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

A. Gran-Scheuch, F. Aalbers, Y. Woudstra, L. Parra, M.W. Fraaije

Research output: Chapter in Book / Report / Conference proceedingChapterAcademicpeer-review


© 2021 Elsevier Inc.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.
Original languageEnglish
Title of host publicationMethods in Enzymology
EditorsM. Merkx
PublisherAcademic Press Inc.
ISBN (Print)9780128208182
Publication statusPublished - 1 Jan 2021
Externally publishedYes

Publication series

NameMethods in Enzymology
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988


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