A kinetic and molecular study of the lipase from Geobacillus thermoleovorans GE-7

[摘要] English: Geobacillus thermoleovorans GE-7 was isolated from the West-Driefontein goldmine in South Africa. It is a Gram positive rod showing optimal growth at 65°C. Lipase production by the organism cna be induced using tributyrin, Tween 80 and stearic acid. We were able to partially purify this thermophilic lipase (native lipase) using hydrophobic (Phenyl-Toyopearl), anionic (Super-Q and DEAE-Toyoperal) and size exclusion (Sephacryl S-100/200-HR) chromatographic resins. Using triburyrin zymograms, the size of the protein was estimated to be 40 kDa with a pl of 7. The lipase open reading frame (1251 bp), coding for the signal peptide and mature lipase, could be successfully amplified from genomic DNA using polymerase chain reaction and the PCR product was cloned into the pGEM®T-easy cloning vector (pGEMGtherm-LipA; LipA). The gene was modified with PCR and cloned into the pET 28a expression vector to yield a hexa histidine tag at the C-(pETGtherm-LipA(C)) and N-therminus (pETGtherm-LipA(N)) of the lipase gene. All three clones could be successfully expressed in E. coli JM109(DE3), with expression levels of the N-tagged lipase 23.4 times higher when compared to optimised lipase production by G. thermoleovorans GE-7. The N-tagged and C-tagged lipase could be successfully purified using affinity chromatography (Ni-NTA resin) whilst the LipA lipase was only partially purified using the same purification protocols used for the native lipase. The N-terminal His6 tag was removed using thrombin targeting the incorporated thrombin cleavage site, resulting in the Detagged lipase which could be separated from the cleaved His6 tag using the Ni-NTA resin. The N- and Detagged lipase was stable during storage but the C-tagged lipase has to be stored in with 1 mM CaCl2 and 1 mM ZnSO4to retain activity. The different lipase sizes and pl's were comparable, ranging from 42 kDa (pl 7.3) for the C-tagged lipase, 41.1 kDa (pl 7.1) for the N-tagged lipase, 39 kDa (pl 6.8) for the Detagged lipase and 41 kDa for LipA. The optimum temperatures for the lipases varied from 55°C (C-tagged) to 60°C (N-tagged, Detagged, native, LipA) but could be increased to 65°C with the addition of 1mM CaCl2 (Detagged and N-tagged lipase). All the lipases showed an optimum pH of 9-10 and had a preference for the shorter chain saturated for the shorter chain saturated fatty acids. In the presence of 1 mM CaCl2 the N-tagged lipase had a half-life of 1 h at 70°C and 23 h at 65°C while the Detagged lipase had a half-life of 19 min at 70°C and 2.7 h at 65°C. The C-tagged lipase had a half-life of only 2 min at this temperature. Initial interest was in the production of a lipase by an extremophillic bacterium, but addition of the N-terminal histidine tag also lead to a higher enzyme stability at higher temperatures. The activity of the C-. N- and Detagged lipase was enhanced by te presence of Triton X-100 and CHAPS, but was inhibited by SDS and cetrimide, and were stable in up to 20% (v/v) of various alcohols. Ca2+, M2+, and Mn2+ (up to 5 mM) enhanced the activity of the C- and N-tagged lipase but slightly inhibited the Detagged lipase. All three enzymes showed slight inhibition in the presence of Zn2+ but was inhibited by Al2+ and Hg2+. EDTA had an inhibitory effect on lipase activity, but TPEN showed no effect. The N-tagged and Detagged lipases showed preference for sn-1,3-dicaprin isomer with monolayer experiments but did not exhibit any phospho- or galactolipase activity.

[发布机构] University of the Free State