Overcoming emerging challenges in biocatalysis

In the last few decades, biocatalysis has emerged as a promising path towards more cost-effective and sustainable chemical synthesis in the path of developing more environmentally friendly strategies. Biocatalysis offers a beneficial approach but its widespread application is hampered by the drawbacks associated with the use of enzymes; especially, their stability under industrial requirements and their reusability. New, more robust, biocatalysts have been identified from extremophiles and in parallel, protein immobilisation has advanced, permitting the reuse of the biocatalysts, facile separation from the reaction bulk and their application in continuous processes. In this work, several aspects linked to the use of biocatalysts in flow reactors have been investigated to solve relevant challenges and facilitate the application of biocatalytic methods for chemical synthesis. In this sense, a new method for the immobilisation of challenging enzymes has been developed and applied to a carboxylesterase for the hydrolytic deracemization of naproxen butyl ester in flow. Moreover, substrate insolubility has been resolved with the addition of surfactants (Chapter 4). Transaminases, and further optimization of their stability and applications, have also been investigated. Specifically, residues involved in PLP binding have been studied identifying a poorly conserved asparagine as a key amino acid in PLP stabilization in dimeric transaminases (Chapter 5). Novel synthetic methods involving transaminases have also been developed, specifically looking at the combination of a novel halo adapted alanine dehydrogenase to shift the equilibrium and reduce the amount of amino donor generally required (Chapter 6). Finally, the deamination of lysine was investigated for the synthesis of pipecolic acid. Here, the use of a transaminase was explored as well as a redox neutral cascade. This last system was co-immobilised in the same support and successfully applied in flow, achieving a volumetric yield of up to 2.5 g/h/L, 10-fold improvement from the fermentation-based production used up to date (Chapter 7)

Overcoming emerging challenges in biocatalysis

In the last few decades, biocatalysis has emerged as a promising path towards more cost-effective and sustainable chemical synthesis in the path of developing more environmentally friendly strategies. Biocatalysis offers a beneficial approach but its widespread application is hampered by the drawbacks associated with the use of enzymes; especially, their stability under industrial requirements and their reusability. New, more robust, biocatalysts have been identified from extremophiles and in parallel, protein immobilisation has advanced, permitting the reuse of the biocatalysts, facile separation from the reaction bulk and their application in continuous processes. In this work, several aspects linked to the use of biocatalysts in flow reactors have been investigated to solve relevant challenges and facilitate the application of biocatalytic methods for chemical synthesis. In this sense, a new method for the immobilisation of challenging enzymes has been developed and applied to a carboxylesterase for the hydrolytic deracemization of naproxen butyl ester in flow. Moreover, substrate insolubility has been resolved with the addition of surfactants (Chapter 4). Transaminases, and further optimization of their stability and applications, have also been investigated. Specifically, residues involved in PLP binding have been studied identifying a poorly conserved asparagine as a key amino acid in PLP stabilization in dimeric transaminases (Chapter 5). Novel synthetic methods involving transaminases have also been developed, specifically looking at the combination of a novel halo adapted alanine dehydrogenase to shift the equilibrium and reduce the amount of amino donor generally required (Chapter 6). Finally, the deamination of lysine was investigated for the synthesis of pipecolic acid. Here, the use of a transaminase was explored as well as a redox neutral cascade. This last system was co-immobilised in the same support and successfully applied in flow, achieving a volumetric yield of up to 2.5 g/h/L, 10-fold improvement from the fermentation-based production used up to date (Chapter 7)

Combined chemoenzymatic strategy for sustainable continuous synthesis of the natural product hordenine

To improve sustainability, safety and cost-efficiency of synthetic methodologies, biocatalysis can be a helpful ally. In this work, a novel chemoenzymatic stategy ensures the rapid synthesis of hordenine, a valuable phenolic phytochemical under mild working conditions. In a two-step cascade, the immobilized tyrosine decarboxylase from Lactobacillus brevis (LbTDC) is here coupled with the chemical reductive amination of tyramine. Starting from the abundant and cost-effective amino acid L-tyrosine, the complete conversion to hordenine is achieved in less than 5 minutes residence time in a fully-automated continuous flow system. Compared to the metal-catalyzed N,N-dimethylation of tyramine, this biocatalytic approach reduces the process environmental impact and improves its STY to 2.68 g/(L·h)

