Halophilic enzymes in tandem flow reactions

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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.