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


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UZUNER U. , ÇANAKÇI S. , Bektas K. I. , SAPMAZ M. T. , BELDÜZ A. O.

BIOCHIMIE, cilt.137, ss.174-189, 2017 (SCI İndekslerine Giren Dergi) identifier identifier identifier

Özet

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 Geolnt3 and Geolnt4 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, Geolnt4 retained 60% and 40% of its activity at pH 11.0 and 12.0, respectively. Thus, Geolnt4 stands out as a more competent biocatalyst that is suitable for alkaline environments of diverse industrial applications.