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Motivation Enzymes are complex macromolecules crucial to life on earth. From bacteria to human beings, all organisms use enzymes to catalyse the many thousands of chemical reactions occurring in their cells. Enzyme functions are so diverse that the use of enzymes in industries like pharmaceuticals and agriculture has gained popularity over recent years as \"biocatalysts\". Unfortunately, the confident laboratory-based characterisation of enzyme function has lagged behind a massive increase in sequencing data, slowing down initiatives that look to use biocatalysts as part of their chemical processes. Computational methods for identifying biocatalysts do exist, but often falter due to the complexity of enzymes and sequence bias, leaving much of the catalytic space of enzymes and their families undiscovered. This thesis has two major themes: the development of in silico approaches for curating diverse panels of novel enzyme sequences for experimental characterisation, and of tooling that integrates in silico panel creation and in vitro enzyme characterisation into a unified and iterative framework. Contributions of this thesis The contributions of this thesis can be divided into the two larger themes, starting with the diverse panel selection of sequences from an enzyme family: • A novel type of protein network based on patterns of coevolving residues that can be used to identify functionally-interesting groupings in enzyme families. • The automatic sampling of functionally diverse subsets of enzyme sequences by solving the maximum diversity problem. - i - • A study into the viability of artificially increasing enzyme family diversity through neural networks-based generation of synthetic sequences. The second theme, which deals with built tools for bridging the gap between the in silico and in vitro side of enzyme family exploration: • A platform that integrates the panel selection process and resulting characterisation data to promote an iterative approach to exploring enzyme families. • A repository for storing the metadata generated by the major steps of characterisation assays in the lab.

The catalytical asymmetric reduction of small cyclic prochiral ketones with the formation of optically pure alcohols and amines is a fundamentally important reaction in synthetic organic chemistry. However, achieving selectivity using traditional synthetic methodology is challenging, particularly when the prochiral ketone has minimal discrimination between the two side chains. In light of this, new biocatalytic strategies for the enantioselective preparation of small cyclic molecules starting from readily available and inexpensive prochiral ketones was explored. To this aim, initial efforts focussed on the use of an established and highly efficient solid phase screening methodology for the screening of aminotransferase (ATs) variants. The screen was successfully applied to an AT from Halomonas elongata (HeWT), evolved through rounds of random mutagenesis towards a series of 4 small cyclic ketones, which lead to the identification of a variant with modest improvements in activity (ca. 2-fold) (Chapter 3). The substrate scope of a ketoreductase from Pichia glucozyma (KRED1-Pglu) and a structurally different AT from Pseudomonas fluorescens (PfTA) was subsequently probed using the same panel of small cyclic ketones. Under the reaction conditions tested, KRED1-Pglu failed to produce the target alcohols with a synthetically useful optical purity, while HeWT presented itself as a much more robust biocatalyst for the synthesis of the target amines, with respect to PfTA. To increase the complexity of the system, the development of a tandem enzymatic reaction, combing HeWT with a second biocatalyst, an acyltransferase from Mycobacterium smegmatis (MsAcT), for the synthesis of optically active amides was explored (Chapter 4). Finally, a multi-enzymatic cascade in continuous flow, featuring the immobilised HeWT and MsAcT is presented, demonstrating a fully intensified and industrially relevant biocatalytic process for the synthesis of small cyclic chiral molecules (Chapter 5).

Biocatalysis has widened its scope and relevance since new molecular tools, including improved expression systems for proteins, protein and metabolic engineering, and rational techniques for immobilization, have become available. However, applications are still sometimes hampered by low productivity and difficulties in scaling up. A practical and reasonable step to improve the performances of biocatalysts (including both enzymes and whole-cell systems) is to use them in flow reactors. This review describes the state of the art on the design and use of biocatalysis in flow reactors. The encouraging successes of this enabling technology are critically discussed, highlighting new opportunities, problems to be solved and technological advances. Biocatalyzed reactions with different classes of enzymes can be implemented with the integration of flow reactor technology, potentially leading to sustainable and highly productive continuous processes. The combination of biocatalysis and flow chemistry opens the door to extensive application in cascade reactions. Biocatalyzed flow reactions can occur either in monophasic flow or in segmented (slug) flow, where two or more immiscible phases are present. Limitation of substrate/product inhibition effects, in-line purification with easy recovery of the product, and no mechanical mixing are among the most distinctive advantages of flow-based biocatalysis. Automated machines and devices for in-line product recovery are now available at relatively low prices, making flow-based biocatalysis an easy-to-use technology.

