title: High level expression and characterization of a novel thermostable, organic solvent tolerant, 1,3-regioselective lipase from Geobacillus sp. strain ARM.
abstract: The mature ARM lipase gene was cloned into the pTrcHis expression vector and over-expressed in Escherichia coli TOP10 host. The optimum lipase expression was obtained after 18 h post induction incubation with 1.0mM IPTG, where the lipase activity was approximately 1623-fold higher than wild type. A rapid, high efficient, one-step purification of the His-tagged recombinant lipase was achieved using immobilized metal affinity chromatography with 63.2% recovery and purification factor of 14.6. The purified lipase was characterized as a high active (7092 U mg(-1)), serine-hydrolase, thermostable, organic solvent tolerant, 1,3-specific lipase with a molecular weight of about 44 kDa. The enzyme was a monomer with disulfide bond(s) in its structure, but was not a metalloenzyme. ARM lipase was active in a broad range of temperature and pH with optimum lipolytic activity at pH 8.0 and 65°C. The enzyme retained 50% residual activity at pH 6.0-7.0, 50°C for more than 150 min.
authors.affiliation: Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. a_ebrahimpour@yahoo.com
authors.forename: Afshin
authors.initials: A
authors.lastname: Ebrahimpour
authors.forename: Raja Noor Zaliha Raja Abd
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: D000222:Adaptation, Physiological / Q000187:drug effects*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005798:Genes, Bacterial / Q000235:genetics; D056506:Geobacillus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007477:Ions; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009930:Organic Chemicals / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000302:isolation & purification; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D012997:Solvents / Q000494:pharmacology*; D013237:Stereoisomerism; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D007477:Ions; D008670:Metals; D009930:Organic Chemicals; D011994:Recombinant Proteins; D012997:Solvents; D008049:Lipase