High-level heterologous expression and properties of a novel lipase from Ralstonia sp. M1
Quyen. D. T Le. T. T. G Nguyen. T. T Oh. T. -K Lee. J. -K
Protein Expression and Purification ; 2005 [Vol.39] Pages:97-106
Abstract
The mature lipase LipA and its 56aa-truncated chaperone LipBhis (with 6£his-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 NiNTA purified chaperone LipBhis in molar ratio 1:1 at 4°C for 24 hours in H2O. The in vitro refolded lipase LipA had an optimal activity in the temperature range of 5055°C and was stable up to 45°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 8.010.5 with more than 52% activity retention. The enzyme was found to be highly resistant to many organic solvents especially induced by ethanolamine (remaining activity 137334%), but inhibited by 1-butanol and acetonitrile (4086%). 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 3065% and Fe3+ to a lesser degree (activity retention of 7786%). Tween 80, Tween 60, and Tween 40 induced the activation of the lipase LipA (222330%) and 0.21% (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 3189%. The inhibitors including DEPC, EDTA, PMSF, and 2-mercaptoethanol (0.110mM) inhibited moderately the lipase with remaining activity of 57105%. 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%).
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