‣ catalysis . ¹¹ ^, ¹² ^, ¹³ The assembly of the naturally homopentameric enzyme and its function were studied in E . coli , revealing that a functional CdLDI oligomer was formed only in the periplasm , while cytosolic expression resulted in a distinctly different assembly and complete loss of activity . ¹³ This implies that periplasmic translocation is of utmost importance for the formation of the correct reduction – oxidation state of all four essential cysteines in the CdLDI protein . ¹¹ ^, ¹⁴ CdLDI catalyses the reversible ( de ) hydration and isomerisation of ( S )-(+)-linalool, resulting in the formation of β-myrcene and geraniol respectively ( Figure 3 ). ¹⁴ In the reverse direction , ( S )-(+)- linalool is produced from β-myrcene with high stereoselectivity ( ee ≥95 %), but the thermodynamic equilibrium is not favourable enough to achieve the desired high conversion rates . ⁹ Yet , LDI is already applied in the production of dienes , such as isoprene and butadiene , at industrial scale , with market values of billions of dollars . ¹¹ Fatty acids constitute one of the most sustainable natural feedstocks . Consequently , fatty acid hydratases ( FAHYs ) and especially oleate hydratases ( OHYs ) have been intensively studied since the 1960s . A substantial part of these works has been performed on the oleate hydratase from EmOhyA . ¹ ^, ³ ^, ¹⁵ ^, ¹⁶ This enzyme catalyses the regio- and stereo-selective hydration of oleic acid ( OA ), yielding ( R )-10-hydroxy stearic acid with an ee of ≥98 % and without

15 , 17 , 18

the need for co-factor recycling . ( R )-10-hydroxy stearic acid is already marketed by DSM as Beautactive . ¹⁹ Moreover , this compound can be trimmed by fungal β-oxidation to yield in δ-dodecalactone , which is of interest in the dairy industry . The elucidation of the protein structure of EmOhyA in 2015 , including the essential flavin cofactor , provided a major breakthrough for understanding the process of enzymatic water addition to nonactivated C = C bonds . ¹⁸ For a long time , the use of OHYs and FAHYs in industrial applications has been limited due to a very narrow substrate range , characterised by four paradigms :

1 . Minimum distance of seven carbon atoms between the essential carboxylate and the double bond in cis-configuration

2 . Minimum fatty acid chain length of 11 carbon

3 . Sub-terminal position of the C = C bond

4 . A free carboxylate of the fatty acid

10 , 18 , 19 substrate

Recently , a cleverly devised decoy molecule strategy using hexanoic acid as the dummy substrate enabled EmOhyA to hydrate 1-decene to ( S ) -2- decanol , thus ruling out paradigm 3 . Various terminal and internal alkenes can be addressed along these lines . ²⁰ Biocatalysts should not be too narrow in their substrate tolerance , particularly considering that commercially interesting substrates frequently differ from the natural ones . In this respect , ACIB research has challenged the requirement for a carboxylate head group of the EmOhyA substrates . Head groups with different physiochemical properties regarding size , hydrophobicity and polarisation have been analysed , i . e . amine , amide , hydroxamic acid , alcohol and shortchain esters derived from oleic acid , respectively ( Figure 4A ). Surprisingly , even wild-type EmOhyA can add water to the double bonds of many of these compounds ( Figure 4B ). This shows that a carboxylate function is not strictly required , thus ruling out paradigm . ⁴ ^. ²¹ Rationalising that more hydrophobic and / or larger head groups should require smaller and less polar amino acids in the active site , alanines have been introduced at relevant positions . The basis for the sitedirected mutagenesis approach has been the identification of four amino

Figure 3 – Active site cavity of CdLDI with four essential cysteines of two non-covalently linked enzyme sub-units

48 SPECIALITY CHEMICALS MAGAZINE ESTABLISHED 1981

Speciality Chemicals Magazine MAR / APR 2021 | Page 48