Engineering cotA laccase for acidic pH stability using Bacillus subtilis spore display

Silu Sheng, Han Jia, Sidney Topiol, Edgardo T. Farinas

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


Bacillus subtilis spores can be used for protein display to engineer protein properties. This method overcomes viability and protein-folding concerns associated with traditional protein display methods. Spores remain viable under extreme conditions and the genotype/ phenotype connection remains intact. In addition, the natural sporulation process eliminates protein-folding concerns that are coupled to the target protein traveling through cell membranes. Furthermore, ATP-dependent chaperones are present to assist in protein folding. CotA was optimized as a whole-cell biocatalyst immobilized in an inert matrix of the spore. In general, proteins that are immobilized have advantages in biocatalysis. For example, the protein can be easily removed from the reaction and it is more stable. The aim is to improve the pH stability using spore display. The maximum activity of CotA is between pH 4 and 5 for the substrate ABTS (ABTS = diammonium 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate). However, the activity dramatically decreases at pH 4. The activity is not significantly altered at pH 5. A library of approximately 3,000 clones was screened. A E498G variant was identified to have a half-life of inactivation (t) at pH 4 that was 24.8 times greater compared with wt-CotA. In a previous investigation, a CotA library was screened for organic solvent resistance1/2 and a T480A mutant was found. Consequently, T480A/E498G-CotA was constructed and the t was 62.1 times greater than wt-CotA. Finally, E498G-CotA and T480A/E498G-CotA yielded 3.7- and 5.3-fold more product than did wt-CotA after recycling the biocatalyst seven1/2 times over 42 h.

Original languageEnglish (US)
Pages (from-to)507-513
Number of pages7
JournalJournal of Microbiology and Biotechnology
Issue number3
StatePublished - Mar 2017

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Applied Microbiology and Biotechnology


  • Directed evolution
  • Laccase
  • Protein display
  • Protein stability
  • Spore


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