title: Purification and characterisation of an F16L mutant of a thermostable lipase.
abstract: A mutant of the lipase from Geobacillus sp. strain T1 with a phenylalanine to leucine substitution at position 16 was overexpressed in Escherichia coli strain BL21(De3)pLysS. The crude enzyme was purified by two-step affinity chromatography with a final recovery and specific activity of 47.4 and 6,315.8 U/mg, respectively. The molecular weight of the purified F16L lipase was approximately 43 kDa by 12% SDS-PAGE analysis. The F16L lipase was demonstrated to be a thermophilic enzyme due its optimum temperature at 70 °C and showed stability over a temperature range of 40-60 °C. The enzyme exhibited an optimum pH 7 in phosphate buffer and was relatively stable at an alkaline pH 8-9. Metal ions such as Ca(2+), Mn(2+), Na(+), and K(+) enhanced the lipase activity, but Mg(2+), Zn(2+), and Fe(2+) inhibited the lipase. All surfactants tested, including Tween 20, 40, 60, 80, Triton X-100, and SDS, significantly inhibited the lipolytic action of the lipase. A high hydrolytic rate was observed on long-chain natural oils and triglycerides, with a notable preference for olive oil (C18:1; natural oil) and triolein (C18:1; triglyceride). The F16L lipase was deduced to be a metalloenzyme because it was strongly inhibited by 5 mM EDTA. Moderate inhibition was observed in the presence of PMSF at a similar concentration, indicating that serine residues are involved in its catalytic action. Further, the activity was not impaired by water-miscible solvents, including methanol, ethanol, and acetone.
authors.affiliation: Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. shukuri@biotech.upm.edu.my
authors.forename: Mohd Shukuri Mohamad
authors.initials: MS
authors.lastname: Ali
authors.forename: Chong Chai
authors.initials: CC
authors.lastname: Yun
authors.forename: Adam Leow Thean
authors.initials: AL
authors.lastname: Chor
authors.forename: Raja Noor Zaliha Raja Abdul
authors.initials: RN
authors.lastname: Rahman
authors.forename: Mahiran
authors.initials: M
authors.lastname: Basri
authors.forename: Abu Bakar
authors.initials: AB
authors.lastname: Salleh
mesh_terms: D001426:Bacterial Proteins; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D056506:Geobacillus / Q000737:chemistry / Q000201:enzymology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008670:Metals / Q000737:chemistry; D009154:Mutation; D009930:Organic Chemicals / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013501:Surface-Active Agents / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D008670:Metals; D009930:Organic Chemicals; D011994:Recombinant Proteins; D013501:Surface-Active Agents; D008049:Lipase; C428386:thermostable lipase