The market for industrial enzymes has witnessed constant growth, which is currently around 7% a year, projected to reach $10.5 billion in 2024. Lipases are hydrolase enzymes naturally responsible for triglyceride hydrolysis. They are the most expansively used industrial biocatalysts, with wide application in a broad range of industries. However, these biocatalytic processes are usually limited by the low stability of the enzyme, the half-life time, and the processes required to solve these problems are complex and lack application feasibility at the industrial scale. Emerging technologies create new materials for enzyme carriers and sophisticate the well-known immobilization principles to produce more robust, eco-friendlier, and cheaper biocatalysts. Therefore, this review discusses the trending studies and industrial applications of the materials and protocols for lipase immobilization, analyzing their advantages and disadvantages. Finally, it summarizes the current challenges and potential alternatives for lipases at the industrial level.

By exploiting versatile P450 enzymes, whole‐cell biocatalysis can be performed to synthesize valuable compounds in Escherichia coli. However, the insufficient supply of heme limits the whole‐cell P450 biocatalytic activity. Here a strategy for improving intracellular heme biosynthesis to enhance the catalytic efficiencies of P450s is reported. After comparing the effects of improving heme transport and biosynthesis on P450 activities, intracellular heme biosynthesis is optimized through the integrated expression of necessary synthetic genes at proper ratios and the assembly of rate‐limiting enzymes using DNA‐guided scaffolds. The intracellular heme level is fine‐tuned by the combined use of mutated heme‐sensitive biosensors and small regulatory RNA systems. The catalytic efficiencies of three different P450s, BM3, sca‐2, and CYP105D7, are enhanced through fine‐tuning heme biosynthesis for the synthesis of hydroquinone, pravastatin, and 7,3′,4′‐trihydroxyisoflavone as example products of chemical intermediate, drug, and natural product, respectively. This strategy of fine‐tuned heme biosynthesis will be generally useful for developing whole‐cell biocatalysts involving hemoproteins. Here, the development of an engineered E. coli strain capable of improved heme supply suitable for whole‐cell P450 biocatalysts is reported. The intracellular heme level is fine‐tuned by the combined use of mutated heme‐sensitive biosensors and small regulatory RNA systems. This strategy of fine‐tuned heme biosynthesis will be generally useful for developing whole‐cell biocatalysts involving hemoproteins.

α-Amylases are the oldest and versatile starch hydrolysing enzymes which can replace chemical hydrolysis of starch in industries. It cleaves the α-(1,4)-D-glucosidic linkage of starch and other related polysaccharides to yield simple sugars like glucose, maltose and limit dextrin. α-Amylase covers about 30% shares of the total enzyme market. On account of their superior features, α-amylase is the most widely used among all the existing amylases for hydrolysis of polysaccharides. Endo-acting α-amylase of glycoside hydrolase family 13 is an extensively used biocatalyst and has various biotechnological applications like in starch processing, detergent, textile, paper and pharmaceutical industries. Apart from these, it has some novel applications including polymeric material for drug delivery, bioremediating agent, biodemulsifier and biofilm inhibitor. The present review will accomplish the research gap by providing the unexplored aspects of microbial α-amylase. It will allow the readers to know about the works that have already been done and the latest trends in this field. The manuscript has covered the latest immobilization techniques and the site-directed mutagenesis approaches which are readily being performed to confer the desirable property in wild-type α-amylases. Furthermore, it will state the inadequacies and the numerous obstacles coming in the way of its production during upstream and downstream steps and will also suggest some measures to obtain stable and industrial-grade α-amylase.

