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Additionally , chemical catalysts often lack sufficient stereoselectivity to produce one enantiomer of the product exclusively . Therefore , to purify the desired product stereoisomer , the synthesis is followed by resolution using chiral substances such as D-lactic acid or (+) -phencyphos in the case of NEA to precipitate only the required enantiomer . This step is followed by dissolving the salt and purifying it again to yield the final product . These strategies suffer from low E-factors because of the usage of multiple auxiliary chemicals needed for the resolution and the downstream purification , increasing the processes ’ environmental impact . However , strategies dedicated to greener chemistry prioritise improving the product : waste ratio and reducing the environmental burden of the catalyst and the production process as a whole . Especially for the synthesis of chiral substances , using enzymes as natural , bio-based catalysts is an attractive alternative . Such biocatalysts eliminate the need for transition metal catalysts and , because of their inherent selectivity , no protection , deprotection , or resolution steps are needed — hence , suitable enzymes produce large amounts of the desired stereoisomer .
Yet , nature often does not provide us with enzymes ready to withstand the rigours of the commercial production environment , especially harsh process conditions or utilising challenging non-natural substrates and / or using high substrate concentrations with low water solubility . However , enzyme engineering advancements allow us to quickly evolve an enzyme to achieve the characteristics required for a given process . To evolve enzymes that stand up to the task , we use our proprietary directed evolution technology platform , BioEngine ®, to engineer improved enzyme variants . Specifically , for the synthesis of NEA and PEA , we used a transaminase for the asymmetric reductive amination of the keto function in the substrate molecule . However , the natural enzyme for the synthesis of PEA was inhibited at 5 g / L product concentrations and inactivated at 2 M concentrations of the necessary amine donor iso-propylamine ( IPM ). These limitations are typical when using transaminases and many other enzymes in non-natural synthetic routes , as desired performance levels tend to be limited to water-based reaction conditions . For example , only low product concentrations typically result from the poorly water-soluble ketones acetophenone and 1-acetylnaphthalene when synthesising PEA and NEA in an aqueous reaction set-up , respectively . Most literature describes values of < 10 g / L , resulting in a water / product content of only 1 % ( w / v ). Because of the low product concentrations , productivity is low and isolating the product can be challenging . Specifically , to isolate the desired amine product from the water phase , energy-intensive water distillation and / or environmentally harmful solvent extraction are required . However , possible solutions , such as substrate emulsions or high concentrations of organic co-solvents , significantly decrease enzyme stability . To address these challenges , we increased the stability of a transaminase using our BioEngine ® enzyme evolution platform . In this case , the enzyme was engineered to deliver the target performance after ten rounds of enzyme evolution . Whereas enzymes are usually applied in aqueous reaction environment , after evolution , we used the transaminase in neat substrate conditions with 800 g / L of substrate , and the enzyme was stable for more than 180 hours at 2 M IPM . No additional solvents or reagents were required for the synthesis , and very high space – time yields were obtained , with up to 160 g / L / d in the case of PEA . Overall , enzymes can eliminate costly transition metal catalysts and resource-intensive protection / deprotection or resolution steps for the synthesis of chiral amines and numerous other fine chemicals . But wild-type enzymes are nearly always insufficient for commercial applications and must be engineered to achieve the needed performance . Enzyme evolution is a fruitful and auspicious process to optimise enzymes to perform successfully in efficient , sustainable and more cost-effective API and fine chemical manufacturing . •
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