title: Comparative biochemical characterization and in silico analysis of novel lipases Lip11 and Lip12 with Lip2 from Yarrowia lipolytica.
abstract: Novel lipases lip11 and lip12 from Yarrowia lipolytica MSR80 were cloned and expressed in E. coli HB101 pEZZ18 system along with lip2. These enzymes were constitutively expressed as extracellular proteins with IgG tag. The enzymes were purified by affinity chromatography and analyzed by SDS-PAGE with specific activity of 314, 352 and 198 U/mg for Lip2, Lip11 and Lip12, respectively on olive oil. Biochemical characterization showed that all were active over broad range of pH 4.0-9.0 and temperature 20-80 °C with optima at pH 7 and 40 °C. All the three lipases were thermostable up to 80 °C with varying t(½). Activity on various substrates revealed that they were most active on oils > triacylglycerides > p-np-esters. Relatively Lip2 and Lip11 showed specificity for mid to long chain fatty acids, while Lip12 was mid chain specific. GC analysis of triolein hydrolysis by these lipases revealed that Lip2 and Lip11 are regioselective, while Lip12 is not. Effect of metal ions showed that Lip2 and Lip12 were activated by Ca²⁺ whereas Lip11 by Mg²⁺. All were thiol activated and inhibited by PMSF and N-bromosuccinimide. All were activated by non polar solvents and inhibited by polar solvents. Detailed sequence analysis and structural predictions revealed Lip11 and Lip12 shared 61 and 62 % homology with Lip2 (3O0D) and three dimensional superimposition revealed Lip2 was closer to Lip11 than to Lip12 as was observed during biochemical characterization. Finally, thermostability and substrate specificity has been explained on the basis of detailed amino acid analysis.
authors.affiliation: Department of Microbiology, University of Delhi, South Campus, New Delhi 110021, India.
authors.forename: Arti
authors.initials: A
authors.lastname: Kumari
authors.forename: Ved Vrat
authors.initials: VV
authors.lastname: Verma
authors.forename: Rani
authors.initials: R
authors.lastname: Gupta
mesh_terms: D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D019295:Computational Biology; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D025062:Yarrowia / Q000201:enzymology* / Q000235:genetics*
chemical_list: D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D008049:Lipase