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Purification and properties of the alkaline lipase from Burkholderia cepacia A.T.C.C. 25609
Author(s) -
Dalal Sohel,
Singh Pradeep Kumar,
Raghava Smita,
Rawat Seema,
Gupta Munishwar Nath
Publication year - 2008
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20070186
Subject(s) - lipase , burkholderia , enzyme , molecular mass , extracellular , triacylglycerol lipase , chromatography , biochemistry , strain (injury) , chemistry , pseudomonas , escherichia coli , bacteria , enzyme assay , biology , gene , anatomy , genetics
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 °C and its half‐life ( t 1/2 ) values were 54 and 46 min at 50 and 60 °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% α‐helix, 7.7% β‐sheet, 12.6% β‐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.