Department of Biotechnology, School of Life Sciences, Pondicherry University, Puducherry - 605 014, India.
CAS in Crystallography and Biophysics, University of Madras, Chennai - 600 025, Tamilnadu, India.
Int J Biol Macromol. 2022 Jun 30;211:741-753. doi: 10.1016/j.ijbiomac.2022.04.174. Epub 2022 Apr 30.
The lipase gene from Psychrobacter celer PU3 was cloned into pET-28a(+) expression vector and overexpressed in E. coli BL21 (DE3) pLysS cells. The purified Psychrobacter celer lipase (PCL) was characterized as an alkaline active enzyme and has a molecular mass of around 30 kDa. The PCL was active even at a low temperature and the optimum range was observed between 10 and 40 °C temperatures. MALDI-TOF and phylogenetic analysis ensured that Psychrobacter celer PU3 lipase (PCL) was closely related to P. aureginosa lipase (PAL). MD simulation results suggest that temperature change did not affect the overall structure of PCL, but it might altered the temperature-dependent PCL functional changes. R (129-135 AA) and R (187-191 AA) regions could be important for temperature-dependent PCL function and they fluctuated much at 35 °C temperature. PMSF completely inhibited PCL lipase activity and it demonstrates the presence of serine residues in the active site of PCL. PCL is moderately halophilic and most of the tested organic solvents found to be inhibiting the lipase activity except the solvents ethanol and methanol. PCL activity was increased with surfactants (SDS and CTAB) and bleaching agents (hydrogen peroxide). The effect of different metal ions on PCL resulted that only mercuric chloride was found as the enhancer of the lipase activity. Antibiofilm property of PCL was evaluated against pathogenic Vibrio parahaemolyticus isolated from the diseased shrimp and MIC value was 500 U. PCL significantly altered the morphology and biofilm density of V. parahaemolyticus and the same was observed through scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM) imaging. RT-PCR analysis revealed that the mRNA expression level of biofilm, colony morphology and major toxin-related (aphA, luxS, opaR, tolC, toxR) genes of V. parahaemolyticus were significantly downregulated with PCL treatment.
从冷杆菌 PU3 中克隆了脂肪酶基因到 pET-28a(+)表达载体，并在大肠杆菌 BL21 (DE3) pLysS 细胞中过表达。纯化的冷杆菌脂肪酶（PCL）是一种碱性活性酶，分子量约为 30 kDa。PCL 即使在低温下也具有活性，最佳温度范围在 10 到 40°C 之间。MALDI-TOF 和系统发育分析确保冷杆菌 PU3 脂肪酶（PCL）与金黄杆菌脂肪酶（PAL）密切相关。MD 模拟结果表明，温度变化不会影响 PCL 的整体结构，但可能会改变依赖温度的 PCL 功能变化。R（129-135 AA）和 R（187-191 AA）区域可能对温度依赖的 PCL 功能很重要，它们在 35°C 温度下波动很大。PMSF 完全抑制了 PCL 脂肪酶活性，表明 PCL 活性位点存在丝氨酸残基。PCL 中度嗜盐，除乙醇和甲醇外，大多数测试的有机溶剂都发现抑制脂肪酶活性。PCL 活性随着表面活性剂（SDS 和 CTAB）和漂白剂（过氧化氢）的增加而增加。不同金属离子对 PCL 的影响表明，只有氯化汞被发现是增强脂肪酶活性的物质。PCL 的抗生物膜特性针对从患病虾中分离出的致病性副溶血弧菌进行了评估，其 MIC 值为 500 U。PCL 显著改变了副溶血弧菌的形态和生物膜密度，通过扫描电子显微镜（SEM）和共聚焦激光扫描显微镜（CLSM）成像观察到了同样的情况。RT-PCR 分析表明，PCL 处理后，副溶血弧菌的生物膜、菌落形态和主要毒素相关（aphA、luxS、opaR、tolC、toxR）基因的 mRNA 表达水平显著下调。
