Engineering a de novo internal disulfide bridge to improve the thermal stability of xylanase from Bacillus stearothermophilus No. 236.
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.
Jeong
Mi-Young
MY
School of Life Science and Biotechnology, Korea University, Seoul 136-701, Republic of Korea.
Kim
Sanguk
S
Yun
Cheol-Won
CW
Choi
Yong-Jin
YJ
Cho
Ssang-Goo
SG
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
D004220:Disulfides; D050505:Mutant Proteins; D013438:Sulfhydryl Compounds; D014990:Xylans; D043364:Endo-1,4-beta Xylanases