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Purification, Characterization of Alkaline Cold Active Lipase from Acinetobacter radioresistens PR8 and Development of a New Zymography Method for Lipase Detection

[ Vol. 25 , Issue. 10 ]

Author(s):

Krishna K. Gupta, Sharmili Jagtap*, Priya R. and Krishna Ramadas   Pages 897 - 907 ( 11 )

Abstract:


Background: Bacterial lipases find so many industrial applications. Therefore, new source of lipase suitable for industrial conditions is always required. Lipase zymography methods use costly chromogenic substrates and indicator dyes and are few in numbers.

Objective: The objectives of this work include lipase purification and its characterization from Acinetobacter radioresiens PR8 and development of new zymography method for lipase detection.

Method: The lipase was purified using conventional method and cation exchange chromatography and it was characterized biochemically and analytically. Based on these characterization new in-gel lipase zymography method was developed.

Results: In this present work, an alkalophilic lipase producing bacterium was isolated from soil; screened for extracellular lipase activity and identified to be Acinetobacter radioresistens PR8 (Genbank accession ID: MF073322). Enzyme production kinetics showed maximum production (4.16 U/ml at pH 9) of enzyme after 72 h. The lipase activity was found to be highest in olive oil (1% v/v; 8.1 U/ml). Low molecular weight (27 kDa) alkaline (pH 9) cold active (20 °C) lipase was purified from Acinetobacter radioresistens PR8. Lipase was characterized using PMF, FT-IR and its high conformational stability (Transition temperature: 122.3 °C) was attributed from its DSC spectrum. The importance of magnesium and sodium ions for enhancing lipase activity was obtained from flux balance analysis.

Conclusion: Based on the lipase activating role of Mn2+ and Na+ ions, optimum temperature, pH with no chromogenic substrates and indicator dyes, a new in gel zymography method for lipase detection was developed.

Keywords:

Acinetobacter, alkaline, flux balance analysis, lipase, purification, zymography.

Affiliation:

Centre for Bioinformatics, School of Life Sciences, Pondicherry University, Pondicherry 605014, Department of Microbiology, School of Life Sciences, Pondicherry University, Pondicherry 605014, Department of Microbiology, School of Life Sciences, Pondicherry University, Pondicherry 605014, Centre for Bioinformatics, School of Life Sciences, Pondicherry University, Pondicherry 605014

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