Ana I. Benítez-Mateos1,
Andreas Schneider2,
Eimear Hegarty1,
Bernhard Hauer2,
Francesca Paradisi1
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1 Department of Chemistry, Biochemistry and Pharmaceutical Sciences. University of Bern Freiestrasse 3, 3012 Bern (Switzerland)
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2Institute of Biochemistry and Technical Biochemistry, University of Stuttgart Allmandring 31, 70569 Stuttgart-Vaihingen (Germany)
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.
