title: Engineering a de novo internal disulfide bridge to improve the thermal stability of xylanase from Bacillus stearothermophilus No. 236.
abstract: Improvement of thermal stability of the Bacillus stearthermophilus No. 236 endo-beta-1,4-xylanase (XynA) was tried by engineering a de novo designed disulfide bridge. Disulfide design was performed firstly using the disulfide bond design program (Disulfide by Designtrade mark) to identify residue pairs having the favorable geometric characteristics for disulfide formation. Subsequently, the selected 25 amino acid pairs were filtered with the evolutionarily conserved Cys residues identified by alignment of 34 family 11 mesophilic and thermophilic xylanases, and also by doing inspection of the molecular model of the xylanases. Only one pair (Ser100 and Asn150) was finally chosen, and the respective amino acids were substituted with cysteine residues. The newly designed disulfide bridge increased thermostability of the XynA about 5 degrees C. This improved thermal stability was supported by the increase in the energy barrier for inactivation. As expected, the mutant XynA SNC demonstrated its better use in the hydrolysis of xylan at substantially higher temperatures than permitted by its native counterpart. The mutation had little influence on the catalytic efficiency and other functional properties of the XynA. In conclusion, it is evident that the strategically placed disulfide bridge has made the XynA be more effective in resisting thermal inactivation.
authors.affiliation: School of Life Science and Biotechnology, Korea University, Seoul 136-701, Republic of Korea.
authors.forename: Mi-Young
authors.initials: MY
authors.lastname: Jeong
authors.forename: Sanguk
authors.initials: S
authors.lastname: Kim
authors.forename: Cheol-Won
authors.initials: CW
authors.lastname: Yun
authors.forename: Yong-Jin
authors.initials: YJ
authors.lastname: Choi
authors.forename: Ssang-Goo
authors.initials: SG
authors.lastname: Cho
mesh_terms: D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006358:Hot Temperature; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013816:Thermodynamics; D017186:Titrimetry; D014990:Xylans / Q000378:metabolism
chemical_list: D004220:Disulfides; D050505:Mutant Proteins; D013438:Sulfhydryl Compounds; D014990:Xylans; D043364:Endo-1,4-beta Xylanases