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Two Step Purification of Acinetobacter sp. Lipase and Its Evaluation as a Detergent Additive at Low Temperatures

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Abstract

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°C, respectively. The lipase was active at low temperatures and it retained 86.8% activity at 10°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°C–20°C, respectively.

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Acknowledgements

The authors thank Dr. A.B. Mandal, Director, Central Leather Research Institute, Chennai, for his kind permission to publish this work. The financial assistance extended to N. Saisubramanian and S. Sivasubramanian by CSIR, New Delhi, India, is gratefully acknowledged.

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Authors and Affiliations

  1. Department of Biotechnology, Central Leather Research Institute, Adyar, Chennai, 600 020, India

    N. Saisubramanian, S. Sivasubramanian, N. Nandakumar, N. Amaranath Chaudhary & R. Puvanakrishnan

  2. Department of Pharmacy, National University of Singapore, Science Drive 4, Singapore, 117543, Singapore

    B. Indirakumar

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  1. N. Saisubramanian
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  2. S. Sivasubramanian
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  3. N. Nandakumar
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  6. R. Puvanakrishnan
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Correspondence to R. Puvanakrishnan.

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Saisubramanian, N., Sivasubramanian, S., Nandakumar, N. et al. Two Step Purification of Acinetobacter sp. Lipase and Its Evaluation as a Detergent Additive at Low Temperatures. Appl Biochem Biotechnol 150, 139–156 (2008). https://doi.org/10.1007/s12010-008-8143-1

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