Purification and properties of the alkaline lipase from <i>Burkholderia cepacia</i> A.T.C.C. 25609

Abstract

A Burkholderia cepacia (bacteria) strain, A.T.C.C. 25609, which had been isolated from the bronchial washings of a cystic fibrosis patient, was used to produce lipase. The presence of sodium alginate at an optimal concentration of 8 mg.ml(-1) in the growth medium nearly doubled the production of extracellular lipase activity. The enzyme could be purified with 38-fold purification and 96% activity recovery using a two-step purification protocol. The molecular mass of the purified lipase determined by SDS/PAGE was shown to be 28 kDa. The pH optimum of the purified enzyme was 9 and it was stable up to 12 h at pH 9 and 10. The enzyme has a temperature optimum of 40 degrees C and its half-life (t(1/2)) values were 54 and 46 min at 50 and 60 degrees C respectively. The lipase was found to be stable in the presence of the detergents Tween 20 and Triton X-100. The secondary-structure analysis of lipase by CD spectroscopy showed 52% alpha-helix, 7.7% beta-sheet, 12.6% beta-turn and 27.8% random structure. The lipase was cloned and overexpressed in Escherichia coli. The gene sequence of the cloned lipase was determined and compared with other lipases.