title: Enhanced thermal stability of lichenase from Bacillus subtilis 168 by SpyTag/SpyCatcher-mediated spontaneous cyclization.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Bioengineering and Biotechnology, Huaqiao University, Xiamen, 361021 Fujian China.
authors.forename: Jindan
authors.initials: J
authors.lastname: Wang
authors.affiliation: Biochip Laboratory, Yantai Yuhuangding Hospital Affiliated to Medical College of Qingdao University, Yantai, 264000 Shandong China.
authors.forename: Yilin
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Bioengineering and Biotechnology, Huaqiao University, Xiamen, 361021 Fujian China.
authors.forename: Xinzhe
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Bioengineering and Biotechnology, Huaqiao University, Xiamen, 361021 Fujian China.
authors.forename: Dandan
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Department of Bioengineering and Biotechnology, Huaqiao University, Xiamen, 361021 Fujian China.
authors.forename: Shuyu
authors.initials: S
authors.lastname: Wu
authors.affiliation: Department of Bioengineering and Biotechnology, Huaqiao University, Xiamen, 361021 Fujian China.
authors.forename: Guangya
authors.initials: G
authors.lastname: Zhang
keywords: Cyclization; Lichenase; Non-chromatographic purification; SpyTag/SpyCatcher; Thermal stability