High-level heterologous expression and properties of a novel lipase from Ralstonia sp. M1.
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%).
Quyen
Dinh Thi
DT
Institute of Biotechnology, Vietnamese Academy of Science and Technology 18 Hoang Quoc Viet Road, Distr. Caugiay, 10600 Hanoi, Viet Nam.
Giang Le
Thi Thu
TT
Nguyen
Thi Thao
TT
Oh
Tae-Kwang
TK
Lee
Jung-Kee
JK
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
D001426:Bacterial Proteins; D003902:Detergents; C079999:LipA protein, Bacteria; D018832:Molecular Chaperones; D012997:Solvents