Thermostable adenosine 5'-monophosphate phosphorylase from Thermococcus kodakarensis forms catalytically active inclusion bodies

Catalytically active inclusion bodies (CatIBs) produced in Escherichia coli are an interesting but currently underexplored strategy for enzyme immobilization. They can be purified easily and used directly as stable and reusable heterogenous catalysts. However, very few examples of CatIBs that are naturally formed during heterologous expression have been reported so far. Previous studies have revealed that the adenosine 5'-monophosphate phosphorylase of Thermococcus kodakarensis (TkAMPpase) forms large soluble multimers with high thermal stability. Herein, we show that heat treatment of soluble protein from crude extract induces aggregation of active protein which phosphorolyse all natural 5'-mononucleotides. Additionally, inclusion bodies formed during the expression in E. coli were found to be similarly active with 2-6 folds higher specific activity compared to these heat-induced aggregates. Interestingly, differences in the substrate preference were observed. These results show that the recombinant thermostable TkAMPpase is one of rare examples of naturally formed CatIBs.

Automated summary

Catalytically active inclusion bodies (CatIBs) produced in Escherichia coli are an interesting strategy for enzyme immobilization, with potential applications as stable and reusable catalysts. Research on naturally formed CatIBs, such as the thermostable TkAMPpase, has shown that they exhibit higher specific activity compared to heat-induced aggregates, with differences in substrate preference. This highlights the potential of recombinant thermostable enzymes as naturally formed CatIBs for biocatalysis applications.