SpyTag/SpyCatcher Cyclization Enhances the Thermostability of Firefly Luciferase

Abstract

SpyTag can spontaneously form a covalent isopeptide bond with its protein partner SpyCatcher. Firefly luciferase from Photinus pyralis was cyclized in vivo by fusing SpyCatcher at the N terminus and SpyTag at the C terminus. Circular LUC was more thermostable and alkali-tolerant than the wild type, without compromising the specific activity. Structural analysis indicated that the cyclized LUC increased the thermodynamic stability of the structure and remained more properly folded at high temperatures when compared with the wild type. We also prepared an N-terminally and C-terminally shortened form of the SpyCatcher protein and cyclization using this truncated form led to even more thermostability than the original form. Our findings suggest that cyclization with SpyTag and SpyCatcher is a promising and effective strategy to enhance thermostability of enzymes.

Valency engineering of monomeric enzymes for self-assembling biocatalytic hydrogels

“…Indeed, several doubly labeled enzymes have already been described in the literature, including monomers and SC/ST-modied variants that retain their activity aer labeling. [16][17][18] In our case, however, the resulting Gre2p-SC 2 enzyme revealed only poor polymerization with GDH-ST ( Fig. S1 †).…”

Engineering Potato Virus X Particles for a Covalent Protein Based Attachment of Enzymes

“…The optimal temperature and pH for free and immobilized Cel12A‐SC‐His were 50 °C and pH 6.5, respectively, unlike the discovered pH optimum for plant‐derived Cel12A‐His (pH 5.0), but similar to reported Escherichia coli derived Cel12A (pH 6.0) . Recent studies showed, that the SpyTag/SpyCatcher mediated cyclization is a promising strategy to enhance the structural stability and therefore thermotolerance of enzymes like β‐lactamase, luciferase, or lichenase . If these findings are also applicable to T. reesei endoglucanase Cel12A can be taken into consideration for further work.…”

“…[73] Recent studies showed, that the SpyTag/SpyCatcher mediated cyclization is a promising strategy to enhance the structural stability and therefore thermotolerance of enzymes like β-lactamase, luciferase, or lichenase. [62,74,75] If these findings are also applicable to T. reesei endoglucanase Cel12A can be taken into consideration for further work. During these studies an increased thermotolerance could be shown for the fusion of Cel12A to SC.…”

Power to the protein: enhancing and combining activities using the Spy toolbox

“…63 Similar effects on resilience were found for SpyRing versions of phytase, 64 xylanase 65 and luciferase. 66 Apart from temperature, increased tolerance to organic solvents and denaturant has been found upon SpyRing cyclisation. 67 Another approach to enhance stability and performance has been to encapsulate enzymes in protein cages.…”