Singh Yogesh, Gupta Namita, Verma Ved Vrat, Goel Manisha, Gupta Rani
Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
Department of Biophysics, University of Delhi South Campus, New Delhi 110021, India.
Biochem Biophys Res Commun. 2016 Mar 25;472(1):223-30. doi: 10.1016/j.bbrc.2016.01.189. Epub 2016 Feb 27.
TALipB (33 kDa) is a solvent stable, enantioselective lipase from Trichosporon asahii MSR54. It is cysteine-rich and shows activation in presence of thiol reducing agents. DIANNA server predicted three disulphide bridges C53-C195 (S1), C89-C228 (S2) and C164-C254 (S3) in the enzyme. Selective disruption of disulphide bonds by cysteine to alanine mutations at Cys53 and Cys89 of S1 and S2 bonds resulted in enzyme activation. Mutant mTALipB (S1+S2) showed increase in specific activity by ∼4-fold (834 mM/mg) and improved Vmax of 6.27 μmol/ml/min at 40 °Con pNP caprate. Temperature optima of mTALipB shifted from 50 to 40 °C and activation energy decreased by 0.7 kcal mol(-1). However, the mutant was less thermostable with a t1/2 of 18 min at 60 °C as compared to t1/2 of 38 min for the native enzyme. Mutant also displayed an improved activity on all pNP esters and higher enantiomeric excess (61%) during esterification of (±) 1-phenylethanol. Far-UV CD analysis showed significant changes in secondary structure after S-S bridge disruption with 7.16% decrease in α-helices and 1.31% increase in β-sheets. In silico analysis predicted two lids (α5 and α9) in TALipB. Molecular dynamic simulations at 40 °C and 50 °C revealed that in the mTALipB, both the lids opened at 40 °C with clockwise and anticlockwise rotations in Lid1 and Lid2, respectively. In the native protein, however, the lid was only partially open even at 50 °C. Concomitant to lid flexibility, there was an extension of accessible catalytic triad surface area resulting in improved catalytic efficiency of the mutant enzyme.
TALipB（33 kDa）是一种来自浅黄金色毛孢子菌MSR54的溶剂稳定型对映选择性脂肪酶。它富含半胱氨酸，在硫醇还原剂存在下会被激活。DIANNA服务器预测该酶中有三个二硫键，分别为C53 - C195（S1）、C89 - C228（S2）和C164 - C254（S3）。通过将S1和S2键的半胱氨酸53和半胱氨酸89突变为丙氨酸来选择性破坏二硫键，导致酶被激活。突变体mTALipB（S1 + S2）的比活性提高了约4倍（834 mM/mg），在40℃下对对硝基苯酚癸酸酯的Vmax提高到6.27 μmol/ml/min。mTALipB的最适温度从50℃变为40℃，活化能降低了0.7 kcal mol⁻¹。然而，与天然酶在60℃下38分钟的半衰期相比，该突变体在60℃下的半衰期为18分钟，热稳定性较差。该突变体对所有对硝基苯酚酯也表现出更高的活性，在（±）1 - 苯乙醇的酯化反应中对映体过量更高（61%）。远紫外圆二色性分析表明，二硫键破坏后二级结构发生了显著变化，α - 螺旋减少了7.16%，β - 折叠增加了1.31%。计算机模拟分析预测TALipB中有两个盖子（α5和α9）。40℃和50℃下的分子动力学模拟表明，在mTALipB中，两个盖子在40℃时打开，Lid1和Lid2分别顺时针和逆时针旋转。然而，在天然蛋白质中，即使在50℃时盖子也只是部分打开。与盖子的灵活性相伴的是，可及的催化三联体表面积增加，导致突变酶的催化效率提高。
