Creating cellulose-binding domain fusions of the coenzymea biosynthetic enzymes to enable reactor-based biotransformations
Coenzyme A (CoA) analogues are widely used as tools in chemical biology. Currently, most CoA analogues are prepared by biotransformation of pantothenic acid analogues using the three CoA biosynthetic enzymes (PanK, PPAT, and DPCK) that constitute the CoA salvage pathway. However, because the enzymes are usually lost or destroyed in the solution-based methods that are presently used, we created fusions of the Escherichia coli and Staphylococcus aureus PanK, PPAT, and DPCK enzymes with a cellulose-binding domain (CBD) which can be used to immobilize each of these proteins on cellulose. We show that all the CBD-fusion proteins can be expressed in soluble form, and that instead of impacting negatively on the activity of their enzyme partners, the presence of the CBD improves their kinetic profiles in some cases. We subsequently determined which combinations of the available enzymes are most effective in producing CoA and a typical CoA analogue, and used these to demonstrate that the rate of biotransformation is not severely affected even when the CBD-fusion enzymes are immobilized on cellulose. Finally, we constructed batch and column reactors from cellulose loaded with the CBD enzymes and tested these in the biocatalytic production of a fluorescent CoA analogue often used for protein labeling. Our results show that such reactors can successfully be used, and that the enzymes retain their activities upon storage in this format. This study is the first to showcase the use of a multi-enzyme reactor system based on CBD-fusion proteins in biocatalysis. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.