title: Obtaining cellulose binding and hydrolyzing activity of a family 11 hybrid xylanase by fusion with xylan binding domain.
abstract: The xylan binding domain (XBD) and linker sequences (LS) from thermostable and thermophilic Thermomonospora fusca xylanase A (TfxA) was fused to the carboxyl-terminus of a family 11 hybrid xylanase ATx. The constructed chimera (ATxX) was successfully expressed in Pichia pastoris, partially purified to homogeneity, and then characterized in detail. After 96-h 0.25% methanol induction, the xylanase and cellulose activity of ATxX from pPATxX1 transformant culture medium supernatant were 452.1 U/mg and 19.3 U/mg, respectively. SDS-PAGE analysis revealed that the molecular mass of ATxX was about 33.01 kDa. 3.7% ATxX was bound after incubation with 1% microcrystal cellulose at 25 °C for 3 h, while the ATx did not show cellulose binding-hydrolyzing ability. These results suggested that ATx obtained cellulose binding and hydrolyzing ability by fusing with XBD and LS. Enzymatic studies showed that the temperature and pH optimum of the ATxX xylanase activity were 60 °C and pH 5.0, respectively, which were the same as that of ATx. The temperature and pH optimum of the ATxX cellulase activity were 60 °C and pH 6.0, respectively. The major hydrolytic products released by ATxX from birchwood xylan were xylotriose and xylohexaose. Xylooligosaccharides from xylobiose to xylohexaose could be hydrolyzed by ATxX. Mode of action analysis showed that the chimeric ATxX was an endo-acting enzyme. The XBD and LS plays an important role in the binding and hydrolyzing of xylanase to insoluble substrates.
authors.affiliation: College of Life Science, China Jiliang University, Hangzhou 310018, China. tony_cjlu@yahoo.com.cn
authors.forename: Ming-Qi
authors.initials: MQ
authors.lastname: Liu
authors.forename: Xian-Jun
authors.initials: XJ
authors.lastname: Dai
authors.forename: Guang-Fu
authors.initials: GF
authors.lastname: Liu
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
mesh_terms: D000192:Actinomycetales / Q000201:enzymology*; D002482:Cellulose / Q000737:chemistry; D043364:Endo-1,4-beta Xylanases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D010843:Pichia; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D014990:Xylans / Q000737:chemistry / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D014990:Xylans; D002482:Cellulose; D043364:Endo-1,4-beta Xylanases; C109691:microcrystalline cellulose