Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
Protein J. 2012 Mar;31(3):229-37. doi: 10.1007/s10930-012-9395-8.
A mutant of the lipase from Geobacillus sp. strain T1 with a phenylalanine to leucine substitution at position 16 was overexpressed in Escherichia coli strain BL21(De3)pLysS. The crude enzyme was purified by two-step affinity chromatography with a final recovery and specific activity of 47.4 and 6,315.8 U/mg, respectively. The molecular weight of the purified F16L lipase was approximately 43 kDa by 12% SDS-PAGE analysis. The F16L lipase was demonstrated to be a thermophilic enzyme due its optimum temperature at 70 °C and showed stability over a temperature range of 40-60 °C. The enzyme exhibited an optimum pH 7 in phosphate buffer and was relatively stable at an alkaline pH 8-9. Metal ions such as Ca(2+), Mn(2+), Na(+), and K(+) enhanced the lipase activity, but Mg(2+), Zn(2+), and Fe(2+) inhibited the lipase. All surfactants tested, including Tween 20, 40, 60, 80, Triton X-100, and SDS, significantly inhibited the lipolytic action of the lipase. A high hydrolytic rate was observed on long-chain natural oils and triglycerides, with a notable preference for olive oil (C18:1; natural oil) and triolein (C18:1; triglyceride). The F16L lipase was deduced to be a metalloenzyme because it was strongly inhibited by 5 mM EDTA. Moderate inhibition was observed in the presence of PMSF at a similar concentration, indicating that serine residues are involved in its catalytic action. Further, the activity was not impaired by water-miscible solvents, including methanol, ethanol, and acetone.
来自地芽孢杆菌 T1 菌株的脂肪酶突变体在位置 16 处的苯丙氨酸被亮氨酸取代，在大肠杆菌 BL21(De3)pLysS 中过表达。粗酶通过两步亲和层析纯化，最终回收率和比活分别为 47.4 和 6315.8 U/mg。通过 12%SDS-PAGE 分析，纯化的 F16L 脂肪酶的分子量约为 43 kDa。由于最适温度为 70°C，F16L 脂肪酶被证明是一种嗜热酶，并在 40-60°C 的温度范围内表现出稳定性。该酶在磷酸盐缓冲液中表现出最佳 pH 值 7，在碱性 pH 值 8-9 下相对稳定。Ca(2+)、Mn(2+)、Na(+)和 K(+)等金属离子增强了脂肪酶的活性，但 Mg(2+)、Zn(2+)和 Fe(2+)抑制了脂肪酶。测试的所有表面活性剂，包括 Tween 20、40、60、80、Triton X-100 和 SDS，都显著抑制了脂肪酶的脂肪分解作用。在长链天然油和甘油三酯上观察到高水解速率，对橄榄油（C18:1；天然油）和三油酸甘油酯（C18:1；甘油三酯）表现出明显的偏好。F16L 脂肪酶被推断为金属酶，因为它被 5 mM EDTA 强烈抑制。在类似浓度下存在 PMSF 时观察到适度抑制，表明丝氨酸残基参与其催化作用。此外，活性不受甲醇、乙醇和丙酮等水溶性溶剂的损害。