De novo enzyme design has sought to introduce active sites and substrate-binding pockets that are predicted to catalyse a reaction of interest into geometrically compatible native scaffolds 1 , 2 , but has been limited by a lack of suitable protein structures and the complexity of native protein sequence–structure relationships. Here we describe a deep-learning-based ‘family-wide hallucination’ approach that generates large numbers of idealized protein structures containing diverse pocket shapes and designed sequences that encode them. We use these scaffolds to design artificial luciferases that selectively catalyse the oxidative chemiluminescence of the synthetic luciferin substrates diphenylterazine 3 and 2-deoxycoelenterazine. The designed active sites position an arginine guanidinium group adjacent to an anion that develops during the reaction in a binding pocket with high shape complementarity. For both luciferin substrates, we obtain designed luciferases with high selectivity; the most active of these is a small (13.9 kDa) and thermostable (with a melting temperature higher than 95 °C) enzyme that has a catalytic efficiency on diphenylterazine ( k cat / K m  = 10 6  M −1  s −1 ) comparable to that of native luciferases, but a much higher substrate specificity. The creation of highly active and specific biocatalysts from scratch with broad applications in biomedicine is a key milestone for computational enzyme design, and our approach should enable generation of a wide range of luciferases and other enzymes. A deep-learning-based strategy is used to design artificial luciferases that catalyse the oxidative chemiluminescence of diphenylterazine with high substrate specificity and catalytic efficiency.

The late-stage oxidative functionalisation of C-H bonds can be used to circumvent synthetic route re-design and to generate molecules not easily accessible via conventional methods. Such reactions can be problematic due to a lack of site-selectivity and the poor intrinsic reactivity of the C-H bond. Enzymes may provide a solution to both these issues, and enhancements in activity, selectivity, temperature stability and organic solvent tolerance over the wild-type enzyme are achievable by protein engineering. The stereoselective chlorination of unactivated hydrocarbons is challenging from a synthetic perspective, yet this type of reaction is performed in nature by enzymes of the iron and 2-oxoglutarate (2OG)-dependent halogenase family. By utilising a radical mechanism, these enzymes remove the requirement of existing chlorination biocatalysts for inherent substrate activation (in the form of aromatic or alkene functionalities), and demonstrate the potential for the evolution of new and interesting C-H functionalisation capabilities. Historically, enzymes from the iron and 2OG- dependent halogenase family were unsuitable for biocatalysis due to the necessity of a substrate-bound carrier protein for activity. Identified in 2014, WelO5 was the first enzyme from this family shown to be capable of carrier protein-independent chlorination. This thesis has evaluated the tractability of developing synthetically useful biocatalysts based on the 2OG-dependent halogenase WelO5, with the aim of adding new reaction capabilities to the synthetic toolbox of the future. To achieve this, WelO5 was produced in high yields and purity for characterisation and crystallisation trials. Three novel WelO5 crystal structures were determined, which aided the rational selection of active site residues for replacement. The natural substrate for WelO5, (+)-12-epi-fischerindole U isonitrile, was synthetically produced and used for the development of a screening assay for assessing WelO5 variant activity against substrate-like analogues. A total of 40 WelO5 variants were produced and tested for activity towards the natural substrate and structurally similar analogues. Variant I161A was found to introduce a new hydroxylation activity to the enzyme. Activity was also seen for WelO5 and variants against two structurally unrelated compounds. Overall, the generated variants of WelO5 have shown promise as biocatalysts, with the main limitation to further progress being the throughput of the available screening methods. Novel activities have been discovered which merit further investigation into small molecule 2OG-halogenases as biocatalysts and this thesis provides the tools with which to do so.

Acid anhydrides have been used to carry out the regioselective acylation of primary hydroxyl group in benzyl 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate and 4-fluorobenzyl 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate and deacylation of their diesters in the presence of Lipozyme.sup.® TL IM in diisopropyl ether. Amongst different acid anhydrides used, butanoic anhydride was found to be the best acylating agent as compared to others. Both acylation and deacylation reactions were highly selective and efficient yielding exclusively the monoacylated products in 70-88 % yields.