title: Creation of thermostable and alkaline stable xylanase variants by DNA shuffling.
abstract: Mutant xylanases, G41 and G53, were generated by random mutagenesis of Thermomyces lanuginosus xylanase DSM 5826 (xynA) in a previous study. Incubation at 90 min showed that G41 had 75% activity at 80 °C and G53 had 93% activity at pH 10. In order to create xylanase variants possessing both thermal and alkaline stability in a single enzyme, G41 and G53 served as templates for DNA shuffling using the StEP recombination method. One of the resulting StEP recombinants, S340, retained 54% stability at 80 °C and 60% stability at pH 10 with three resulting amino acid mutations. Another StEP recombinant, S325, displayed 85% stability at 80 °C and 60% stability at pH 10 and DNA sequencing showed that it inherited mutations from both parents. All thermostable variants displayed an increase in arginine content with poor enzyme activity. Thus, the StEP recombination method successfully recombined mutations into two xylanases that were more robust than their parent counterparts. Additionally, the 3D-models of the wild type T. lanuginosus xynA (xyl_ext) and its variants, G41 and S325, were predicted using I-TASSER and then subjected to molecular dynamics (MD) simulations at 300 K for a deeper understanding of their structural features. The results from the predicted 3D models show clearly the presence of α-helical regions in the N-terminal residues of the xyl_ext, G41 and S325. Moreover, the MD analysis suggests that the presence of additional residues (1-31) and point mutation induces slight structural changes with the stability of the protein being evenly distributed over the whole structure.
authors.affiliation: Department of Biotechnology and Food Technology, ML Sultan Campus, Durban University of Technology, Durban, South Africa. Electronic address: dawnestephens@yahoo.com.
authors.forename: Dawn Elizabeth
authors.initials: DE
authors.lastname: Stephens
authors.affiliation: Department of Biotechnology and Food Technology, ML Sultan Campus, Durban University of Technology, Durban, South Africa; Department of Chemistry, Steve Biko Campus, Durban University of Technology, Durban, South Africa.
authors.forename: Faez Iqbal
authors.initials: FI
authors.lastname: Khan
authors.affiliation: Department of Chemistry, Steve Biko Campus, Durban University of Technology, Durban, South Africa.
authors.forename: Parvesh
authors.initials: P
authors.lastname: Singh
authors.affiliation: Department of Chemistry, Steve Biko Campus, Durban University of Technology, Durban, South Africa.
authors.forename: Krishna
authors.initials: K
authors.lastname: Bisetty
authors.affiliation: Department of Biotechnology and Food Technology, ML Sultan Campus, Durban University of Technology, Durban, South Africa.
authors.forename: Suren
authors.initials: S
authors.lastname: Singh
authors.affiliation: Department of Biotechnology and Food Technology, ML Sultan Campus, Durban University of Technology, Durban, South Africa.
authors.forename: Kugen
authors.initials: K
authors.lastname: Permaul
mesh_terms: D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics*; D032621:DNA Shuffling / Q000379:methods*; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D013696:Temperature
chemical_list: D043364:Endo-1,4-beta Xylanases
keywords: DNA shuffling; Stability; Staggered extension process; T. lanuginosus; Xylanase