Purification and Characterization of an Alkali-Thermostable Lipase from Thermophilic Anoxybacillus flavithermus HBB 134

An intracellular lipase from Anoxybacillus flavithermus HBB 134 was purified to 7.4-fold. The molecular mass of the enzyme was found to be about 64 kDa. The maximum activity of the enzyme was at pH 9.0 and 50 degrees C. The enzyme was stable between pH 6.0 and 11.0 at 25 degrees C, 40 degrees C, and 50 degrees C for 24 h. The K-m and V-max of the enzyme for pNPL substrate were determined as 0.084 mM and 500 U/mg, respectively. Glycerol, sorbitol, and mannitol enhanced the enzyme thermostability. The enzyme was found to be highly stable against acetone, ethyl acetate, and diethyl ether. The presence of PMSF, NBS, DTT and beta-mercaptoethanol inhibited the enzyme activity. Hg2+, Fe3+, Pb2+, Al3+, and Zn2+ strongly inhibited the enzyme whereas Li+, Na+, K+, and NH4+ slightly activated it. At least 60% of the enzyme activity and stability were retained against sodium deoxycholate, sodium taurocholate, n-octyl-beta-D-glucopyranoside, and CHAPS. The presence of 1% Triton X-100 caused about 34% increase in the enzyme activity. The enzyme is thought to be a true lipase since it has preferred the long-chain triacylglycerols. The lipase of HBB 134 cleaved triolein at the 1-or 3-position.