Recent advances on halohydrin dehalogenases—from enzyme identification to novel biocatalytic applications

Halohydrin dehalogenases are industrially relevant enzymes that catalyze the reversible dehalogenation of vicinal haloalcohols with formation of the corresponding epoxides. In the reverse reaction, also other negatively charged nucleophiles such as azide, cyanide, or nitrite are accepted besides halides to open the epoxide ring. Thus, novel C-N, C-C, or CO bonds can be formed by halohydrin dehalogenases, which makes them attractive biocatalysts for the production of various β-substituted alcohols. Despite the fact that only five individual halohydrin dehalogenase enzyme sequences have been known until recently enabling their heterologous production, a large number of different biocatalytic applications have been reported using these enzymes. The recent characterization of specific sequence motifs has facilitated the identification of novel halohydrin dehalogenase sequences available in public databases and has largely increased the number of recombinantly available enzymes. These will help to extend the biocatalytic repertoire of this enzyme family and to foster novel biotechnological applications and developments in the future. This review gives a general overview on the halohydrin dehalogenase enzyme family and their biochemical properties and further focuses on recent developments in halohydrin dehalogenase biocatalysis and protein engineering

Multi-Enzymatic Cascades In Vitro

The combination of enzymatic reactions in a simultaneous or sequential fashion by designing artificial synthetic cascades allows for the synthesis of complex compounds from simple precursors. Such multi-catalytic cascade reactions not only bear a great potential to minimize downstream processing steps but can also lead to a drastic reduction of the produced waste. With the growing toolbox of biocatalysts, alternative routes employing enzymatic transformations towards manifold and diverse target molecules become accessible. In vitro cascade reactions open up new possibilities for efficient regeneration of the required cofactors such as nicotinamide cofactors or nucleoside triphosphates. They are represented by a vast array of two-enzyme cascades that have been designed by coupling the activity of a cofactor regenerating enzyme to the product generating enzyme. However, the implementation of cascade reactions requires careful consideration, particularly with respect to whether the pathway is constructed concurrently or sequentially. In this regard, this chapter describes how biocatalytic cascades are classified, and how such cascade reactions can be employed in order to solve synthetic problems. Recent developments in the area of dynamic kinetic resolution or cofactor regeneration and showcases are presented. We also highlight the factors that influence the design and implementation of purely enzymatic cascades in one-pot or multi-step pathways in an industrial setting

Biphasic biocatalytic testosterone dehydrogenation in microfluidic droplets

Two-phase biocatalysis is commonly used for the bioconversion of water-insoluble chemical and pharmaceutical compounds. In research laboratories, such processes often take place in flasks and are driven and limited by slow diffusive processes. In this work, we developed droplet-based microfluidic systems and used them to perform testosterone dehydrogenation. The use of microdroplets as reaction vessels offers the advantage of greatly improved diffusion rates due to significantly increased surface-to-volume ratio. We found that indeed reaction time was reduced from tens of minutes to tens of seconds without sacrificing conversion efficiency. This demonstrates the potential of droplet-based two-phase biocatalysis

