Abstract
BackgroundSpyTag is a peptide that can form an irreversible covalent linkage to its 12 kDa partner SpyCatcher via a spontaneous isopeptide bond. Herein, we fused SpyTag at the N-terminal of lichenase and SpyCatcher at C-terminal so that the termini of lichenase were locked together by the covalent interaction between the partners. In addition, an elastin-like polypeptides tag was subsequently attached to the C-terminus of SpyCatcher, thereby facilitating the non-chromatographic purification of cyclized lichenase.ResultsThe study showed that the optimum temperature of the cyclized lichenase was about 5 °C higher in comparison to its linear counterpart. Moreover, nearly 80 % of the cyclized lichenase activities were retained after 100 °C exposure, whereas the linear form lost almost all of its activities. Therefore, the cyclized variant displayed a significantly higher thermal stability as temperature elevated and was resistant to hyperthermal denaturation. Besides, the Km value of the cyclized lichenase (7.58 ± 0.92 mg/mL) was approximately 1.7-fold lower than that of the linear one (12.96 ± 1.93 mg/mL), indicating a higher affinity with substrates.ConclusionsThis new SpyTag/SpyCatcher cyclization strategy is deemed as a generalized reference for enhancing enzyme stability and can be effectively customized to the cyclization of various enzymes, hence a tremendous potential for successful application in the biocatalytic conversion of biomass to produce fuels and chemicals.
Highlights
SpyTag is a peptide that can form an irreversible covalent linkage to its 12 kDa partner SpyCatcher via a spontaneous isopeptide bond
It is conceivable that SpyTag/SpyCatcher-mediated cyclization method has overcome the defects of other alternative approaches, and it is expected to become a potential staple in enhancing thermal stability and usher in a new era of industrial biocatalysis
Verification for the cyclized conformation of lichenase The crude lysate was subjected to two-round inverse transition cycling (ITC) purification, and the purities of fusion lichenase after each round purification procedure were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) analysis (Fig. 1)
Summary
Verification for the cyclized conformation of lichenase The crude lysate was subjected to two-round ITC purification, and the purities of fusion lichenase after each round purification procedure were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) analysis (Fig. 1). We discarded the insoluble active lichenase when purifying the soluble linear and cyclized lichenase as their purities were ranging from 85 to 90 %, which was not enough for theoretical study here, but they may be suitable for practical applications In this condition, the ELPs fusion tag functioned as a “stabilizer” that was similar to the role carrier plays in the enzyme immobilization. It is anticipated that by tuning the reaction parameters, we could achieve the naturally occurring self-assembling between cyclized lichenase and ELPs tag, immediately after the covalent cyclization of the enzyme Once this was completed, it would enable the time- and cost-efficient integration of lichenase immobilization with purification, and improve the thermal stability and recyclability of the lichenase on the optimum conditions. Our method may further expedite the utilization of biological cyclization strategy in the biocatalytic field, as well as providing solid foundations for the industrial application of biocatalysts
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