FLAVOURS & FRAGRANCES
Figure 2 - Enzymatic aldehyde synthesis in vitro Figure 3 - Application possibilities of CAR enzymes immobilised metal ions . His-tagged CAR enzyme can thus be pulled out of protein mixtures , attached to the solid support and used for reactions , while remaining bound on this support . In this way , a fungal CAR was used as recyclable catalyst to reduce benzoic acid to benzaldehyde . 7 A bacterial CAR was co-immobilised on commercial EziGTM residue , together with two ATP-recycling enzymes for the same purpose . 8 Protein purification and immobilisation are cost factors , therefore the application of enzymes in crude form is preferred . Such crude forms might be enriched enzyme preparations , cell lysates or permeabilised cells , preferably in lyophilised form , in order to allow the chemists the addition of enzyme like any other powdery reagent . The convenience is somewhat offset by lower product selectivity : while CARs deliver the desired aldehydes , other protein and small molecule impurities in lysates and cells tend to consume the reactive aldehyde and form the corresponding alcohol under the applied reductive conditions . The most popular strategy for achieving high aldehyde yields therefore involves in situ product
removal ( ISPR ) to separate the relatively water-insoluble aldehyde from the aqueous system containing the enzymes and hydrophilic reducing equivalents . ISPR is most often described by using a bi-phasic system with a non-water-miscible organic layer , such as toluene , cyclohexane , heptane or hexadecane , when CAR enzymes are involved . 5 , 6 , 9 The other end of the spectrum when using CAR enzymes for aldehyde synthesis is embedded in living cells ( Figure 3 ). In this case , cellular metabolism can be exploited for the supply of ATP and NADPH . When opting for this strategy , ISPR has the double function of protecting the aldehyde product from cellmediated follow-up reactions and protecting the cells from the cytotoxic product compound . High aldehyde yield was achieved for the conversion of octanoic acid to octanal using a recombinant CAR from Mycobacterium marinum in engineered E . coli cells in the presence of n-heptane as ISPR solvent . 9 , 10 Biocompatible solvents like hexadecane hold even more promise , as they are able to sustain cell viability and therefore ATP supply even better .
Aldehydes are challenging chemicals to make , due to their high reactivity : the kinetic preference for aldehyde reduction in comparison to acid reduction is a problem in chemical reduction . CAR enzymes circumvent this problem , because the catalytic mechanism would only allow a bound carboxylate to enter the catalytic reduction site , while an aldehyde cannot be bound . In this sense , CAR enzymes are highly selective for aldehyde formation and a great starting point for aldehyde synthesis . How much aldehyde can eventually be isolated depends on the chemical structure of the substrate acid , the formulation of the catalyst – most significantly , the presence or absence of other active oxidoreductases – and the reaction mode . •
Dr Martin Trinker
DIRECTOR - BUSINESS DEVELOPMENT & FUNDRAISING
ACIB - AUSTRIAN CENTRE OF INDUSTRIAL BIOTECHNOLOGY
k + 43 316 873 9316 J martin . trinker @ acib . at j www . acib . at
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