Purification of lipase from Cunninghamella verticillata by stepwise precipitation and optimized conditions for crystallization T.S. Kumarevel 1, * , ,à , S.C.B. Gopinath 2, ,à , A. Hilda 2 , N. Gautham 1 and M.N. Ponnusamy 1 1 Department of Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai 600 025, Tamil Nadu, India 2 Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai 600 025, Tamil Nadu, India *Author for correspondence: Tel.: +81-298-61-6085, Fax: +81-298-61-6095, E-mail: kumarevel-thirumananseri@ aist.go.jp   Present address: Functional Nucleic Acids Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan à These authors equally contributed to this work Received 13 November 2003; accepted 13 May 2004 Keywords: Crystallization, Cunninghamella verticillata, lipase, purification, stepwise precipitation Summary Lipase from the oil-mill waste isolate Cunninghamella verticillata was purified by stepwise precipitation using acetone, as a sequel to our earlier conventional column chromatographic method [Gopinath et al. (2002) World Journal of Microbiology and Biotechnology 18, 449–458]. The yield of purified lipase was approx. 4-fold higher than by the previous method and the purified lipase was obtained with 70–80% acetone saturations. The enzyme was resolved as a single band with homogeneity both by native and by SDS–PAGE. The optimum condition for the lipase to crystallize was 5 lg of enzyme in 0.05 M sodium phosphate buffer (pH 6.5) with 5 mM FeCl 2 and 10% 2-methyl 2,4-pentanediol (MPD). Introduction Lipases [triacylglycerol acylhydrolases (EC 3.1.1.3)] are water-soluble enzymes, but they act on non-soluble substrates. They catalyse the hydrolysis of fats and oils at the oil–water interface to glycerol and free fatty acids. Lipases are among the most useful and most studied enzymes in the fats and oil industry (Malcata et al. 1990). Microbial lipases have attracted considerable attention owing to their biotechnological potential, ranging from use in laundry detergents to stereospecific biocatalysis (Jaeger et al. 1994). Microbial lipases today occupy a place of prominence among biocatalysts due to their ability to catalyse a wide variety of reactions in aqueous and non-aqueous media. Their chemo-, regio- and enantio-specific behaviour has caused tremendous interest among scientists and industrialists (Saxena et al. 2003). A variety of lipases of microbial origin with different properties and specificities have been described and characterized (Adlercreutz et al. 2002; Gopinath et al. 2002, 2003). Knowledge of the three-dimensional structure of lipases plays an important role in designing and engi- neering lipases for specific purposes. More than 12 lipases from various sources have been crystallized, and extensive information on lipase engineering has been documented (Acharya & Rao 2003). The structures of lipases from diverse sources, ranging from microbes to mammalian enzymes, conform to the a/b hydrolase fold (Rubin 1994; Schrag et al. 1997), a structural motif common to a wide variety of hydrolases. In all the cases studied so far, the catalytic centre contains a triad of amino acids (Ser-His-Asp/Glu) and this triad is respon- sible for the nucleophilic attack on the carbonyl carbon of the scissile ester bond (Derewenda et al. 1994). The interfacial activation with a lid covering the active site is today an area of extensive research involving X-ray crystallographers, biochemists, molecular biologists, chemists and biochemical engineers (Saxena et al. 2003). At present, many traditional purification strategies have low yields and long time periods as main con- straints. Industries today look for purification strategies that are inexpensive, rapid, high yielding and amenable to large-scale operations. In the present study, we have attempted to purify the lipase from C. verticillata by stepwise precipitation using acetone. This method leads to a purification with high purity and yield along with high enzymatic activity. World Journal of Microbiology & Biotechnology 2005 21: 23–26 DOI: 10.1007/s11274-004-1005-2 Ó Springer 2005