title: Improvement of the thermostability and catalytic activity of a mesophilic family 11 xylanase by N-terminus replacement.
abstract: To improve the thermostability and catalytic activity of Aspergillus niger xylanase A (AnxA), its N-terminus was substituted with the corresponding region of Thermomonospora fusca xylanase A (TfxA). The constructed hybrid xylanase, named ATx, was overexpressed in Pichia pastoris and secreted into the medium. After 96-h 0.25% methanol induction, the activity of the ATx in the culture supernatant reached its peak, 633 U/mg, which was 3.6 and 5.4 times as high as those of recombinant AnxA (reAnxA) and recombinant TfxA (reTfxA), respectively. Studies on enzymatic properties showed that the temperature and pH optimum of the ATx were 60 degrees C and 5.0, respectively. The ATx was more thermostable, when it was treated at 70 degrees C, pH 5.0, for 2 min, the residual activity was 72% which was higher than that of reAnxA and similar to that of reTfxA. The ATx was very stable over a broader pH range (3.0-10.0) and much less affected by acid/base conditions. After incubation at pH 3.0-10.0, 25 degrees C for 1 h, all the residual activities of the ATx were over 80%. These results revealed that the thermostability and catalytic activity of the AnxA were enhanced. The N-terminus of TfxA contributed to the observed thermostability of itself and the ATx, and to the high activity of the ATx. Replacement of N-terminus between mesophilic eukaryotic and thermostable prokaryotic enzymes may be a useful method for constructing the new and improved versions of biologically active enzymes.
authors.affiliation: Microbiology Division, Feed Science Institute, College of Animal Science, Zhejiang University, Hangzhou, China. jysun@zju.edu.cn
authors.forename: Jian-Yi
authors.initials: JY
authors.lastname: Sun
authors.forename: Ming-Qi
authors.initials: MQ
authors.lastname: Liu
authors.forename: Ying-Lei
authors.initials: YL
authors.lastname: Xu
authors.forename: Zi-Rong
authors.initials: ZR
authors.lastname: Xu
authors.forename: Lin
authors.initials: L
authors.lastname: Pan
authors.forename: Hui
authors.initials: H
authors.lastname: Gao
mesh_terms: D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D011993:Recombinant Fusion Proteins; D043364:Endo-1,4-beta Xylanases