Jain Rahul, Pandey Anita, Pasupuleti Mukesh, Pande Veena
Biotechnological Applications, G B Pant National Institute of Himalayan Environment and Sustainable Development, Kosi-Katarmal, Almora, Uttarakhand, 263 643, India.
Department of Microbiology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, 226 031, India.
Mol Biotechnol. 2017 Jan;59(1):34-45. doi: 10.1007/s12033-016-9989-z.
Pseudomonas, being the common inhabitant of colder environments, are suitable for the production of cold-active enzymes. In the present study, a newly isolated strain of Pseudomonas from cold desert site in Indian Himalayan Region, was investigated for the production of cold-active lipase. The bacteria were identified as Pseudomonas proteolytica by 16S rDNA sequencing. Lipase production by bacteria was confirmed by qualitative assay using tributyrin and rhodamine-B agar plate method. The bacterium produced maximum lipase at 25 °C followed by production at 15 °C while utilizing olive, corn, as well as soybean oil as substrate in lipase production broth. Enzyme produced by bacteria was partially purified using ammonium sulphate fractionation. GBPI_Hb61 showed aggregation behaviour which was confirmed using several techniques including gel filtration chromatography, dynamic light scattering, and native PAGE. Molecular weight determined by SDS-PAGE followed by in-gel activity suggested two lipases of nearly similar molecular weight of ~50 kDa. The enzyme showed stability in wide range of pH from 5 to 11 and temperature up to 50 °C. The enzyme from GBPI_Hb61 exhibited maximum activity toward p-nitrophenyldecanoate (C10). The stability of enzyme was not affected with methanol while it retained more than 75% activity when incubated with ethanol, acetone, and hexane. The bacterium is likely to be a potential source for production of cold-active lipase with efficient applicability under multiple conditions.
假单胞菌是寒冷环境中的常见居民，适合用于生产冷活性酶。在本研究中，对从印度喜马拉雅地区寒冷沙漠地点新分离出的一株假单胞菌进行了冷活性脂肪酶生产的研究。通过16S rDNA测序将该细菌鉴定为解蛋白假单胞菌。使用丁酸甘油酯和罗丹明B琼脂平板法通过定性测定确认了细菌产生的脂肪酶。该细菌在25℃时产生的脂肪酶最多，其次是在15℃时产生的脂肪酶，同时在脂肪酶生产肉汤中使用橄榄油、玉米油以及大豆油作为底物。使用硫酸铵分级分离法对细菌产生的酶进行了部分纯化。GBPI_Hb61表现出聚集行为，这通过包括凝胶过滤色谱法、动态光散射法和非变性聚丙烯酰胺凝胶电泳在内的多种技术得到了证实。通过SDS-PAGE测定分子量，随后进行凝胶内活性测定，结果表明有两种分子量相近的脂肪酶，约为50 kDa。该酶在pH值5至11的宽范围内以及温度高达50℃时都具有稳定性。来自GBPI_Hb61的酶对癸酸对硝基苯酯（C10）表现出最大活性。该酶的稳定性不受甲醇影响，而与乙醇、丙酮和己烷一起孵育时仍保留超过75%的活性。该细菌可能是生产冷活性脂肪酶的潜在来源，在多种条件下具有高效适用性。
