Saisubramanian N, Sivasubramanian S, Nandakumar N, Indirakumar B, Chaudhary N Amaranath, Puvanakrishnan R
Department of Biotechnology, Central Leather Research Institute, Adyar, Chennai 600 020, India.
Appl Biochem Biotechnol. 2008 Aug;150(2):139-56. doi: 10.1007/s12010-008-8143-1. Epub 2008 Feb 20.
Acinetobacter sp. lipase was purified to homogeneity by a two-step process. The crude enzyme (along with biomass) was subjected to partial purification by aqueous two phase system (ATPS), avoiding centrifugation and filtration steps. Conditions for lipase partitioning by ATPS were optimized by response surface methodology (RSM) and a combination of 29.45% polyethylene glycol 8000, 15.5% phosphate, and a pH of 7.0 resulted in an optimal partition coefficient. Partially pure lipase was further purified by a modified batch process using Octyl Sepharose CL-4B in a vacuum filtration apparatus. This two-step process resulted in a purified lipase with a yield of 74.6% having a specific activity of 88.8 U/mg of protein and a purification fold of 14.92. The homogeneity of the lipase preparation obtained by the purification process was confirmed by reversed phase high performance liquid chromatography profile. The molecular weight of the purified lipase was found to be around 32 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified lipase exhibited pH and temperature optima of 8.5 and 37 degrees C, respectively. The lipase was active at low temperatures and it retained 86.8% activity at 10 degrees C. It also displayed other features such as stability over a broad range of pH (3.0-9.0) as well as stability in the presence of hydrogen peroxide and commercial detergents. Based on these characteristics, the potential of this lipase as an additive in laundry detergent formulation was evaluated under low temperature wash conditions. The results indicated that Acinetobacter sp. lipase increased the washing efficiency of the detergent Nirma by 21-24% at 15 degrees C-20 degrees C, respectively.
通过两步法将不动杆菌属脂肪酶纯化至同质。粗酶（连同生物质）通过双水相系统（ATPS）进行部分纯化，避免了离心和过滤步骤。采用响应面法（RSM）优化了ATPS对脂肪酶分配的条件，29.45%聚乙二醇8000、15.5%磷酸盐和pH 7.0的组合产生了最佳分配系数。部分纯的脂肪酶在真空过滤装置中使用辛基琼脂糖凝胶CL-4B通过改进的分批法进一步纯化。这两步法得到了纯化的脂肪酶，产率为74.6%，比活性为88.8 U/mg蛋白质，纯化倍数为14.92。通过反相高效液相色谱图证实了纯化过程中获得的脂肪酶制剂的同质性。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，纯化脂肪酶的分子量约为32 kDa。纯化的脂肪酶的最适pH和温度分别为8.5和37℃。该脂肪酶在低温下具有活性，在℃时保留86.8%的活性。它还表现出其他特性，如在很宽的pH范围（3.0-9.0）内稳定，以及在过氧化氢和市售洗涤剂存在下稳定。基于这些特性，在低温洗涤条件下评估了这种脂肪酶作为洗衣洗涤剂配方添加剂的潜力。结果表明，不动杆菌属脂肪酶在15℃-20℃时分别将洗涤剂Nirma的洗涤效率提高了21%-24%。
