title: Enhanced thermostability of a mesophilic xylanase by N-terminal replacement designed by molecular dynamics simulation.
abstract: BACKGROUND
  Xylanases have attracted much attention owing to their potential applications. The applicability of xylanases, however, was bottlenecked by their low stabilities at high temperature or extreme pH. The purpose of this work was to enhance the thermostability of a mesophilic xylanase by N-terminal replacement.
  
  
  RESULTS
  The thermostability of AoXyn11, a mesophilic family 11 xylanase from Aspergillus oryzae, was enhanced by replacing its N-terminal segment with the corresponding one of EvXyn11(TS) , a hyperthermotolerant family 11 xylanase. A hybrid xylanase with high thermostability, NhXyn11⁵⁷, was predicted by molecular dynamics (MD) simulation. An NhXyn11⁵⁷-encoding gene, Nhxyn11⁵⁷, was then constructed as designed theoretically, and overexpressed in Pichia pastoris. The temperature optimum of recombinant NhXyn11⁵⁷ (re-NhXyn11⁵⁷) was 75 °C, much higher than that of re-AoXyn11. Both xylanases were thermostable at 65 and 40 °C, respectively. Additionally, the pH optimum and stability of re-NhXyn11⁵⁷ were 5.5 and at a range of 4.0-8.5. Its activity was not significantly affected by metal ions tested and EDTA, but strongly inhibited by Mn²⁺ and Ag⁺.
  
  
  CONCLUSIONS
  This work obviously enhanced the thermostability of a mesophilic xylanase, making re-NhXyn11⁵⁷ a promising candidate for industrial processes. It also provided an effective technical strategy for improving thermostabilities of other mesophilic enzymes.
authors.affiliation: School of Biotechnology and Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Yin
authors.forename: Jian-Fang
authors.initials: JF
authors.lastname: Li
authors.forename: Jun-Qing
authors.initials: JQ
authors.lastname: Wang
authors.forename: Cun-Duo
authors.initials: CD
authors.lastname: Tang
authors.forename: Min-Chen
authors.initials: MC
authors.lastname: Wu
mesh_terms: D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000302:isolation & purification; D002681:China; D043364:Endo-1,4-beta Xylanases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005511:Food Handling; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008345:Manganese / Q000494:pharmacology; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012834:Silver / Q000494:pharmacology; D012988:Soil Microbiology
chemical_list: D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012834:Silver; D008345:Manganese; D043364:Endo-1,4-beta Xylanases
keywords: Aspergillus oryzae; N-terminal replacement; molecular dynamics simulation; thermostability; xylanase