Purification of lipase from Cunninghamella verticillata and optimization of enzyme activity using response surface methodology

  • S.C.B. Gopinath
  • A. Hilda
  • T. Lakshmi priya
  • G. Annadurai


The fungus Cunninghamella verticillata was selected from isolates of oil-mill waste as a potent lipase producer as determined by the Rhodamine-B plate method. The lipase was purified from C. verticillata by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The purified enzyme was formed from a monomeric protein with molecular masses of 49 and 42 kDa by SDS–PAGE and gel filtration, respectively. The optimum pH at 40 °C was 7.5 and the optimum temperature at pH 7.5 was 40 °C. The enzyme was stable between a pH range of 7.5 and 9.0 at 30 °C for 24 h. The enzyme activity was strongly inhibited by AgNO3, NiCl2, HgCl2, CdCl2 and EDTA. However, the presence of Ca2+, Mn2+ and Ba2+ ions enhanced the activity of the enzyme. The activity of purified lipase with respect to pH, temperature and salt concentration was optimized using a Box–Behnken design experiment. A polynomial regression model used in analysing this data, showed a significant lack of fitness. Therefore, quadratic terms were incorporated in the regression model through variables. Maximum lipase activity (100%) was observed with 2 mM CaCl2, (pH 7.5) at a temperature of 40 °C. Regression co-efficient correlation was calculated as 0.9956.

Box–Behnhen design experiment Cunninghamella verticillata enzyme characterization lipase Rhodamine-B vegetable oil 


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Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  • S.C.B. Gopinath
    • 1
  • A. Hilda
    • 1
  • T. Lakshmi priya
    • 2
  • G. Annadurai
    • 3
  1. 1.Centre for Advanced studies in BotanyUniversity of Madras, Guindy Campus, ChennaiTamil NaduIndia
  2. 2.Department of MathematicsSBK College, AruppukottaiTamil NaduIndia
  3. 3.Department of Chemical EngineeringYuan-Ze UniversityTaoyuanTaiwan, ROC

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