Directed evolution of transaminases is a widespread technique in the development of highly sought-after biocatalysts for industrial applications.

Significant progress has been made in establishing transaminases as robust biocatalysts for the green and scalable synthesis of a diverse range of chiral amines. However, very few examples on the amination of small cyclic ketones have been reported. Cyclic ketones are particularly challenging for transaminase enzymes because they do not display the well-defined small and large substituent areas that are characteristic for the bio- catalytic mechanism. In this work, we exploited the broad substrate scope of the (S)-selective transaminase from Halomonas elongata (HeWT) to develop an efficient biocatalytic system in continuous flow to generate a range of small cyclic amines which feature very often in pharmaceuticals and agrochemicals. [3] Tetrahydrofuran-3-one and other challenging prochiral ketones were rapidly (5–45 min) transformed to their corresponding amines with excellent molar conversion (94–99%) and moderate to excellent ee.

Squalene-hopene cyclases are a highly valuable and attractive class of membrane-bound enzymes as sustainable biotechnological tools to produce aromas and bioactive compounds at industrial scale. However, their application as whole-cell biocatalysts suffer from the outer cell membrane acting as a diffusion barrier for the highly hydrophobic substrate/product, while the use of purified enzymes leads to dramatic loss of stability. Here we present an unexplored strategy for biocatalysis: the application of squalene-hopene-cyclase spheroplasts. By removing the outer cell membrane, we produce stable and substrate-accessible biocatalysts. These spheroplasts exhibit up to 100-fold higher activity than their whole-cell counterparts for the biotransformations of squalene, geranyl acetone, farnesol, and farnesyl acetone. Their catalytic ability is also higher than the purified enzyme for all high molecular weight terpenes. In addition, we introduce a concept for the carrier-free immobilization of spheroplasts via crosslinking, crosslinked spheroplasts. The crosslinked spheroplasts maintain the same catalytic activity of the spheroplasts, offering additional advantages such as recycling and reuse. These timely solutions contribute not only to harness the catalytic potential of the squalene-hopene cyclases, but also to make biocatalytic processes even greener and more cost-efficient.

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Squalene-hopene cyclases (SHCs) are a highly valuable and attractive class of membrane-bound enzymes as sustainable biotechnological tools to produce aromas and bioactive compounds at industrial scale. However, their application as whole-cell biocatalysts suffer from the outer cell membrane acting as a diffusion barrier for the highly hydrophobic substrate/product, while the use of purified enzymes leads to dramatic loss of stability. Here we present an unexplored strategy for biocatalysis: the application of SHC spheroplasts. By removing the outer cell membrane, we produced stable and substrate-accessible biocatalysts. SHC spheroplasts exhibited up to 100-fold higher activity than their whole-cell counterparts for the biotransformations of squalene, geranyl acetone, farnesol, and farnesyl acetone. Their catalytic ability was also higher than the purified enzyme for all high molecular weight terpenes. In addition, we introduce a new concept for the carrier-free immobilization of spheroplasts via crosslinking, CLS (crosslinked spheroplasts). The CLS maintained the same catalytic activity of the spheroplasts, offering additional advantages such as recycling and reuse. These timely solutions contribute not only to harness the catalytic potential of the SHCs, but also to make biocatalytic processes even greener and more cost-efficient.