Graduate Program of Bioengineering, Seoul National University, Seoul 151-742, South Korea.
Appl Biochem Biotechnol. 2013 Jun;170(4):925-33. doi: 10.1007/s12010-013-0237-8. Epub 2013 Apr 28.
The activity of Candida antarctica lipase B was improved by mutation of the area surrounding the active site. We changed the edges of four helices surrounding the active site to flexible amino acids. Two mutants, V139E and I255E, obtained as a result of Pichia pastoris expression, showed enhanced specific activity of 9.9 and 8.1 U/mg while that of wild type was 2.3 U/mg for p-nitrophenyl caprylate hydrolysis. It was nearly 5.4-fold and 3.5-fold, respectively. The stability of both mutants on organic solvent was slightly decreased but almost similar with that of wild type. In the kinetic assay, k(cat) values were shown as dominant factor for the enhancement of catalytic efficiency, k(cat)/K(m), since it was 4.1-fold and 3.8-fold, respectively.
通过突变活性位点周围区域，提高了南极假丝酵母脂肪酶 B 的活性。我们将围绕活性位点的四条螺旋的边缘改变为灵活的氨基酸。通过毕赤酵母表达获得的两个突变体 V139E 和 I255E，其对 p-硝基苯辛酸酯水解的比活性分别提高了 9.9 和 8.1 U/mg，而野生型的比活性为 2.3 U/mg。分别提高了近 5.4 倍和 3.5 倍。两种突变体在有机溶剂中的稳定性略有下降，但与野生型几乎相同。在动力学测定中，kcat 值是提高催化效率的主要因素，kcat/Km，分别提高了 4.1 倍和 3.8 倍。