Novel halohydrin dehalogenases by protein engineering and database mining

Eén enkele bacteriële cel bevat duizenden enzymen die vele conversies uitvoeren aan een grote diversiteit van chemische verbindingen. Dit etaleert dat enzymen een verbluffende selectiviteit ten toon spreiden. De huidige synthetische chemie maakt gebruik van deze selectiviteit, onder andere in de productie van zogenaamde fine chemicals, zoals farmaceutische producten of hen pre-cursors. Wat dat betreft zijn halohydrine dehalogenases uitermate geschikte biokatalysatoren, omdat ze in staat zijn om diverse chemisch uiteenlopende verbindingen met nieuwe koolstof-koolstof, -stikstof of -zuurstof bindingen te vormen. De selectieve vorming van nieuwe koolstof-koolstof bindingen door deze enzymen is vooral interessant, omdat de traditionele organisch chemische reacties erg ingewikkeld zijn. Wild-type enzymen zoals HheC van Agrobacterium radiobacter AD1 zijn vaak niet direct toepasbaar en moeten daarom door protein engineering geschikt gemaakt worden voor industriële processen. In dit proefschrift worden twee sterk gemodificeerde HheC varianten met 37 mutaties gekarakteriseerd en gekrystalliseerd om de effecten van de mutaties op de activiteit en stabiliteit te verklaren. In een succesvolle poging om een hogere koolstof-koolstof binding vormende activiteit te bereiken, werd een enkele mutatie in HheC geïntroduceerd die in andere enzymen verondersteld wordt hogere activaties te veroorzaken. Daarnaast werden een aantal varianten gecreëerd met verbeterde of tegenovergestelde selectiviteit ten opzichte van het wild-type enzym. Naast mutagenese strategieën werd de natuurlijke diversiteit van deze enzym familie onderzocht en dit resulteerde in de ontdekking van 37 nieuwe enzymen. Samenvattend laten de resultaten beschreven in dit proefschrift zien dat nieuwe enzymen kunnen worden gevonden door zowel protein engineering of door gebruik te maken van de natuurlijke diversiteit

Exploring glutathione lyases as biocatalysts: paving the way for enzymatic lignin depolymerization and future stereoselective Applications

Glutathione-dependent β-etherases and glutathione lyases are key-enzymes for the biocatalytic depolymerization of lignin. In the first step, the nucleophilic attack of glutathione to the common β-O-4-aryl-ether motif in lignin is catalyzed by β-etherases and afterwards the glutathione is removed again by the action of glutathione lyases. Given their potential impact for lignin valorization, in this paper novel glutathione lyases are reported and biocatalytically characterized based on lignin model compounds. As a result, an enzyme exhibiting increased thermostability and lowered enantioselectivity - key features for implementation of glutathione lyases in enzymatic lignin depolymerization processes - was identified. Furthermore, first mutational studies of these enzymes revealed the possibility to further alter the activity as well as enantioselectivity of glutathione lyases by means of protein engineering. From a practical perspective, one-pot multi-step processes combining β-etherases and glutathione lyases are successfully set-up, giving hints on the potential that the implementation of these biocatalysts may bring for biorefinery purposes

The Depth-Dependent Mechanical Behavior of Anisotropic Native and Cross-Linked HheG Enzyme Crystals

Enzymes are able to catalyze various specific reactions under mild conditions and can, therefore, be applied in industrial processes. To ensure process profitability, the enzymes must be reusable while ensuring their enzymatic activity. To improve the processability and immobilization of the biocatalyst, the enzymes can be, e.g., crystallized, and the resulting crystals can be cross-linked. These mechanically stable and catalytically active particles are called CLECs (cross-linked enzyme crystals). In this study, the influence of cross-linking on the mechanical and catalytic properties of the halohydrin dehalogenase (HheG) crystals was investigated using the nanoindentation technique. Considering the viscoelastic behavior of protein crystals, a mechanical investigation was performed at different indentation rates. In addition to the hardness, for the first time, depth-dependent fractions of elastic and plastic deformation energies were determined for enzyme crystals. The results showed that the hardness of HheG enzyme crystals are indentation-rate-insensitive and decrease with increases in penetration depth. Our investigation of the fraction of plastic deformation energy indicated anisotropic crystal behavior and higher irreversible deformation for prismatic crystal faces. Due to cross-linking, the fraction of elastic energy of anisotropic crystal faces increased from 8% for basal faces to 68% for prismatic crystal faces. This study demonstrates that mechanically enhanced CLECs have good catalytic activity and are, therefore, suitable for industrial use

Film media and nationalism in Thailand : comparative studies of film media propaganda in Thailand and its influence on Thai nationalism, political ideology, and class structure since the enactment of the Thai Constitution of 1997.

