title: Engineering hyperthermostability into a mesophilic family 11 xylanase from Aspergillus oryzae by in silico design of N-terminus substitution.
abstract: A mesophilic xylanase from Aspergillus oryzae CICC40186 (abbreviated to AoXyn11A) belongs to glycoside hydrolase family 11. The thermostability of AoXyn11A was significantly improved by substituting its N-terminus with the corresponding region of a hyperthermostable family 11 xylanase, EvXyn11(TS) . The suitable N-terminus of AoXyn11A to be replaced was selected by the comparison of B-factors between AoXyn11A and EvXyn11(TS) , which were generated and calculated after a 15 ns molecular dynamic (MD) simulation process. Then, the predicted hybrid xylanase (designated AEx11A) was modeled, and subjected to a 2 ns MD simulation process for calculating its total energy value. The N-terminus substitution was confirmed by comparing the total energy value of AEx11A with that of AoXyn11A. Based on the in silico design, the AEx11A was constructed and expressed in Pichia pastoris GS115. After 72 h of methanol induction, the recombinant AEx11A (reAEx11A) activity reached 82.2 U/mL. The apparent temperature optimum of reAEx11A was 80°C, much higher than that of reAoXyn11A. Its half-life was 197-fold longer than that of reAoXyn11A at 70°C. Compared with reAoXyn11A, the reAEx11A displayed a slight alteration in K(m) but a decrease in V(max).
authors.affiliation: School of Medicine and Pharmaceutics, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, PR China.
authors.forename: Shu-Juan
authors.initials: SJ
authors.lastname: Gao
authors.forename: Jun-Qing
authors.initials: JQ
authors.lastname: Wang
authors.forename: Min-Chen
authors.initials: MC
authors.lastname: Wu
authors.forename: Hui-Min
authors.initials: HM
authors.lastname: Zhang
authors.forename: Xin
authors.initials: X
authors.lastname: Yin
authors.forename: Jian-Fang
authors.initials: JF
authors.lastname: Li
mesh_terms: D019943:Amino Acid Substitution; D001236:Aspergillus oryzae / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D005800:Genes, Fungal; D005818:Genetic Engineering; D006358:Hot Temperature; D007700:Kinetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases