Thermostable adenosine 5\u2032-monophosphate phosphorylase from Thermococcus kodakarensis forms catalytically active inclusion bodies

Sarah Kamel,Miriam C Walczak,Peter Neubauer,Sarah Westarp,Anke Kurreck,Felix Kaspar

doi:10.1038/s41598-021-96073-5

Sarah Kamel, Miriam C Walczak + Show 4 more

Open Access

https://doi.org/10.1038/s41598-021-96073-5

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Abstract

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.

Highlights

Abbreviations AMP Adenosine 5′-monophosphate TkAMPpase Thermococcus kodakarensis Adenosine 5′-monophosphate phosphorylase Catalytically active inclusion bodies (CatIBs) Catalytically active inclusion body cytidine 5′-monophosphate (CMP) Cytidine 5′-monophosphate GMP Guanosine 5′-monophosphate heat-induced aggregates (HIA) Heat-induced aggregate IB Inclusion body NMP Nucleoside 5′-monophosphate dNMP 2′-Deoxy-nucleoside 5′-monophosphate R15P Ribose-1,5-bisphosphate UMP Uridine 5′-monophosphate
Heterologous expression of genes in Escherichia coli often leads to intracellular aggregation of the target overproduced protein which are called inclusion bodies (IBs)
While we were studying this enzyme in more detail, we discovered that it is prone to aggregation both during expression and after exposure to heat in cell crude extract which led us to develop a purification method to obtain the insoluble protein as CatIBs

Summary

Introduction

Abbreviations AMP Adenosine 5′-monophosphate TkAMPpase Thermococcus kodakarensis Adenosine 5′-monophosphate phosphorylase CatIB Catalytically active inclusion body CMP Cytidine 5′-monophosphate GMP Guanosine 5′-monophosphate HIA Heat-induced aggregate IB Inclusion body NMP Nucleoside 5′-monophosphate dNMP 2′-Deoxy-nucleoside 5′-monophosphate R15P Ribose-1,5-bisphosphate UMP Uridine 5′-monophosphate. Heterologous expression of genes in Escherichia coli often leads to intracellular aggregation of the target overproduced protein which are called inclusion bodies (IBs). For this reason, intentional aggregation of the target protein and purification of the resulting IB is a widely used strategy for product concentration and crude purification in the pharmaceutical industry. Active inclusion bodies (CatIBs) offer a valuable alternative as they can be produced in E. coli without the need for laborious solubilization. The application of CatIBs offers several advantages compared to soluble or synthetically immobilized enzymes. These include compatibility with aqueous and non-aqueous media, straightforward and cheap purification, reusability and no loss of activity due to immobilization[1]

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