Ma'ruf Ilma Fauziah, Widhiastuty Made Puspasari, Moeis Maelita Ramdani
Biochemistry Research Group, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Indonesia.
Genetic and Molecular Biotechnology Research Group, School of Life Sciences and Technology, Institut Teknologi Bandung, Indonesia.
Biotechnol Rep (Amst). 2021 Jan 16;29:e00590. doi: 10.1016/j.btre.2021.e00590. eCollection 2021 Mar.
Mutant of lipase at oxyanion hole (H110 F) was constructed. The gene was highly expressed in BL21 (DE3) and the recombinant protein was purified using Ni-NTA affinity chromatography. The activity of mutant enzyme was significantly increased compared to that the wild type. Further comparison showed that both of the enzymes exhibited same optimum pH and temperature, and showed highest lipolytic activity on pNP-decanoate (C10). The wild type appeared lost of activity on C14 and C16 substrates meanwhile the mutant still showed activity up to 20 %. In the presence of non polar organic solvent such as n-hexane, the wild type became inactive enzyme meanwhile the mutant still remained 50 % of its activity. The results suggested that mutation at oxyanion hole (H110 F) caused enzyme-substrate interaction change resulting on elevation of activity, better activity toward longer carbon chain substrate and improving the activity in the present of non polar organic solvent.
构建了氧阴离子空穴处脂肪酶的突变体（H110F）。该基因在BL21(DE3)中高表达，重组蛋白通过镍-氮三乙酸亲和层析法纯化。与野生型相比，突变酶的活性显著增加。进一步比较表明，两种酶都表现出相同的最适pH和温度，并且对对硝基苯癸酸酯（C10）表现出最高的脂解活性。野生型在C14和C16底物上失去活性，而突变体仍表现出高达20%的活性。在正己烷等非极性有机溶剂存在的情况下，野生型变成无活性的酶，而突变体仍保留其50%的活性。结果表明，氧阴离子空穴处的突变（H110F）导致酶-底物相互作用发生变化，从而提高了活性，对更长碳链底物具有更好的活性，并提高了在非极性有机溶剂存在下的活性。
