Purification and preliminary characterization of the extracellular lipase of Bacillus subtilis 168, an extremely...

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.