Jeong Mi-Young, Kim Sanguk, Yun Cheol-Won, Choi Yong-Jin, Cho Ssang-Goo
School of Life Science and Biotechnology, Korea University, Seoul 136-701, Republic of Korea.
J Biotechnol. 2007 Jan 1;127(2):300-9. doi: 10.1016/j.jbiotec.2006.07.005. Epub 2006 Jul 16.
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.
通过设计一个全新的二硫键来提高嗜热脂肪芽孢杆菌236号内切β-1,4-木聚糖酶（XynA）的热稳定性。首先使用二硫键设计程序（Disulfide by Designtrade mark）进行二硫键设计，以确定具有形成二硫键有利几何特征的残基对。随后，通过对34种11家族嗜温和嗜热木聚糖酶进行比对所鉴定出的进化保守半胱氨酸残基，以及对木聚糖酶分子模型进行检查，对所选的25个氨基酸对进行筛选。最终仅选择了一对（Ser100和Asn150），并将各自的氨基酸替换为半胱氨酸残基。新设计的二硫键使XynA的热稳定性提高了约5摄氏度。失活能垒的增加支持了这种热稳定性的提高。正如预期的那样，突变体XynA SNC在比其天然对应物所允许的温度高得多的温度下对木聚糖水解表现出更好的适用性。该突变对XynA的催化效率和其他功能特性影响很小。总之，显然策略性地引入的二硫键使XynA在抵抗热失活方面更有效。
