title: Thermostable lipase from Geobacillus sp. Iso5: bioseparation, characterization and native structural studies.
abstract: 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.
authors.affiliation: Department of PG Studies and Research in Biochemistry, Jnana Sahyadri, Kuvempu University, Shankaraghatta, Karnataka, India.
authors.forename: Gurumurthy D
authors.initials: GD
authors.lastname: Mahadevan
authors.forename: Shivayogeeswar E
authors.initials: SE
authors.lastname: Neelagund
mesh_terms: D001426:Bacterial Proteins; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D020536:Enzyme Activators / Q000032:analysis; D004791:Enzyme Inhibitors / Q000032:analysis; D004795:Enzyme Stability; D056506:Geobacillus / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D017433:Protein Structure, Secondary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D008049:Lipase; C428386:thermostable lipase
keywords: Circular dichromism; FTIR and Raman spectroscopy; Geobacillus; Lipase; MALDI-TOF MS/MS