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 19000 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 preferantially cleaved the 1(3)-position ester bond. No interfacial activation effect was observed with triacetyl glycerol as substrate.