Wang Qin, Xia Tao
College of Life Science and Biotechnology, Shanghai Jiaotong University, 800 Rm. 211, No. 3 Biopharmacology Building, 800 Dongchuan Road, Shanghai, 200240, P.R. China.
Biotechnol Lett. 2008 May;30(5):937-44. doi: 10.1007/s10529-007-9508-1. Epub 2008 Feb 22.
Directed evolution has been used to enhance the catalytic activity and alkaline pH stability of Thermobifida fusca xylanase A, which is one of the most thermostable xylanases. Under triple screened traits of activity, alkaline pH stability and thermostability, through two rounds of random mutagenesis using DNA shuffling, a mutant 2TfxA98 with approximately 12-fold increased k(cat)/K(m) and 4.5-fold decreased K(m) compared with its parent was obtained. Moreover, the alkaline pH stability of 2TfxA98 is increased significantly, with a thermostability slightly lower than that of its parent. Five amino acid substitutions (T21A, G25P, V87P, I91T, and G217L), three of them are near the catalytic active site, were identified by sequencing the genes encoding this evolved enzyme. The activity and stabilizing effects of each amino acid mutation in the evolved enzyme were evaluated by site-directed mutagenesis. This study shows a useful approach to improve the catalytic activity and alkaline pH stability of T. fusca xylanase A toward the hydrolysis of xylan.
定向进化已被用于提高嗜热栖热放线菌木聚糖酶A的催化活性和碱性pH稳定性，该酶是最耐热的木聚糖酶之一。在活性、碱性pH稳定性和热稳定性这三个筛选性状下，通过两轮使用DNA改组的随机诱变，获得了一个突变体2TfxA98，其k(cat)/K(m)比其亲本增加了约12倍，K(m)降低了4.5倍。此外，2TfxA98的碱性pH稳定性显著提高，热稳定性略低于其亲本。通过对编码这种进化酶的基因进行测序，鉴定出五个氨基酸取代（T21A、G25P、V87P、I91T和G217L），其中三个位于催化活性位点附近。通过定点诱变评估了进化酶中每个氨基酸突变的活性和稳定作用。这项研究展示了一种提高嗜热栖热放线菌木聚糖酶A对木聚糖水解的催化活性和碱性pH稳定性的有效方法。
