Redesigning pH optimum of Geobacillus sp. TF16 endoxylanase through in silico designed DNA swapping strategy

Biochimie. 2017 Jun:137:174-189. doi: 10.1016/j.biochi.2017.03.017. Epub 2017 Mar 27.

Abstract

Thermoalkaliphilic xylanases are highly desired and of great importance due to their vast potential in paper pulp and bleaching processes. Here, we report rapid, cost-effective, and result-oriented combinatorial potential of in silico DNA swapping strategy to engineer the pH optimum of industrially crucial enzymes, particularly engineering of Geobacillus sp. TF16 endoxylanase for alkaline environments. The 3D structures of Geobacillus sp. TF16 and donor Bacillus halodurans C-125 endoxylanases were firstly predicted, analyzed, and compared for their similarities before any in silico design of mutants. Reasonably, to improve its alkaline pH tolerance, the corresponding regions in Geobacillus sp.TF16 endoxylanase were further engineered by swapping with negatively-charged amino acid-rich regions from B. halodurans C-125 endoxylanase. Through only two of four in silico-designed mutants, the optimum pH of GeoTF16 endoxylanase was improved from 8.5 to 10.0. Moreover, as compared to GeoTF16 parental enzyme, both GeoInt3 and GeoInt4 mutants revealed (i) enhanced biobleaching performance, (ii) improved adaptability to alkaline conditions, and (iii) better activity for broader pH range. Unlike GeoTF16 losing activity at pH 11.0 completely, GeoInt4 retained 60% and 40% of its activity at pH 11.0 and 12.0, respectively. Thus, GeoInt4 stands out as a more competent biocatalyst that is suitable for alkaline environments of diverse industrial applications. The current study represents an efficient protein engineering strategy to adapt industrial catalysts to diverse processing conditions. Further comprehensive and fine-tuned research efforts may result in biotechnologically more promising outcomes.

Keywords: Alkaline pH; DNA swapping; Endoxylanase; Geobacillus sp. TF16; In silico design.

MeSH terms

  • Amino Acid Sequence
  • Computer Simulation
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics*
  • Endo-1,4-beta Xylanases / chemistry*
  • Endo-1,4-beta Xylanases / genetics*
  • Endo-1,4-beta Xylanases / metabolism
  • Enzyme Stability
  • Geobacillus / enzymology*
  • Hydrogen-Ion Concentration
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Protein Engineering*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid

Substances

  • DNA, Bacterial
  • Mutant Proteins
  • Recombinant Proteins
  • Endo-1,4-beta Xylanases