SpyRing interrogation: analyzing how enzyme resilience can be achieved with phytase and distinct cyclization chemistries

Abstract

Enzymes catalyze reactions with exceptional selectivity and rate acceleration but are often limited by instability. Towards a generic route to thermo-resilience, we established the SpyRing approach, cyclizing enzymes by sandwiching between SpyTag and SpyCatcher (peptide and protein partners which lock together via a spontaneous isopeptide bond). Here we first investigated the basis for this resilience, comparing alternative reactive peptide/protein pairs we engineered from Gram-positive bacteria. Both SnoopRing and PilinRing cyclization gave dramatic enzyme resilience, but SpyRing cyclization was the best. Differential scanning calorimetry for each ring showed that cyclization did not inhibit unfolding of the inserted β-lactamase. Cyclization conferred resilience even at 100 °C, where the cyclizing domains themselves were unfolded. Phytases hydrolyze phytic acid and improve dietary absorption of phosphate and essential metal ions, important for agriculture and with potential against human malnutrition. SpyRing phytase (PhyC) resisted aggregation and retained catalytic activity even following heating at 100 °C. In addition, SpyRing cyclization made it possible to purify phytase simply by heating the cell lysate, to drive aggregation of non-cyclized proteins. Cyclization via domains forming spontaneous isopeptide bonds is a general strategy to generate resilient enzymes and may extend the range of conditions for isolation and application of enzymes.

Engineering a Rugged Nanoscaffold To Enhance Plug-and-Display Vaccination

“… 66 However, SpyCatcher has shown the ability to enhance protein thermal resilience in the context of cyclizing enzymes. 67 , 68 …”

New Routes and Opportunities for Modular Construction of Particulate Vaccines: Stick, Click, and Glue

“…Immobilization of enzymes onto potato virus X VLPs via SpyCatcher/SpyTag technology also suggests improved heat tolerance up to 50°C ( 91 ). Intramolecular cyclization via “sandwiching” enzymes using Tag/Catcher can impart high resilience to heat inactivation and even allow purification of an enzyme via boiling ( 89 , 96 ). One could contemplate harnessing this resilience for purification and sterilization for cheap production of monomeric antigens ( 16 , 29 ).…”

Autocatalytic association of proteins by covalent bond formation: a Bio Molecular Welding toolbox derived from a bacterial adhesin

“…This pioneering strategy developed by the Howarth’s group consists in splitting a pilin into two separate protein-peptide pairs and reconstituting specifically the globular domain through the covalent association between the two partners. This protein-peptide tag system has been fused to various proteins-of-interest for several applications, including polymerization of linear, branched or combinatorial polyproteins via iterative sequential reactions on solid-phase 14 , design of new protein architectures 15 , protein cyclization to resist unfolding 16 and vaccination 17 .…”