title: High-level heterologous expression and properties of a novel lipase from Ralstonia sp. M1.
abstract: The mature lipase LipA and its 56aa-truncated chaperone DeltaLipBhis (with 6xhis-tag) from Ralstonia sp. M1 were over-expressed in Escherichia coli BL21 under the control of T7 promoter with a high level of 70 and 12mg protein per gram of wet cells, respectively. The simply purified lipase LipA was effectively refolded by Ni-NTA purified chaperone DeltaLipBhis in molar ratio 1:1 at 4 degrees C for 24 hours in H2O. The in vitro refolded lipase LipA had an optimal activity in the temperature range of 50-55 degrees C and was stable up to 45 degrees C with more than 84% activity retention. The maximal activity was observed at pH 10.75 for hydrolysis of olive oil and found to be stable over alkaline pH range 8.0-10.5 with more than 52% activity retention. The enzyme was found to be highly resistant to many organic solvents especially induced by ethanolamine (remaining activity 137-334%), but inhibited by 1-butanol and acetonitrile (40-86%). Metal ions Cu2+, Sn2+, Mn2+, Mg2+, and Ca2+ stimulated the lipase slightly with increase in activity by up to 22%, whereas Zn2+ significantly inhibited the enzyme with the residual activity of 30-65% and Fe3+ to a lesser degree (activity retention of 77-86%). Tween 80, Tween 60, and Tween 40 induced the activation of the lipase LipA (222-330%) and 0.2-1% (w/v) of Triton X-100, X-45, and SDS increased the lipase activity by up to 52%. However, 5% (w/v) of Triton X-100, X-45, and SDS inhibited strongly the activity by 31-89%. The inhibitors including DEPC, EDTA, PMSF, and 2-mercaptoethanol (0.1-10mM) inhibited moderately the lipase with remaining activity of 57-105%. The lipase LipA hydrolyzed a wide range of triglycerides, but preferentially short length acyl chains (C4 and C6). In contrast to the triglycerides, medium length acyl chains (C8 and C14) of p-nitrophenyl (p-NP) esters were preferential substrates of this lipase. The enzyme preferentially catalyzed the hydrolysis of cottonseed oil (317%), cornoil (227%), palm oil (222%), and wheatgerm oil (210%) in comparison to olive oil (100%).
authors.affiliation: Institute of Biotechnology, Vietnamese Academy of Science and Technology 18 Hoang Quoc Viet Road, Distr. Caugiay, 10600 Hanoi, Viet Nam.
authors.forename: Dinh Thi
authors.initials: DT
authors.lastname: Quyen
authors.forename: Thi Thu
authors.initials: TT
authors.lastname: Giang Le
authors.forename: Thi Thao
authors.initials: TT
authors.lastname: Nguyen
authors.forename: Tae-Kwang
authors.initials: TK
authors.lastname: Oh
authors.forename: Jung-Kee
authors.initials: JK
authors.lastname: Lee
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D003902:Detergents; D004795:Enzyme Stability; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D018832:Molecular Chaperones / Q000096:biosynthesis / Q000235:genetics*; D043367:Ralstonia / Q000201:enzymology*; D012997:Solvents; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D003902:Detergents; C079999:LipA protein, Bacteria; D018832:Molecular Chaperones; D012997:Solvents