title: Redesigning pH optimum of Geobacillus sp. TF16 endoxylanase through in silico designed DNA swapping strategy.
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.
authors.affiliation: Department of Molecular Biology and Genetics, Faculty of Science, Karadeniz Technical University, 61080, Trabzon, Turkey; Institute for Plant Genomics and Biotechnology, Texas A&M University, 2123 TAMU, College Station, TX, 77840, USA. Electronic address: uguruzuner@tamu.edu.
authors.forename: Ugur
authors.initials: U
authors.lastname: Uzuner
authors.affiliation: Department of Biology, Faculty of Science, Karadeniz Technical University, 61080, Trabzon, Turkey.
authors.forename: Sabriye
authors.initials: S
authors.lastname: Canakci
authors.affiliation: Department of Molecular Biology and Genetics, Faculty of Science, Karadeniz Technical University, 61080, Trabzon, Turkey.
authors.forename: Kadriye Inan
authors.initials: KI
authors.lastname: Bektas
authors.affiliation: Department of Biology, Faculty of Science, Karadeniz Technical University, 61080, Trabzon, Turkey.
authors.forename: Merve Tuncel
authors.initials: MT
authors.lastname: Sapmaz
authors.affiliation: Department of Biology, Faculty of Science, Karadeniz Technical University, 61080, Trabzon, Turkey.
authors.forename: Ali Osman
authors.initials: AO
authors.lastname: Belduz
mesh_terms: D000595:Amino Acid Sequence; D003198:Computer Simulation; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D056506:Geobacillus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D004269:DNA, Bacterial; D050505:Mutant Proteins; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases
keywords: Alkaline pH; DNA swapping; Endoxylanase; Geobacillus sp. TF16; In silico design