Investigation of aldehyde dehydrogenase from thermus thermophilus: biochemical characterisation and use in biocatalytic enzymatic reactors
The use of enzymes in biochemical processes is of interest due to their ability to work under mild conditions while attaining high reaction rates. Enzyme based biocatalysts play an important role in an array of applications in organic synthesis and industry, e.g., fine chemicals, food, energy, textiles, agricultural, cosmetics, medicine and pharmaceuticals, and allow for a greener production route compared to traditional chemical synthesis. However, the use of soluble enzymes is generally hampered by their lack of long term stability and inefficient reuse. Immobilization of enzymes plays a prominent role in overcoming these drawbacks and is commonly employed in industry. While hydrolases, lipases and laccases are readily used in industrial processes, the use of dehydrogenase enzymes is less so due to their requirement for costly cofactors. This is a key area that needs addressing, through cofactor regeneration mechanisms, as the products produced through dehydrogenase reactions can be interesting and relevant. In light of this, this project investigated the thermophilic aldehyde dehydrogenase from Thermus thermophilus, carrying out biochemical characterisation and identification of its substrate and product scope. Additionally, supports for immobilization were screened for development of enzymatic reactors using ALDHTt in both batch and continuous flow mode, also placing a focus on coupled enzymatic cofactor regeneration.
The ALDHTt has been recombinantly expressed in an E. coli host and subsequently purified allowing for in-depth biochemical characterisation of the enzyme in terms of both the dehydrogenase and esterase reaction mechanisms. A wide range of aldehyde substrates were screened for catalytic activity with the ALDHTt, consisting of aliphatics, aromatics and cyclics. This screening allowed for development of a product scope of carboxylic acid based materials that could potentially be synthesised via the biocatalytic route of ALDHTt. Out of the twelve substrates screened, eight could be catalysed efficiently. Most notably, hexanal, benzaldehyde, terephthalaldehyde and p-tolualdehyde exhibited reasonable rates of oxidation, resulting in hexanoic, benzoic, terephthalic and p-toluic acid formation. The latter two were of significant interest due to their role in pharmaceutical, polyethylene terephthalate and dye manufacture.
As target products were identified, the development of batch and continuous flow enzymatic reactors employing immobilized ALDHTt was performed. Metal organic frameworks have recently been a prime target for the in-situ immobilization of enzymes due to their associated rapid and facile immobilization process. This support was targeted for the development of a batch mode enzymatic reactor, however the hydrolytic instability of MOFs was considered. This lead to the screening of five MOFs, with different chemical compositions in order to identify the most stable support for subsequent immobilization of enzymes. The MOFs were analysed for stability in different aqueous buffered solutions typically used in enzymatic reactions, (citrate, sodium acetate, potassium phosphate and Tris-HCl). This allowed for identification of MOF-buffer combinations that resulted in the highest stability of the support, so not to hamper the biocatalytic system. Polymer coating of MOFs was also investigated for enhanced stability, to which polyacrylic acid allowed for heightened stability of the MOF while maintaining catalytic activity of an immobilized lipase. Further ALDHTt and an L-lactate dehydrogenase were immobilized, but diminished enzymatic activity resulted.
As previously mentioned, dehydrogenases cannot be readily used in applications without an efficient cofactor regeneration system. Additionally, the MOF reactors posed significant diffusion limitations, due to entrapment immobilization. Furthermore surface attachment of the ALDHTt and LDH to agarose supports for implementation in a flow reactor was explored. The coupled immobilized packed-bed reactor consisted of ALDHTt immobilized on Ni2+ activated sepharose via affinity binding and LDH covalently attached to glyoxyl agarose. An active, highly stable and reusable enzymatic flow reactor was eveloped employing cofactor regeneration of NAD+. Operation of the bienzymatic reactor allowed for increased production of carboxylic acid products up to 7-fold, resulting in approx. 900 μM of hexanoic, benzoic and p-toluic acid, from an NAD+starting concentration of 250 μM.
- Faculty of Science and Engineering
First supervisorEdmond Magner
Second supervisorTewfik Soulimane
Other Funding informationThe EU Horizon 2020 research and innovation program, Oyster, under which this project was funded.
Department or School
- Chemical Sciences