Optimizing the linker length for fusing an alcohol dehydrogenase with a cyclohexanone monooxygenase

Alejandro Gran-Scheuch, Friso Aalbers, Yannick Woudstra, Loreto Parra, Marco W Fraaije*

*Bijbehorende auteur voor dit werk

OnderzoeksoutputAcademicpeer review

2 Citaten (Scopus)


The use of enzymes in organic synthesis is highly appealing due their remarkably high chemo-, regio- and enantioselectivity. Nevertheless, for biosynthetic routes to be industrially useful, the enzymes must fulfill several requirements. Particularly, in case of cofactor-dependent enzymes self-sufficient systems are highly valuable. This can be achieved by fusing enzymes with complementary cofactor dependency. Such bifunctional enzymes are also relatively easy to handle, may enhance stability, and promote product intermediate channeling. However, usually the characteristics of the linker, fusing the target enzymes, are not thoroughly evaluated. A poor linker design can lead to detrimental effects on expression levels, enzyme stability and/or enzyme performance. In this chapter, the effect of the length of a glycine-rich linker was explored for the case study of ɛ-caprolactone synthesis through an alcohol dehydrogenase-cyclohexanone monooxygenase fusion system. The procedure includes cloning of linker variants, expression analysis, determination of thermostability and effect on activity and conversion levels of 15 variants of different linker sizes. The protocols can also be used for the creation of other protein-protein fusions.

Originele taal-2English
TitelLinkers in Biomacromolecules
RedacteurenMaarten Merkx
Aantal pagina's37
ISBN van geprinte versie978-0-12-820818-2
StatusPublished - 2021

Publicatie series

NaamMethods in Enzymology
ISSN van geprinte versie0076-6879

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