title: Engineering CotA Laccase for Acidic pH Stability UsingBacillus subtilisSpore Display.
abstract: Bacillus subtilisspores 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 (t1/2) 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 resistance and a T480A mutant was found. Consequently, T480A/E498G-CotA was constructed and thet1/2was 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 seven times over 42 h.
authors: New Jersey Institute of Technology, Department of Chemistry and Environmental Science, University Heights, Newark, NJ 07102, USA.
authors: Silu
authors: S
authors: Sheng
authors: New Jersey Institute of Technology, Department of Chemistry and Environmental Science, University Heights, Newark, NJ 07102, USA.
authors: Han
authors: H
authors: Jia
authors: Center for Healthcare Innovation, Stevens Institute of Technology, Hoboken, NJ 07030, USA.
authors: Sidney
authors: S
authors: Topiol
authors: New Jersey Institute of Technology, Department of Chemistry and Environmental Science, University Heights, Newark, NJ 07102, USA.
authors: Edgardo T
authors: ET
authors: Farinas
mesh_terms: D001412:Bacillus subtilis / Q000502:physiology*; D061505:Cell Surface Display Techniques; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D055550:Protein Stability; D013171:Spores, Bacterial
chemical_list: D042845:Laccase
keywords: Protein display; directed evolution; laccase; protein stability; spore