The goal of this proposal is to assemble a combined enzymatic system that will allow two sequential reactions to take place in flow. We will use this system to generate amines from alcohols via a carbonyl intermediate. Amines are key functional groups of numerous intermediates for pharma and agrochemical applications, and enzymatic synthesis will offer a ‘green’ alternative to traditional methodologies. To overcome the limitations of mesophilic enzymes such as low stability in organic solvents, we will design a novel system based on two halophilic biocatalysts that offer remarkable stability and excellent substrate scope.
The advantages of flow chemistry over batch reactions include efficient mixing, high throughput, and assembly of multi-step reactions. (Bio)catalysts can be immobilized on a solid matrix through which the reagents are pumped, generating a very high ratio of catalyst/reagent with significant economic benefits, since enzymatic immobilization improves the stability of the biocatalyst and catalytic performance over time.
Both enzymes (HvADH2 and HvAAT1) are from the halophilic archaeon Haloferax volcanii. HvADH2 is exceptionally tolerant to organic solvents, has an unusually broad substrate scope, and shows enhanced stability upon covalent immobilization. This makes it an ideal candidate for flow applications. HvAAT1 has been expressed in H. volcanii. The gene for HvAAT1 (gabT1) is genetically linked to the adh2 gene, raising the possibility that alcohol substrates converted to aldehydes and ketones by HvADH2 may be acting as amino acceptor for HvAAT1. Such a combined system, where ADH and AAT can be easily coupled, would yield the first completely compatible tandem reaction system using halophilic enzymes.
To achieve the goals of this project, two parallel approaches will be developed.
1) Sequential immobilization of HvADH2 and HvAAT1 HvADH2 and HvAAT1 will be covalently immobilized and connected sequentially. While amino transaminases steadily regenerate their bound cofactor, HvADH2 is NADP(H)-dependent and the cofactor reacts stoichiometrically with the substrates. NADP(H) is generally added as a reagent in the reaction mix. In this proposal we will covalently co-immobilize the cofactor with HvADH2 on epoxy resin for steady availability and use mildly oxidative conditions in the media to regenerate NADP+ in situ.
2) H. volcanii whole cell immobilization (co-expression of HvADH2 and HvAAT1) We will co-express both enzymes in H. volcanii and assemble a whole-cell bioreactor where the cells are immobilized on nitrocellulose membrane. The retention of the enzymes within the cell system has been proven to be efficient and high-yielding. To maintain cell integrity, the reaction will be studied in various salt concentrations. We will expand the scope to the use of ionic liquids, which is of high relevance for industrial applications.