Khazaal Kadhim Almansoori Ammar, Reddy Nidyaletchmy Subba, Abdulfattah Mustafa, Ismail Sarah Solehah, Abdul Rahim Rashidah
School of Biological Sciences, Universiti Sains Malaysia, Gelugor, Penang, Malaysia.
Department of Medical Laboratory Techniques, Al-Mustaqbal University, Hillah, Babylon, Iraq.
PLoS One. 2024 Dec 17;19(12):e0314556. doi: 10.1371/journal.pone.0314556. eCollection 2024.
This study focuses on a novel lipase from Bacillus licheniformis IBRL-CHS2. The lipase gene was cloned into the pGEM-T Easy vector, and its sequences were registered in GenBank (KU984433 and AOT80658). It was identified as a member of the bacterial lipase subfamily 1.4. The pCold I vector and E. coli BL21 (DE3) host were utilized for expression, with the best results obtained by removing the enzyme's signal peptide. Optimal conditions were found to be 15°C for 24 h, using 0.2 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). The His-tagged lipase was purified 13-fold with a 68% recovery and a specific activity of 331.3 U/mg using affinity purification. The lipase demonstrated optimal activity at 35°C and pH 7. It remained stable after 24 h in 25% (v/v) organic solvents such as isooctane, n-hexane, dimethyl sulfoxide (DMSO), and methanol, which enhanced its activity. Chloroform and diethyl ether inhibited the lipase. The enzyme exhibited the highest affinity for p-nitrophenol laurate (C12:0) with a Km of 0.36 mM and a Vmax of 357 μmol min-1 mg-1. Among natural oils, it performed best with coconut oil and worst with olive oil. The lipase was stable in the presence of 1 mM and 5 mM Ca2⁺, K⁺, Na⁺, Mg2⁺, and Ba2⁺, but its activity decreased with Zn2⁺ and Al3⁺. Non-ionic surfactants like Triton X-100, Nonidet P40, Tween 20, and Tween 40 boosted activity, while Sodium Dodecyl Sulfate (SDS) inhibited it. This lipase's unique properties, particularly its stability in organic solvents, make it suitable for applications in organic synthesis and various industries.
本研究聚焦于地衣芽孢杆菌IBRL-CHS2产生的一种新型脂肪酶。该脂肪酶基因被克隆到pGEM-T Easy载体中，其序列已在GenBank注册（KU984433和AOT80658）。它被鉴定为细菌脂肪酶亚家族1.4的成员。使用pCold I载体和大肠杆菌BL21 (DE3) 宿主进行表达，去除酶的信号肽后获得最佳结果。发现最佳条件为15°C孵育24 h，使用0.2 mM异丙基-β-D-硫代半乳糖苷（IPTG）。通过亲和纯化，His标签脂肪酶的纯化倍数为13倍，回收率为68%，比活性为331.3 U/mg。该脂肪酶在35°C和pH 7时表现出最佳活性。在25%（v/v）的有机溶剂（如异辛烷、正己烷、二甲基亚砜（DMSO）和甲醇）中24 h后仍保持稳定，这些有机溶剂还增强了其活性。氯仿和乙醚抑制该脂肪酶。该酶对月桂酸对硝基苯酯（C12:0）表现出最高亲和力，Km为0.36 mM，Vmax为357 μmol min-1 mg-1。在天然油脂中，它对椰子油的作用最佳，对橄榄油的作用最差。该脂肪酶在1 mM和5 mM的Ca2⁺、K⁺、Na⁺、Mg2⁺和Ba2⁺存在下稳定，但其活性随Zn2⁺和Al3⁺而降低。非离子表面活性剂如Triton X-100、Nonidet P40、吐温20和吐温40可提高活性，而十二烷基硫酸钠（SDS）则抑制活性。这种脂肪酶的独特性质，特别是其在有机溶剂中的稳定性，使其适用于有机合成和各种工业应用。
