Coupled immobilized bi-enzymatic flow reactor employing cofactor regeneration of NAD+ using a thermophilic aldehyde dehydrogenase and lactate dehydrogenase.

The use of enzymes in biochemical processes is of interest due to their ability to work under mild conditions while attaining high reaction rates. A limitation in the use of enzymes such as oxidoreductases on a large scale lies with their requirement for costly cofactors, e.g. NAD⁺, in stoichiometric quantities. Cofactor regeneration mechanisms using bienzymatic recycling systems is an attractive way to increase productivity and efficiency. The thermophilic enzyme aldehyde dehydrogenase (ALDH_Tt) was immobilized directly from E. coli cell lysate, containing the expressed enzyme, onto Ni²⁺ activated Sepharose®. The system displayed a rate of conversion of approx. 63% NAD⁺ with reuse achievable for up to 5 cycles and residual activity of the enzyme upon storage of 93% after 7 days. L -Lactate dehydrogenase was immobilized in a second reactor module downstream of ALDH_Ttvia two different methods, electrochemical entrapment in poly(3,4-ethylenedioxypyrrole) (PEDOP) and covalent attachment on glyoxyl agarose. Both reactors allowed for up to 100% conversion of NADH, however LDH@agarose proved superior in terms of reuse and storage. LDH@agarose displayed no reduction in activity after 6 cycles of use and retained 98% activity following 56 days storage. A coupled reactor containing immobilized ALDH_Tt–LDH was operated with the substrates hexanal, benzaldehyde, terephthalaldehyde and p-tolualdehyde. A particular advantage of the system is its ability to preferentially oxidise a single aldehyde group in substrates containing two aldehyde functional groups. The reactor demonstrated efficient cofactor regeneration under continual operation for up 24 h, with enhanced product yields. [ABSTRACT FROM AUTHOR]