Kamondi Szilárd, Szilágyi András, Barna László, Závodszky Péter
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Pf. 7, H-1518 Budapest, Hungary.
Biochem Biophys Res Commun. 2008 Oct 3;374(4):725-30. doi: 10.1016/j.bbrc.2008.07.095. Epub 2008 Jul 27.
A possible approach to generate enzymes with an engineered temperature optimum is to create chimeras of homologous enzymes with different temperature optima. We tested this approach using two family-10 xylanases from Thermotoga maritima: the thermophilic xylanase A catalytic domain (TmxAcat, T(opt)=68 degrees C), and the hyperthermophilic xylanase B (TmxB, T(opt)=102 degrees C). Twenty-one different chimeric constructs were created by mimicking family shuffling in a rational manner. The measured temperature optima of the 16 enzymatically active chimeras do not monotonically increase with the percentage of residues coming from TmxB. Only four chimeras had a higher temperature optimum than TmxAcat, the most stable variant (T(opt)=80 degrees C) being the one in which both terminal segments came from TmxB. Further analysis suggests that the interaction between the N- and C-terminal segments has a disproportionately high contribution to the overall thermostability. The results may be generalizable to other enzymes where the N- and C-termini are in contact.
一种产生具有工程化最适温度的酶的可能方法是构建具有不同最适温度的同源酶的嵌合体。我们使用来自嗜热栖热菌的两种10家族木聚糖酶测试了这种方法：嗜热木聚糖酶A催化结构域（TmxAcat，T（opt）=68℃）和超嗜热木聚糖酶B（TmxB，T（opt）=102℃）。通过合理模拟家族改组创建了21种不同的嵌合构建体。16种具有酶活性的嵌合体的实测最适温度并非随着来自TmxB的残基百分比单调增加。只有四种嵌合体的最适温度高于TmxAcat，最稳定的变体（T（opt）=80℃）是两个末端片段均来自TmxB的嵌合体。进一步分析表明，N端和C端片段之间的相互作用对整体热稳定性的贡献 disproportionately 高。这些结果可能适用于其他N端和C端相互接触的酶。
