Halophilic enzymes in tandem flow reactions

Chemical reactions are often multi step processes in which a starting material is converted into a first intermediate, then another and so on till the final molecule is synthesized. Traditionally these reactions are done sequentially, in separate flasks and each step requires a work-up and often purification.

When the syntheses are scaled up for an industrial setting, each step must be optimized and costs become a critical factor. Where possible, reactions are catalyzed to minimize reaction times, and purification is avoided if impurities generated in a particular reaction can be carried forward without causing issues in the next step.

In an effort to streamline production and maximize throughput, the development of flow-chemistry, where a reaction is carried out in a continuous flow through a reactor rather than in a batch, is rapidly expanding. In this set up a catalyst can be immobilized on a solid matrix and the reagents are passed through in a controlled flow allowing for the product to be synthesized continuously.

In recent years the use of enzymes to replace chemical catalysts has also evolved significantly, with major pharmaceuticals now looking at biotechnology for the production of drugs with a more environmentally friendly approach. Moreover, biocatalytic processes reduce the contamination of products with toxic impurities such as (transition) metals, a major cost and time factor in other catalytic processes. The application of biocatalysis to flow chemistry is therefore an excellent target biotechnology also. Furthermore, multiple enzymes can be immobilized in sequence into contiguous bioreactors for cascade reactions to be developed, effectively assembling an artificial biosynthetic sequence where an enzyme recognizes as substrate the product generated by the previous enzyme and so on. This can be achieved either in a cell free system, or the artificial pathway can be constructed inside a biological host which becomes itself a bioreactor.

In this project we are looking at developing a cascade reaction system utilizing enzymes from an organism isolated from the Dead Sea. Haloferax volcanii grows in very high salt environments and its enzymes can catalyze reactions not only in the presence of high salts, but more interestingly they can do this in the presence of organic solvents. Enzymes are normally unstable in non-aqueous media and this makes them often incompatible with chemical reactions where many molecules are insoluble in water. Haloferax enzymes are therefore ideal biocatalysts for chemical synthesis. We have previously investigated the characteristics of an alcohol dehydrogenase from Haloferax (HvADH1) and this enzyme is capable of transforming a very broad range of alcohols into aldehydes or ketones. We also know that we can easily immobilize HvADH1 irreversibly on commercially available beads further increasing its stability and activity. We now want to combine HvADH1 with a second enzyme, also from Haloferax, to transform the aldehydes and ketones produced in the first step to amines, achieving overall a functional group inter-conversion starting from alcohols to generate amines. Amines are very common intermediates in the synthesis of pharmaceuticals and agrochemicals and we will generate them from readily available alcohols. We will test two different systems, one in which the enzymes are individually produced, isolated, immobilized, and the flow optimized for substrate conversion, and a second system in which the enzymes will be over-produced simultaneously inside Haloferax cells which will be itself immobilized and used as a self contained reactor where both steps will take place.

Functional group inter-conversion is a common strategy in synthetic organic chemistry. We will generate a tandem biocatalytic system to convert alcohols into amines using two enzymes from the halophilic archaeon Haloferax volcanii; HvADH2 (gene adh2), an NADP-dependent alcohol dehydrogenase, and HvAAT1 (gene gabT1), an amine aminotransferase. Interestingly, the gene gabT1 is genetically linked to adh2, implying that the products generated by the first enzyme can be substrates for the second one.

Two approaches will be followed to achieve the alcohol to amine conversion:

1) Immobilization of isolated enzymes packaging in sequential columns

HvADH2 and HvAAT1 will be covalently immobilized on different scaffolds spanning from epoxy resins to renewable material. Both enzymes will be optimized individually for activity and stability and subsequently combined in sequential columns. We will address the recycling of NADP+ cofactor needed by HvADH2 by exploring a tailored co-immobilization and the use a mild oxidant such as sodium hypochlorite in the reaction medium, and, in parallel, via the introduction of a third enzyme in the system (NADPH oxidase). This system will be followed by a second reactor in which AvAAT1 will be contained. The amino donor required for the second step will be added either at the beginning or fed exclusively to the second reactor.

2) immobilization of whole cells of H. volcanii expressing both enzymes

Co-expression of both HvADH1 and HvAAT1 can be achieved in H. volcanii and direct immobilization of cells will be performed on nitrocellulose to study the efficiency of alcohol-amine inter-conversion within the cellular system.

A variety of substrates such as primary, secondary and aromatic alcohols will be screened for this system. The generation of mutants enzymes will also be considered to expand the substrate scope of one or both enzymes, if needed.

Beneficiaries of this research

This proposal will have a significant impact on the industrial production of fine chemicals by offering the industry a 'green' alternative to traditional methodologies for synthesizing amine containing molecules, key functional groups found in pharmaceuticals and agrochemicals.Enzymes are utilized more and more as catalysts for biotransformation in the production of small molecules. However, the great majority of such reactions are conducted in batch - the biocatalyst is recovered only is some instances, and separation can be problematic. Flow chemistry will allow continuous production without any concern for the separation of the catalysts. Outcomes from the proposed research will show that it is possible to tune multiple enzymatic steps for the synthesis of relevant intermediates. Therefore, our project delivers economic and societal impact by reducing industrial costs associated with manufacturing pharmaceuticals and agrochemicals whilst delivering environmental benefits minimizing harmful waste.
In particular, the proposed work has significant implications for the pharmaceutical industry. By focusing our work on enzymes from the archaeon H. volcanii, we eliminate the concern that the biocatalysts may be contaminated by bacterial endotoxins; this will have benefits for the direct synthesis of active pharmaceutical ingredients (APIs). Thus, the biotechnology implications of this work fit within the BBSRC's responsive mode priority "New strategic approaches to industrial biotechnology"

How will they benefit from this research?

We will combine two different enzymes for functional group interconversion. However, each enzyme will be optimized as a single biocatalyst, offering a platform to industrial partners who may have a specific interest in just one biotransformation. The key innovation will be the application of flow strategy to biocatalytic processes, which addresses several industrial goals such as the automation of manufacturing, reduced cost, and reduced environmental impact.
We have established collaborations with GSK, Pfizer, and now Johnson Matthey which have a keen interest in alternative sources for biocatalysts and have been directly involved in Dr. Paradisi's research since 2013; we will keep a close interaction throughout the project. If commercially viable outcomes arise, steps towards exploitation will be taken with The University of Nottingham Business Engagement and Innovation Services.

Communication and Engagement

As well as Open Access publications, presentations at conferences and direct communication with the industrial contacts, we will ensure that outcomes from this project will be highlighted on our web pages, the University of Nottingham's Communications and Marketing Unit, Nottingham's Café Scientifique and BioCity, and the BBSRC media office. Throughout the project we will also expand our interaction with industrial partners such as Johnson Matthey and Pfizer, and exploit BBSRC NIBBs such as BioCatNet and CBMNet.

Professional development

The project offers significant opportunities for the PDRA to broaden his/her skills. The work is highly interdisciplinary, encompassing chemistry, enzymology and microbiology. The PDRA will be exposed to a variety of techniques within the laboratories of PIs and collaborators. We will encourage the development of the PDRA scientific communication skills by regularly presenting our research to peer academic audiences and the general public (e.g. Nottingham's Café Scientifique or local schools). The University of Nottingham has a well-established Science Outreach Programme, which will be key in the training of the PDRA in addressing a lay audience. Careers advice for the PDRA will be provided by the University of Nottingham, which runs training courses on career planning, effective CV writing workshops and interview practice for early career scientists.