Abo-Kamer Amal M, Abdelaziz Ahmed A, Elkotb Esraa S, Al-Madboly Lamiaa A
Department of Microbiology and Immunology, Faculty of Pharmacy, Tanta University, Tanta, Egypt.
Microb Cell Fact. 2025 Mar 8;24(1):58. doi: 10.1186/s12934-025-02671-7.
This study aimed to isolate and optimize a high-yield lipase-producing Pseudomonas aeruginosa strain from biological samples, enhance enzyme production through random mutagenesis, and evaluate its potential anticancer activity. Fifty-one biological samples (blood, urine, sputum, wound pus) were screened, and three isolates demonstrated significant lipase activity. The isolate with the highest activity, identified as P. aeruginosa (GenBank accession number PP436388), was subjected to ethidium bromide-induced mutagenesis, resulting in a two-fold increase in lipase activity (312 U/ml). Lipase production was optimized using submerged fermentation, with critical factors identified statistically as Tween 80, peptone, and substrate concentration. The enzyme was purified via ammonium sulfate precipitation and Sephadex G-100 chromatography, and its molecular weight (53 kDa) was confirmed by SDS-PAGE.
Optimal conditions for enzyme production included a pH of 9, temperature of 20 °C, and a 24-h incubation period. The partially purified enzyme exhibited high stability at pH values up to 10 and storage temperatures of 4 °C. Anticancer activity was evaluated using the MTT assay, revealing an IC of 78.21 U/ml against human hepatocellular carcinoma using HepG-2 cells, with no cytotoxicity observed against Vero cells. Flow cytometry confirmed that the enzyme's anticancer potential was mediated through apoptosis and necrosis. QRT-PCR data revealed that the expression of the Bcl-2 gene was significantly downregulated by 62% (P < 0.05) following the treatment of HepG-2 cells with the lipase enzyme. These findings suggest that lipase from P. aeruginosa holds promise as a novel therapeutic agent for hepatocellular carcinoma, addressing the limitations of current treatments.
本研究旨在从生物样本中分离并优化高产脂肪酶的铜绿假单胞菌菌株，通过随机诱变提高酶产量，并评估其潜在的抗癌活性。对51份生物样本（血液、尿液、痰液、伤口脓液）进行了筛选，有3株分离菌表现出显著的脂肪酶活性。活性最高的分离菌被鉴定为铜绿假单胞菌（GenBank登录号PP436388），对其进行溴化乙锭诱导诱变，脂肪酶活性提高了两倍（312 U/ml）。采用深层发酵优化脂肪酶生产，经统计学分析确定关键因素为吐温80、蛋白胨和底物浓度。通过硫酸铵沉淀和Sephadex G-100柱层析对酶进行纯化，经SDS-PAGE确认其分子量为53 kDa。
酶生产的最佳条件包括pH值为9、温度为20℃和培养24小时。部分纯化的酶在pH值高达10和4℃储存温度下表现出高稳定性。使用MTT法评估抗癌活性，结果显示对HepG-2细胞的人肝细胞癌的IC50为78.21 U/ml，对Vero细胞未观察到细胞毒性。流式细胞术证实该酶的抗癌潜力是通过凋亡和坏死介导的。QRT-PCR数据显示，用脂肪酶处理HepG-2细胞后，Bcl-2基因的表达显著下调了62%（P<0.05）。这些发现表明，铜绿假单胞菌脂肪酶有望成为治疗肝细胞癌的新型治疗剂，解决当前治疗方法的局限性。
