Spheroplasts preparation boosts the catalytic potential of a terpene cyclase

Abstract

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.

Tailoring the squalene-hopene cyclase for stereoconvergent and efficient cationic cyclization cascades

“…[37,72] However, an apparent drawback is the long time period of the presented process, which could be overcome by increasing the mass transfer across the biomembrane using membrane transporter enzymes [73] or spheroplast preparation. [74] Furthermore, the herein presented in vitro approach could certainly be fine-tuned by changing deteregent, detergent concentration or buffer, however, was beyond the scope of this study.…”