During periods of official nationalism in Thailand, the Thai media played a significant role in promoting government policy. The Thai media during those periods was a government orientated enterprise. However, the Thai parliament approved a new constitution in 1997 which aims to minimise state control over the media. It is for this reason that this thesis chose to study current events and the current situation. Once the media was opened up, the patterns of ownership started to change. The media, post 1997, began to change from a government orientated enterprise towards a market-orientated private enterprise. This thesis investigates the interaction between media and nationalism in Thailand. It looks at the relationship between media owners and political leaders in general. This thesis also looks at the signs, symbols, costumes, and messages that generate nationalistic feelings among audiences. This research was carried out by analysing the film contents. This analysis of content is used to demonstrate the hypothesis, which states that the new patterns of ownership of the media have led to new techniques for shaping nationalism. In order to do so, this thesis employs a media and nationalism framework which is created by using numerous theories on media and nationalism. The results have indicated that, although the primary motive of film-making may now be turning a profit, films which aim to promote the nationalism or political agendas still exist. It appears in several case studies that the private media owners and the political leaders share common interests. The filmmakers do not hesitate to use the symbols, cultures, and traditions, which are invented by the leaders. Symbols that represent power and legitimacy of the political leaders are promoted by an attempt of the filmmaker to persuade audiences that those practices and tradition, and by extension, the leaders are righteous. Individual films target different classes in Thai society, which vary by their beliefs, culture, and practices. The filmmakers are either making the film according to the culture of those classes to strengthen the nationalistic awareness from the audiences, or they are using their medium to persuade the audiences to accept their class values. Either way, it can be demonstrated that since the end of the period of official nationalism, nationalism is still promoted through film. This media support of political agendas can be seen as the new style to promote nationalism

Asymmetric azidohydroxylation of styrene derivatives mediated by a biomimetic styrene monooxygenase enzymatic cascade

Enantioenriched azido alcohols are precursors for valuable chiral aziridines and 1,2-amino alcohols, however their chiral substituted analogues are difficult to access. We established a cascade for the asymmetric azidohydroxylation of styrene derivatives leading to chiral substituted 1,2-azido alcohols via enzymatic asymmetric epoxidation, followed by regioselective azidolysis, affording the azido alcohols with up to two contiguous stereogenic centers. A newly isolated two-component flavoprotein styrene monooxygenase StyA proved to be highly selective for epoxidation with a nicotinamide coenzyme biomimetic as a practical reductant. Coupled with azide as a nucleophile for regioselective ring opening, this chemo-enzymatic cascade produced highly enantioenriched aromatic α-azido alcohols with up to >99% conversion. A bi-enzymatic counterpart with halohydrin dehalogenase-catalyzed azidolysis afforded the alternative β-azido alcohol isomers with up to 94% diastereomeric excess. We anticipate our biocatalytic cascade to be a starting point for more practical production of these chiral compounds with two-component flavoprotein monooxygenases. A one-pot enzymatic cascade for the asymmetric azidohydroxylation of styrenes leads to chiral 1,2-azido alcohols with up to two stereocenters

From gene to biorefinery: microbial ß-etherases as promising biocatalysts for lignin valorization

The set-up of biorefineries for the valorization of lignocellulosic biomass will be core in the future to reach sustainability targets. In this area, biomass-degrading enzymes are attracting significant research interest for their potential in the production of chemicals and biofuels from renewable feedstock. Glutathione-dependent ß-etherases are emerging enzymes for the biocatalytic depolymerization of lignin, a heterogeneous aromatic polymer abundant in nature. They selectively catalyze the reductive cleavage of ß-O-4 aryl-ether bonds which account for 45–60% of linkages present in lignin. Hence, application of ß-etherases in lignin depolymerization would enable a specific lignin breakdown, selectively yielding (valuable) low-molecular-mass aromatics. Albeit ß-etherases have been biochemically known for decades, only very recently novel ß-etherases have been identified and thoroughly characterized for lignin valorization, expanding the enzyme toolbox for efficient ß-O-4 aryl-ether bond cleavage. Given their emerging importance and potential, this mini-review discusses recent developments in the field of ß-etherase biocatalysis covering all aspects from enzyme identification to biocatalytic applications with real lignin samples