Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
Enzyme Microb Technol. 2013 Dec 10;53(6-7):386-90. doi: 10.1016/j.enzmictec.2013.08.004. Epub 2013 Sep 19.
In lipases, oxyanion hole has crucial role in the stabilisation of enzyme-substrate complex. Majority of lipases from Yarrowia lipolytica consist of two oxyanion hole residues viz.; Thr and Leu. However, Lip12 has Phe instead of Leu at second oxyanion hole residue. It was observed that Lip12 has lower specific activity and catalytic efficiency than other lipases of Yarrowia. In silico analysis of Phe to Leu mutation revealed improved binding energy of Lip12 for p-np palmitate. This was validated by Phe148 to Leu point mutation where, specific activity of mutant was 401U/mg on olive oil, which was two fold higher in comparison to wild-type. Kcat, remained unaltered, while decrease in Km was predominant for all the substrates used in the study. Improved catalytic efficiency of mutant was a function of chain length in case of p-np esters, with 73% improvement for p-np stearate. However, hydrolysis of triacylglycerides improved by 20%, irrespective of chain length. Decrease in activation energy for all the substrates, was observed in mutant in comparison to wild-type, indicating better stabilisation of transition state complex. Further, unaltered differential activation energy for mutant depicts that substrate specificity of enzyme remained same after mutation.
在脂肪酶中，氧阴离子空穴在稳定酶-底物复合物方面起着至关重要的作用。大多数来自假丝酵母的脂肪酶由两个氧阴离子空穴残基组成，即 Thr 和 Leu。然而，Lip12 在第二个氧阴离子空穴残基处有 Phe 而不是 Leu。研究发现，Lip12 的比活性和催化效率均低于假丝酵母的其他脂肪酶。对 Phe 到 Leu 突变的计算机分析表明，Lip12 与 p-np 棕榈酸的结合能得到了改善。通过对突变体的 Phe148 到 Leu 点突变进行验证，突变体在橄榄油上的比活性为 401U/mg，比野生型高两倍。Kcat 保持不变，而 Km 的降低在研究中使用的所有底物中均占主导地位。突变体的催化效率提高是 p-np 酯的链长的函数，p-np 硬脂酸的提高了 73%。然而，三酰基甘油的水解提高了 20%，与链长无关。与野生型相比，突变体的所有底物的活化能均降低，表明过渡态复合物的稳定性更好。此外，突变体的未改变的差分活化能表明，酶的底物特异性在突变后保持不变。
