Purification and preliminary characterization of the extracellular lipase of Bacillus subtilis 168, an extremely basic pH-tolerant enzyme.
The extracellular lipase of Bacillus subtilis 168 was purified from the growth medium of an overproducing strain by ammonium sulfate precipitation followed by phenyl-Sepharose and hydroxyapatite column chromatography. The purified lipase had a strong tendency to aggregate. It exhibited a molecular mass of 19,000 Da by SDS-PAGE and a pI of 9.9 by chromatofocusing. The enzyme showed maximum stability at pH 12 and maximum activity at pH 10. The lipase was active toward p-nitrophenyl esters and triacylglycerides with a marked preference for esters with C8 acyl groups. Using trioleyl glycerol as substrate, the enzyme preferentially cleaved the 1(3)-position ester bond. No interfacial activation effect was observed with triacetyl glycerol as substrate.
Laboratoire de Génétique microbienne, Université Catholique de Louvain, Belgium.
E
E
Lesuisse
K
K
Schanck
C
C
Colson
D000645:Ammonium Sulfate; D001412:Bacillus subtilis / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D002855:Chromatography, Thin Layer; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D006882:Hydroxyapatites; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D014280:Triglycerides / Q000378:metabolism
D006882:Hydroxyapatites; D014280:Triglycerides; D010664:Phenylmethylsulfonyl Fluoride; D017886:Durapatite; D008049:Lipase; D000645:Ammonium Sulfate; D002118:Calcium