Purification and properties of the alkaline lipase from Burkholderia cepacia A.T.C.C. 25609.
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.
Chemistry Department, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India.
Sohel
S
Dalal
Pradeep Kumar
PK
Singh
Smita
S
Raghava
Seema
S
Rawat
Munishwar Nath
MN
Gupta
D001483:Base Sequence; D016956:Burkholderia cepacia / Q000201:enzymology*; D002942:Circular Dichroism; D003550:Cystic Fibrosis / Q000201:enzymology*; D003902:Detergents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006207:Half-Life; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017830:Octoxynol / Q000737:chemistry; D011136:Polysorbates / Q000737:chemistry; D017433:Protein Structure, Secondary; D013696:Temperature
D003902:Detergents; D011136:Polysorbates; D017830:Octoxynol; D008049:Lipase