Department of Chemistry, The University of Azad Jammu and Kashmir, Muzaffarabad, Pakistan.
Department of Zoology, The University of Azad Jammu and Kashmir, Muzaffarabad, Pakistan.
PLoS One. 2022 Aug 30;17(8):e0273368. doi: 10.1371/journal.pone.0273368. eCollection 2022.
Five Bacillus cereus strains including B. cereus AVP12, B. cereus NC7401, B. cereus BDBCO1, B. cereus JF70 and B. specie JL47 isolated from the diesel fuel polluted soil adhered to the roots of Tagetes minuta were screened for lipase production with phenol red agar method. B. cereus NC7401 strain successfully expressing and secreting lipase with maximal lipolytic activity was subjected to a submerged fermentation process with five different carbon (starch, glucose, maltose, fructose, and lactose) and five different nitrogen (tryptone, ammonium nitrate, peptone, urea, yeast extract) sources to produce lipase enzyme. Maximum enzyme activity was found with starch (30.6 UmL-1), maltose (40 UmL-1), and tryptone (38.6 UmL-1), and the lipases produced using these sources were named lipase A, B, and C respectively. The total protein content of 8.56, 8.86, and 2.75 μg mL-1 were obtained from B. cereus NC7401 cultured using starch, maltose, and tryptone respectively. Lipase was stable between temperature range 30-80°C and pH 5-10 whereas optimally active at 55°C and pH 8.0. The enzyme was relatively stable for 10 days at 4°C and its optimum reaction time with the substrate was 30 minutes. It was tolerant to 1.5% (v/v) methanol as an organic solvent, 1.5% (v/v) Triton X-100 as a media additive and 1.5% (w/v) Ni2+ as a metal ion. SDS, n-hexane, and Ag+ inhibited lipolytic activity. Oil stains were removed from cotton fabric which showed oil removal efficiency enhancement in the presence of a lipase. Fat hydrolysis of 20, 24, and 30% was achieved following 6 hours of incubation of the fat particles with lipase A, B, and C respectively at a concentration of 20 mg mL-1. To as best of our knowledge, this study on lipases extracted from bacteria of Azad Kashmir, Pakistan origin has never been reported before.
从柴油污染土壤中附着在 Tagetes minuta 根部的 5 株蜡状芽孢杆菌（包括 B. cereus AVP12、B. cereus NC7401、B. cereus BDBCO1、B. cereus JF70 和 B. specie JL47）中筛选出了具有脂肪酶生产能力的菌株，采用苯酚红琼脂法进行筛选。成功表达和分泌脂肪酶并具有最大脂肪酶活性的 B. cereus NC7401 菌株在五种不同的碳（淀粉、葡萄糖、麦芽糖、果糖和乳糖）和五种不同的氮（胰蛋白胨、硝酸铵、蛋白胨、尿素和酵母提取物）源的浸出发酵过程中产生脂肪酶。在使用淀粉、麦芽糖和胰蛋白胨作为碳源时，脂肪酶的活性最高，分别为 30.6 UmL-1、40 UmL-1 和 38.6 UmL-1，分别将这三种来源产生的脂肪酶命名为脂肪酶 A、B 和 C。从 B. cereus NC7401 培养液中分别获得了 8.56、8.86 和 2.75 μg mL-1 的总蛋白含量。脂肪酶在 30-80°C 的温度范围内和 pH 5-10 的条件下稳定，而在 55°C 和 pH 8.0 时活性最佳。在 4°C 下，该酶可稳定保存 10 天，与底物反应的最佳时间为 30 分钟。它可以耐受 1.5%（v/v）甲醇作为有机溶剂、1.5%（v/v）Triton X-100 作为培养基添加剂和 1.5%（w/v）Ni2+作为金属离子。SDS、正己烷和 Ag+抑制脂肪酶的脂肪水解活性。脂肪酶处理过的棉织物上的油斑被去除，显示出在脂肪酶存在下去污效率提高。脂肪颗粒在浓度为 20 mg mL-1 的条件下分别与脂肪酶 A、B 和 C 孵育 6 小时后，脂肪水解率分别达到 20%、24%和 30%。据我们所知，这是首次报道从巴基斯坦阿扎德克什米尔地区细菌中提取的脂肪酶的研究。
