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.
Dalal
Sohel
S
Chemistry Department, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India.
Singh
Pradeep Kumar
PK
Raghava
Smita
S
Rawat
Seema
S
Gupta
Munishwar Nath
MN
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