Smart cosubstrate

A smart cosubstrate is a type of cosubstrate used for cofactor regeneration to yield greater productivity and lower environmental impact (E-factor). A good example of a '''smart cosubstrate is a lactonizable diol.

In redox biocatalysis, the nicotinamide cofactor (NAD(P)H or NAD(P)+) can act as an electron donor or acceptor by releasing or accepting a hydride. The cofactor must be used in the reaction either in stoichiometric amounts leading to inhibition and economic issues, or in catalytic amounts coupled with an in situ regeneration system. A common approach catalytic amounts is excess use of sacrificial organic molecules such as isopropanol or ethanol. This approach, however, leads to stoichiometric amounts of waste.

The use of 1,4-butanediol as a smart cosubstrate for cofactor regeneration was the next step towards more sustainable redox biocatalysis (Scheme 1). The formation of a thermodynamically stable gamma-butyrolactone as a co-product drives the reaction to completion while yielding higher reaction rates. The use of 1,4-butanediol as an intelligent cosubstrate has also been validated in non-aqueous media using a commercial ADH.



Double-smart cosubstrate


Biocatalytic cascade reactions currently fall into four different categories: However, only two types of redox-neutral cascades have been reported for the in situ regeneration of the cofactors: parallel cascades (i.e., bi-substrate — no intermediate — bi- or tri-product) and linear cascades (i.e., single substrate — single intermediate — single product).
 * 1) Linear, which is biocatalytic
 * 2) Orthogonal
 * 3) Parallel
 * 4) Cyclic

The concept of a smart cosubstrate was developed further through the design of a new class of redox-neutral "convergent cascade" reactions. Convergent cascade reactions involve a bi-substrate and a single product without the formation of an intermediate and were developed for the production of epsilon-caprolactone, which consists of a Baeyer-Villiger monooxygenase; for the oxidation of cyclohexanone; an alcohol dehydrogenase for oxidation of the "double-smart cosubstrate" 1,6-hexanediol; and for simultaneous regeneration of the nicotinamide cofactor. In 2016, two-step optimization of the convergent cascade by Design-of-Experiments and a biphasic system was reported.

Smart cosubstrates are an elegant solution for thermodynamically limited redox reactions and have many advantages:
 * 1) Less conventional cosubstrates (e.g., isopropanol, ethanol) that negatively affect the enzymes’ activities need to be used.
 * 2) Less waste is generated.
 * 3) Reactions are faster, which could be caused by the absence of acetone or acetaldehyde as a coproduct, which lead to reduced enzyme activities.