to the buffer solution. The acid induced chemical and R. glutinis EH mediated enzymatic reaction rate was correlated to both the Hammett constant as well as the Mulliken charge distributions. The Mulliken charge distribution over the protonated epoxides was correlated to the acid induced chemical hydrolysis rates, while the Mulliken charge distribution over the neutral epoxides could be correlated to the enzymatic reaction rates. An increase in the electron-donating properties of the styrene oxide substituent groups was correlated to an increase in both the chemical as well as the R. glutinis EH mediated hydrolysis reaction rates of the styrene oxide derivatives. Docking of the possible conformers of the (R)- and (S)-enantiomers of these meta and para substituted styrene oxides into the EH binding site of the closely related Aspergillus niger displayed a closer and more preferential fit of the (R)-epoxides which is the faster reacting enantiomerfor both A. niger and R. glutinis EHs. The proven relationship between R. glutinis EH activity and selectivity and the electronic properties of substituent groups, as well as the relationship between spatial arrangement of the epoxide hydrolase binding site and the enantioselectivity of the enzyme, could open up the possibility to correctly predict both the enantioselectivity as well as the activity of R. glutinis EH, and possibly other red yeasts, towards more complex epoxide substrates without the need of time consuming screenings. / Thesis (Ph.D. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2008.

Cellulosic biomass is an incredibly abundant resource and a capable feedstock for production of energy, biofuels, and commodity chemicals. Current technologies for bioprocessing of cellulose utilize a three-step process in which enzymes capable of cellulose hydrolysis are expressed and purified, cellulose is hydrolyzed, and then product is formed in separate processes. This multi-step processing increase costs. As such, one approach to lowering these costs it to develop on consolidated system in which all three of these processes occur in a single step. Toward this aim, the three main goals of this dissertation are (1) characterization of a new hydrolytic enzyme and its application to fermentation of relevant sugars, (2) selection of proteins capable of intracellular cellobiose transport, and (3) development of a minimal set of cellulases capable of extensive hydrolysis under physiological conditions. A mixture of cellodextrins is produced by enzymatic hydrolysis of cellulose and Ced3A, a cellodextrinase, was shown to hydrolyze all of these completely to glucose and confer the ability to metabolize these sugars to E. coli when expressed. Activity on cellobiose, however, was lower than on other species. Co-expression of Cep94A, a cellobiose phosphorylase, and Ced3A was shown to improve the cellobiose metabolism of E. coli. In order to facilitate conversion of cellobiose to glucose by Cep94A, cellobiose must be transported into the cytoplasm. Three cellobiose permease enzymes, LacY, CP1, and CP2, were expressed in E. coli. It was shown that each protein has affinity for cellobiose transport and expression of each 126 allowed fermentation of cellobiose by E. coli strains expressing a cytoplasmic cellobiase. All three proteins are likely suitable for cellobiose transport during a consolidated bioprocess. Finally, a system of three cellulase enzymes Cel5H, Cel9R, and Cel48S were evaluated at E. coli physiological conditions and it was shown that extensive hydrolysis occurred at over half of the compositions tested. Additionally, when strains expressing cellulases were grown in binary culture with strains previously engineered for cellodextrin metabolism substantial product formation was observed, representing suitable performance of a consolidated cellulose bioprocess. This dissertation presents successful performance of all three components necessary for consolidated bioprocessing both individually and when working in tandem. Furthermore, the technologies developed in this dissertation demonstrate the capacity for consolidated bioprocessing of cellulose.

Optically pure aromatic D-amino acids, such as D-phenylalanine (D-Phe) and its derivatives, are high-value building blocks for the pharmaceutical industry. These compounds can be prepared using biocatalytic methods relying on various enzymes, including aminotransferases (ATs). ATs, also called transaminases (EC 2.6.1.X), are a subclass of pyridoxal 5′-phosphate-dependent enzymes that catalyze the transfer of the amino group from a donor substrate to a ketone acceptor. Synthesis of optically-pure amino acids using whole-cell biocatalytic cascades based on ATs possess several advantages compared to traditional chemical methods, including excellent enantioselectivity and increased process and step efficiency, which is achieved through the catalysis of multiple steps in one-pot reactions without requirement for intermediate work-ups, cofactor recycling, or toxic metals. However, enzyme biocatalysts typically need to be engineered to alter their substrate specificity or to increase their catalytic efficiency, which has limited their industrial application. Therefore, to facilitate the engineering process of ATs broadly and to produce aromatic D-amino acids, we developed a high-throughput assay for the testing of a broad range of ATs against libraries of potential substrates, and developed a biocatalytic cascade to produce optically pure aromatic D-amino acids.