Enhanced thermal stability of lichenase from Bacillus subtilis 168 by SpyTag/SpyCatcher-mediated spontaneous cyclization

• Published in: Biotechnology for biofuels Vol. 9; no. 76; p. 79
• Main Authors: Wang, Jindan, Wang, Yilin, Wang, Xinzhe, Zhang, Dandan, Wu, Shuyu, Zhang, Guangya
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 31.03.2016; BioMed Central
• Subjects: Analysis; Bacillus subtilis; Chemical synthesis; Chromatography; Denaturation; Enzymes; Molecular weight; Physiological aspects; Proteins; Temperature; Lichenase; Cyclization; Non-chromatographic purification; Thermal stability; SpyTag/SpyCatcher
• ISSN: 1754-6834; 1754-6834
• DOI: 10.1186/s13068-016-0490-5

SpyTag 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. The 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. This 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.