Efficient Amino Donor Recycling in Amination Reactions: Development of a New Alanine Dehydrogenase in Continuous Flow and Dialysis Membrane Reactors

Transaminases have arisen as one of the main biocatalysts for amine production but despite their many advantages, their stability is still a concern for widespread application. One of the reasons for their instability is the need to use an excess of the amino donor when trying to synthesise amines with unfavourable equilibria. To circumvent this, recycling systems for the amino donor, such as amino acid dehydrogenases or aldolases, have proved useful to push the equilibria while avoiding high amino donor concentrations. In this work, we report the use of a new alanine dehydrogenase from the halotolerant bacteria Halomonas elongata which exhibits excellent stability to different cosolvents, combined with the well characterised CbFDH as a recycling system of L-alanine for the amination of three model substrates with unfavourable equilibria. In a step forward, the amino donor recycling system has been co-immobilised and used in flow with success as well as re-used as a dialysis enclosed system for the amination of an aromatic aldehyde

Sustainable synthesis of L-phenylalanine derivatives in continuous flow by immobilized phenylalanine ammonia lyase

The application of phenyl ammonia lyases for the amination of a variety of cinnamic acids has been shown to be a cost-efficient method to produce a variety of phenylalanine analogues. Nonetheless, as many other biocatalytic tools, the process intensification, especially due to the high equivalents of ammonia needed, and the cost-efficiency of the catalyst production and use have been key points to further prove their usefulness. Here, we investigated the use of previously characterized PALs (AvPAL and PbPAL) for the amination of a series of substituted cinnamic acids. To enhance the process scalability and the reusability of the catalyst, we investigated the use of covalent immobilization onto commercially available supports, creating a heterogeneous catalyst with good recovered activity (50%) and excellent stability. The immobilized enzyme was also incorporated in continuous flow for the synthesis of 3-methoxy-phenyl alanine and 4-nitro-phenylalanine, which allowed for shorter reaction times (20 mins of contact time) and excellent conversions (88 ± 4% and 89 ± 5%) respectively, which could be maintained over extended periods of time, up to 24h. This work exemplifies the advantages that the combination of enzyme catalysis with flow technologies can have not only in the reaction kinetics, but also in the productivity, catalyst reusability and downstream processing

A novel thymidine phosphorylase to synthesize (halogenated) anticancer and antiviral nucleoside drugs in continuous flow

Four pharmaceutically relevant nucleoside analogues (5-fluoro-2’-deoxyuridine, 5-chloro-2’-deoxyuridine, 5-bromo-2’-deoxyuridine, and 5-iodo-2’-deoxyuridine) have been synthesized by using a novel thymidine phosphorylase from H. elongata (HeTP). Following enzyme immobilization on microbeads, the biocatalyst was implemented as a packed-bed reactor for the continuous production of halogenated nucleosides, achieving up to 90% conversion at the 10 mM scale with 30 min residence time. Taking the synthesis of Floxuridine (5-fluoro-2’-deoxyuridine) as study case, we obtained the highest space-time yield (5.5 g/L/h) reported to date. In addition, bioinformatic tools such as MD analysis and CapiPy have contributed to shine light on the catalytic performance of HeTP as well as its immobilization, respectively

Genetically fused T4L acts as a shield in covalent enzyme immobilisation enhancing the rescued activity

Enzyme immobilisation is a common strategy to increase enzymes resistance and reusability in a variety of excellent ‘green’ applications. However, the interaction with the solid support often leads to diminished specific activity, especially when non-specific covalent binding to the carrier takes place which affects the delicate architecture of the enzyme. Here we developed a broadly applicable strategy where the T4-lysozyme (T4L) is genetically fused at the N-terminus of different enzymes and used as inert protein spacer which directly attaches to the carrier preventing shape distortion of the catalyst. Halomonas elongata aminotransferase (HEWT), Bacillus subtilis engineered esterase (BS2m), and horse liver alcohol dehydrogenase (HLADH) were used as model enzymes to elucidate the benefits of the spacer. While HEWT and HLADH activity and expression were diminished by the fused T4L, both enzymes retained almost quantitative activity after immobilisation. In the case of BS2m, the protective effect of the T4L effectively was important and led to up to 10-fold improvement in the rescued activit