A current
challenge in catalysis is the development of methodologies
for the production of bulk chemicals needed at levels of tens and
hundreds of thousands of tons per year with the requirement to be
produced at very low costs often being in the single-digit US dollar
range. At the same time, such methodologies should address challenges
raised by current manufacturing processes. Within this research area,
a cyanide-free approach toward aliphatic nitriles used as industrial
chemicals was developed starting from readily accessible n-alkenes as starting materials available in bulk quantities. This
chemoenzymatic process concept is exemplified for the synthesis of
nonanenitrile (as an n-/iso-mixture)
and runs in water at low to moderate temperatures without the need
for any types of cyanide sources. The process is based on a combination
of a metal-catalyzed hydroformylation as the world-leading production
technology for alkyl aldehydes with an emerging enzyme technology,
namely, the recently developed transformation of aldoximes into nitriles
through dehydration by means of aldoxime dehydratases. As a missing
link, an efficient aldoxime formation with subsequent removal of remaining
traces of hydroxylamine as an enzyme-deactivating component was found,
which enabled the merging of these three steps, hydroformylation,
aldoxime formation, and enzymatic dehydration, toward a nitrile synthesis
without the need for purification of intermediates.
In this contribution, the unique and unprecedented stereochemical phenomenon of an aldoxime dehydratase‐catalyzed enantioselective dehydration of racemic E‐ and Z‐aldoximes with selective formation of both enantiomeric forms of a chiral nitrile is rationalized by means of molecular modelling, comprising in silico mutations and docking studies. This theoretical investigation gave detailed insight into why with the same enzyme the use of racemic E‐ and Z‐aldoximes leads to opposite forms of the chiral nitrile. The calculated mutants with a larger or smaller cavity in the active site were then prepared and used in biotransformations, showing the theoretically predicted decrease and increase of the enantioselectivities in these nitrile syntheses. This validated model also enabled the rational design of mutants with a smaller cavity, which gave superior enantioselectivities compared to the known wild‐type enzyme, with excellent E‐values of up to E>200 when the mutant OxdRE‐Leu145Phe was utilized.
Dedicated to Dr. Yasuhisa Asano on the occasion of his 70th birthdayRecently, the capability of the aldoxime dehydratase from Bacillus sp. (OxdB) for the transformation of fatty aldoximes into fatty nitriles with impressive substrate loadings is reported. However, the substrate scope of this biocatalyst turned out to be limited in terms of the chain length with decanal oxime being the substrate with the longest well tolerated n-alkyl chain. Besides the increased bulkiness of the long-chain aldoximes, their strongly decreased water solubility represents a further hurdle for an efficient biotransformation. Addressing this challenge of an expanded substrate spectrum comprising long-chain fatty aldoximes, this work investigates the substrate solubility and enzyme kinetics in combination with molecular modeling in order to find an enzyme mutant being suitable for C12-to C16-aldoximes. Both, fatty aldoxime solubility in water and the active site of the wild-type enzyme OxdB are identified as critical issues for an efficient biotransformation of these substrates. The activity issue is addressed by a rational design of a mutant using a homology modeling as well as a molecular modeling software suitable for enzymes. With the resulting double mutant OxdB-F289A/L293A, this report can achieve successful biotransformations with the C12-to C16-aldoximes at substrate concentrations of 250 × 10 −3 to 1000 × 10 −3 m. For example, an excellent conversion of >99% is obtained with tetradecanal oxime. Practical applications: Fatty nitriles with a prolonged chain length of C12 or more are of high interest in industry due to their use for the production of fatty amines on large technical scale. As an alternative route, fatty nitriles can be generated from their aldoximes by means of an aldoxime dehydratase (Oxd) as biocatalyst. The conversion of long-chain fatty aldoximes, however, remained a challenge up to now. This work describes the optimization of the aldoxime dehydratase OxdB from Bacillus sp. for the dehydration of nonsoluble bulky fatty aldoximes. The created variant can convert long chain fatty aldoximes toward the corresponding nitrile as demonstrated for C12-to C16-nitriles. In addition, high conversion (of up to >99%) is achieved when operating at high substrate concentrations of up to 1000 × 10 −3 m, thus making this approach interesting for industrial applications.
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