Thermostable lipase from Geobacillus sp. Iso5: bioseparation, characterization and native structural studies.

The extracellular thermoalkaline lipase from Geobacillus sp. Iso5 was purified to homogeneity by ultrafiltration, 6% cross-linked agarose and Phenyl spehrose HIC column chromatography. The final purified lipase resulted in 8.7-fold with 6.2% yield. The relative molecular weight of the enzyme was determined to be a monomer of 47 kDa by SDS-PAGE and MALDI-TOF MS/MS spectroscopy. The purified enzyme exhibit optimum activity at 70 °C and pH 8.0. The enzyme retained above 90% activity at temperatures of 70 °C and about 35% activity at 85 °C for 2 h. However, the stability of the enzyme decreased at the temperature over 90 °C. The enzyme activity was promoted in the presence of Ca(2+) and Mg(2+) and strongly inhibited by HgCl2 , PMSF, DTT, K(+) , Co(2+) , and Zn (2+) . EDTA did not affect the enzyme activity. The secondary structure of purified lipase contains 36% α-helix and 64% β-sheet which was determined by Circular dichromism, FTIR, and Raman Spectroscopy.