The mature lipase LipA and its 56aa-truncated chaperone DeltaLipBhis (with 6 x 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 12 mg 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 4degreesC for 24 hours in H2O. The in vitro refolded lipase LipA had an optimal activity in the temperature range of 50-55degreesC and was stable up to 45degreesC 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. PM S F. and 2-mercaptoethanol (0.1-10 mM) 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%). (C) 2004 Elsevier Inc. All rights reserved.