Dr Tamara Wriessnegger and Professor Harald Pichler of ACIB introduce hydratases as biocatalysts for oxy-functionalisation
Enzymatic – thus regio- and
stereo-selective – oxyfunctionalisation has long been of prime interest in biocatalytic approaches and processes . The most prominent enzyme family in these endeavours , cytochrome P450 enzymes , is hampered by the need to regenerate the active moiety at the expense of NAD ( P ) H co-factor . A viable alternative , obviating the need for co-factor regeneration , is the selective hydration of C = C bonds through hydratase action . Regarding reaction mechanisms , hydration reactions can be classified by the substrate properties . Adding water to an electron-deficient , activated C = C bond in α , β- unsaturated carbonyls is achieved via nucleophilic Michael addition . Water addition to electron-rich , isolated C = C bonds follows Markovnikov ’ s rule . 1 , 2 , 3 In the corresponding chemocatalysis , problems lie mainly in the activation of water as a nucleophile . Compared to carbon or nitrogen nucleophiles , oxygencontaining molecules are known to be bad nucleophiles . To improve the reactivity of water for addition to C = C bonds , strong activation is necessary , leading to the infrequent use of acid- or basecatalysed chemical hydrations . This is associated with harsh reaction conditions , the formation of unfavourable side products and / or a complete lack of selectivity . 2 Nature has developed excellent strategies for the activation of water as a nucleophile , which makes
Figure 1 – Hydratases catalysing water addition to non-activated double bonds
hydratases highly attractive for green industrial applications . 4 Hydratases use water as a substrate and provide the active site for the synthesis of enantiomerically pure primary , secondary and tertiary alcohols in a very efficient manner . 1 A remarkable feature of hydratases is their high substrate selectivity , which is important for their natural functions in metabolic pathways . However , for industrial applications , high enantioselectivity and a broad substrate spectrum are required . Thus , extending their substrate scope for industrial production is a central task of hydratase R & D work . Exceptions are fumarase , malease and enoyl-CoA hydratases , which are employed on industrial scales in wellestablished processes despite their narrow substrate profile . Oxy-functionalisation of nonactivated C = C bonds through
( engineered ) hydratases has been a vivid research field lately illustrating biocatalytic access to otherwise hardly accessible secondary and tertiary alcohols ( Figure 1 ). Recent work at ACIB in Graz , Austria , on three promising hydratases and hydratase families illustrate the potential of this enzyme class . These are :
• Kievitone hydratase ( KHS ) from Nectria hematococca ( NhKHS )
• Linalool ( de ) hydratase-isomerase from Castellaniella defragrans ( CdLDI )
• Oleate hydratase from Elizabethkingia meningoseptica ( EmOhyA ) In counteracting the plant phytoalexin kievitone , Fusarium species , having infected Phaseolus vulgaris ( French bean ), use KHS for the detoxification and formation of hydroxy-kievitone by the addition of
46 SPECIALITY CHEMICALS MAGAZINE ESTABLISHED 1981