SpyTag is a peptide which spontaneously forms an amide bond to its protein partner SpyCatcher. Here we fused SpyTag at the N-terminus of β-lactamase and SpyCatcher at the C-terminus, so the partners could cyclize to lock together the termini of the enzyme. Wild-type enzyme aggregates above 37 °C, with irreversible loss of activity. Cyclized β-lactamase was soluble even after heating at 100 °C; after cooling, the catalytic activity was restored. SpyTag/SpyCatcher-cyclization achieved > 60 °C increase in stability, much larger than from point mutation or alternative approaches to cyclization. Cyclized dihydrofolate reductase was similarly resilient. Analyzing unfolding calorimetrically and via mutants, cyclization did not increase the unfolding temperature of β-lactamase, but facilitated refolding after thermal stress. SpyTag and SpyCatcher sandwiching represents a simple and efficient route for enzyme cyclization, with potential to greatly enhance the robustness of biocatalysts.

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.

SpyTag is a peptide which spontaneously forms an amide bond to its protein partner SpyCatcher. Here we fused SpyTag at the N-terminus of β-lactamase and SpyCatcher at the C-terminus, so the partners could cyclize to lock together the termini of the enzyme. Wild-type enzyme aggregates above 37 °C, with irreversible loss of activity. Cyclized β-lactamase was soluble even after heating at 100 °C; after cooling, the catalytic activity was restored. SpyTag/SpyCatcher-cyclization achieved > 60 °C increase in stability, much larger than from point mutation or alternative approaches to cyclization. Cyclized dihydrofolate reductase was similarly resilient. Analyzing unfolding calorimetrically and via mutants, cyclization did not increase the unfolding temperature of β-lactamase, but facilitated refolding after thermal stress. SpyTag and SpyCatcher sandwiching represents a simple and efficient route for enzyme cyclization, with potential to greatly enhance the robustness of biocatalysts.

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