Biocatalysis is emerging as an alternative approach to chemical synthesis of industrially relevant complex molecules. To obtain suitable yields of compounds in a cost-effective manner, biocatalytic reaction cascades must be efficient, robust, and self-sufficient. One approach is to immobilize biocatalysts on a solid support, stabilizing the enzymes and providing optimal microenvironments for reaction sequences. Protein-based scaffolds can be designed as immobilization platforms for biocatalysts, enabling the genetically encoded spatial organization of single enzymes and multistep enzyme cascades. Additionally, protein scaffolds are versatile, are easily adapted, and remain robust under different reaction conditions. In this chapter, we describe methods for the design and production of a self-assembling protein scaffold system for in vitro coimmobilization of biocatalytic cascade enzymes. We provide detailed methods for the characterization of the protein scaffolds, as well as approaches to load biocatalytic cargo enzymes and test activity of immobilized cascades. In addition, we also discuss methods for the development of a scaffold building block toolbox with different surface properties, which could be adapted for a diversity of biocatalysts requiring alternative microenvironments for function.
|Original language||English (US)|
|Title of host publication||Methods in Enzymology|
|Publisher||Academic Press Inc.|
|Number of pages||40|
|State||Published - 2019|
|Name||Methods in Enzymology|
Bibliographical noteFunding Information:
Research on the development of self-assembling protein materials in the Schmidt-Dannert laboratory is supported by a Defense Threat Reduction Agency Grant HDTRA-15-0004, Defense Advanced Research Projects Agency Contract HR0011-17-0038 and Grand Challenges research award from the University of Minnesota.
© 2019 Elsevier Inc.
- Enzyme cascade
- Protein scaffold