Abstract The present study aims to exploit microbial potential from colder region to produce lipase enzyme stable at low temperatures. A newly isolated bacterium GBPI_508 from Himalayan environment, was investigated for the production of cold-active lipase emphasizing on its aggregation properties. Plate based assays followed by quantitative production of enzyme was estimated under different culture conditions. Further characterization of partially purified enzyme was done for molecular weight determination and activity and stability under varying conditions of pH, temperature, and in presence of organic solvents, inhibitors, and metal ions. The psychrotolerant bacterium was identified as Pseudomonas palleroniana following 16S rRNA gene sequencing. Maximum lipase production by GBPI_508 was recorded in 7 days at 25 °C utilizing yeast extract as nitrogen source and olive oil as substrate in the lipase production medium. Triton X-100 (1%) in the medium as emulsifier significantly enhanced the lipase production. Lipase produced by bacterium showed aggregation which was confirmed by dynamic light scattering and native PAGE. SDS-PAGE followed by zymogram analysis of partially purified enzyme showed two active bands of ∼50 kDa and ∼54 kDa. Optimum activity of partially purified enzymatic preparation was recorded at 40 °C while the activity remained nearly consistent from pH 7.0 to 12.0, whereas, maximum stability was recorded at pH values 7.0 and 11.0 at 25 °C. Interestingly, lipase in the partially purified fraction retained 60% enzyme activity at 10 °C. Medium chain pNP ester (C10) was the most preferred substrate for the lipase of GBPI_508. The lipase possessed >50% residual activity when incubated with different organic solvents (25% v/v) except toluene and dichloromethane which inhibited the activity below 50%. Partially purified enzyme was also stable in the presence of metal ions and inhibitors. The study suggests applicability of GBPI_508 lipase in low temperature conditions such as cold-active detergent formulations and cold bioremediation. Graphical Abstract

中文翻译：

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由巴勒罗尼亚假单胞菌 (GBPI_508) 耐冷菌株产生的冷活性脂肪酶的聚集特性

摘要 本研究旨在利用来自较冷地区的微生物潜力来生产在低温下稳定的脂肪酶。研究了从喜马拉雅环境中新分离出的细菌 GBPI_508 以生产冷活性脂肪酶，重点是其聚集特性。在不同的培养条件下估计基于板的测定，然后是酶的定量产生。对部分纯化的酶进行了进一步表征，用于在不同的 pH 值、温度以及有机溶剂、抑制剂和金属离子的存在下测定分子量以及活性和稳定性。经过 16S rRNA 基因测序，该耐冷细菌被鉴定为 Pseudomonas palleroniana。使用酵母提取物作为氮源，橄榄油作为脂肪酶生产培养基中的底物，GBPI_508 的最大脂肪酶产量在 25°C 下在 7 天内记录。培养基中的 Triton X-100 (1%) 作为乳化剂显着提高了脂肪酶的产生。细菌产生的脂肪酶显示聚集，这通过动态光散射和天然 PAGE 得到证实。SDS-PAGE 随后对部分纯化的酶进行酶谱分析，显示出两个约 50 kDa 和约 54 kDa 的活性条带。部分纯化酶制剂的最佳活性在 40 °C 下记录，而活性在 pH 7.0 至 12.0 之间几乎保持一致，而最大稳定性记录在 pH 值 7.0 和 11.0 时，25 °C。有趣的是，部分纯化的部分中的脂肪酶在 10 °C 时保留了 60% 的酶活性。中链 pNP 酯 (C10) 是 GBPI_508 脂肪酶最优选的底物。当与除甲苯和二氯甲烷以外的不同有机溶剂 (25% v/v) 一起温育时，脂肪酶具有 >50% 的残留活性，这些溶剂的活性抑制低于 50%。在金属离子和抑制剂存在的情况下，部分纯化的酶也是稳定的。该研究表明 GBPI_508 脂肪酶在低温条件下的适用性，例如冷活性洗涤剂配方和冷生物修复。图形概要 该研究表明 GBPI_508 脂肪酶在低温条件下的适用性，例如冷活性洗涤剂配方和冷生物修复。图形概要 该研究表明 GBPI_508 脂肪酶在低温条件下的适用性，例如冷活性洗涤剂配方和冷生物修复。图形概要