title: A unique disulfide bridge of the thermophilic xylanase SyXyn11 plays a key role in its thermostability.
abstract: Based on the hyperthermostable family 11 xylanase (EvXyn11(TS)) gene sequence (EU591743), the gene Syxyn11 encoding a thermophilic xylanase SyXyn11 was synthesized with synonymous codons biasing towards Pichia pastoris. The homology alignment of primary structures among family 11 xylanases revealed that, at their N-termini, only SyXyn11 contains a disulfide bridge (Cys5-Cys32). This to some extent implied the significance of the disulfide bridge of SyXyn11 to its thermostability. To confirm the correlation between the N-terminal disulfide bridge and thermostability, a SyXyn11(C5T)-encoding gene, Syxyn11(C5T), was constructed by mutating the Cys5 codon of Syxyn11 to Thr5. Then, the genes for the recombinant xylanases, reSyXyn11 and reSyXyn11(C5T), were expressed in P. pastoris GS115, yielding xylanase activity of about 35 U per ml cell culture. Both xylanases were purified to homogeneity with specific activities of 363 and 344 U/mg, respectively. The temperature optimum and stability of reSyXyn11(C5T) decreased to 70 and 50°C from 85 and 80°C of reSyXyn11, respectively. There was no obvious change in pH characteristics.
authors.affiliation: School of Biotechnology and Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China. yinxinat2008@126.com.
authors.forename: X
authors.initials: X
authors.lastname: Yin
authors.forename: Y
authors.initials: Y
authors.lastname: Yao
authors.forename: M C
authors.initials: MC
authors.lastname: Wu
authors.forename: T D
authors.initials: TD
authors.lastname: Zhu
authors.forename: Y
authors.initials: Y
authors.lastname: Zeng
authors.forename: Q F
authors.initials: QF
authors.lastname: Pang
mesh_terms: D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D004220:Disulfides; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases