Molecular and kinetic properties of recombinant Bacillus lipase

[摘要] English: Lipases (EG 3.1.1.3) catalyze the hydrolysis of triacylglycerols and occur widelyin nature. The lipase-catalysed reaction is reversible and a wide range of transandinteresterification reactions can be catalyzed. These enzymes could be usedto manufacture products which could not be obtained by conventional chemicalprocesses, and as the advantages of the use of lipases relative to traditionalchemical processes are more recognized, lipases may be expected to gain evenmore importance in the enzyme market. For these purposes, new lipases with awide range of novel characteristics are required.Bacterial isolates collected mainly from alkaline dairy washes were screened forlipase production on agar plates containing Rhodamine B/olive oil, TweenBO/GaGb and tributyrin. Isolates that showed lipase production on agar plateswere investigated for lipase production in liquid cultures in the presence ofdifferent carbon sources. Bacillus licheniformis and Bacillus pumilus wereidentified as best producers of lipase on the basis of relative activity, pH andtemperature optimum using p-Nitrophenyl palmitate as the assay substrate.The production of extracellular lipase by Bacillus licheniformis in the presence ofselected carbohydrates and lipidic substrates was investigated. Themicroorganism could not grow in mineral medium containing Tween 20, TweenBD and caproic and caprylic acids. Although the mineral medium supplementedwith tributyrin, triolein, olive oil, tricaprylin, glycerol and glucose supported growthof the microorganism, no lipase production was detected; probably as a result ofthe lack of the lipase inducing factor. When the microorganism was grown onnutrient broth, lipase activity of about 600 units/I was achieved. This indicated thepresence of the lipase producing components in the rich nutrient broth medium.Production of extracellular lipase was repressed by the addition oftriacylglycerols, free fatty acids, glycerol and glucose. The repressive effects oftriacylglycerols were found to be more rapid and pronounced as compared to theeffects of free fatty acids and glycerol alone. The increased repressive effects oftriacylglycerols could have occurred as a result of compounded effects of glyceroland free fatty acids, which are the hydrolytic products when the lipase hydrolysesthe triacylglycerols. These observations suggested synergistic effect or thepresence of two independent pathways by which free fatty acids and glycerolrepress the production of lipase by Bacillus licheniformis.Addition of detergents Tween 20 and Tween 80 enhanced the production oflipases by the microorganism. The extracellular lipase activity increased to levelsof about 2000 units/I in the presence of the detergent.The purification of the lipase from Bacillus licheniformis was attempted using acombination of ion-exchange chromatography, hydrophobic interactionchromatography, hydroxylapatite and size exclusion chromatography. However,aggregation of lipase protein, lack of interaction, and irreversible interaction withchromatography resins resulted in only partially pure lipase preparations.The partially purified lipases showed biochemical properties similar to lipasesproduced by Bacillus pumilus and Bacillus subtilis. The three lipases arethermolabile, alkali tolerant and function optimally in alkaline pH conditions. Thecloning and sequencing of the lipase gene from Bacillus pumilus isolate followedby amino acid analysis revealed high sequence homology suggesting similarprotein folds. This led to the hypothesis that the mature lipase secreted byBacillus Iicheniformis could have significant homology with the mature lipasesecreted by the other two Bacillus species.Degenerate primers were consequently designed based on the sequences ofmature lipases secreted by Bacillus pumilus and Bacillus subtilis. The primersamplified a DNA fragment of 560 bp encoding lipase activity with BacillusIicheniformis genomic DNA as the template. The DNA fragment encoding themature lipase of Bacillus Iicheniformis was subcloned into the pET 20b(+)expression vector to construct a recombinant lipase protein containing 6 histidineresidues at the C-terminal. High-level expression of the lipase by Escherichia colicells harbouring the lipase gene-containing expression vector was observedupon induction with IPTG at 30°C. A one step purification of the recombinantlipase was achieved with Ni-NTA resin. The histidine tag was removed bycreating a 6X His-tag at the N-terminal of the protein followed by a rTEV proteasecleavage site. The lipase protein was purified by Ni-NTA affinity chromatographyfollowed by cleavage with rTEV protease to remove the histidine tag. The CterminalHis-tagged and the non-tagged lipase proteins were characterized.The specific activity of the purified enzyme was about 130 units/mg with pnitrophenyl-palmitate as substrate. The enzyme showed maximum activity at pH9.5-11.5 and was remarkably stable at alkaline pH values up to 20 hours. The Cterminalhistidine tag was found to enhance the specific activity of the lipase inpH conditions between 10-11.5. The enzyme showed maximal activities towardp-nitrophenyl esters and triacylglycerols containing C6 and Ca fatty acyl groups.The metals that affected the lipase significantly were divalent Co, Zn and Hgwhich decreased the activity to less than 30% with Hg abolishing all the lipaseactivity. The enzyme was not inhibited by ethylenediaminetetraacetic acid(EDTA), dithiothreitol (DTT), or mercaptoethanol, while the classical serineprotease inhibitor phenylmethylsulfonyl fluoride (PMSF) decreased the lipaseactivity to 40% at 1 mM concentration.The amino acid sequence of the lipase shows striking similarities to lipases fromBacillus subtilis and Bacillus pumilus. Based on the amino acid identity andbiochemical characteristics, we classified Bacillus licheniformis lipase as amember of Family 1.4 lipases, together with lipolytic enzymes produced byBacillus subtilis and Bacillus pumilus. The consensus sequence of the lipasesaround the nucleophilic Ser deviated from the canonical Gly-X-Ser-X-Gly. In thethree Bacillus lipases, the consensus sequence was found to be Ala-His-Ser-XGly.This motif is shared with another group of larger lipases produced bythermophilic Bacillus species.Site-directed mutagenesis of the Ala and His residues near the nucleophilic Sersuggested that the lipase enzymes acquired the amino acids during evolution foroptimal activity and enhanced thermal stability. Asp133, His 156 together withSer?? were identified by site-directed mutagenesis as residues that form thecatalytic triad of the lipase enzyme. This was confirmed by the three-dimensionalstructure model built using Bacillus subtilis Lipase A as the template.While in pursuit of cloning the promoter region of Bacillus licheniformis lipasegene, a 3.5 kb DNA fragment that showed lipolytic activity on tributyrin agar platewas obtained. Sequence analysis of the cloned DNA fragment revealed that anew carboxyl esterase gene with an open reading frame encoding a protein of484 amino acids with estimated molecular mass of 53 kDa and a pi of 5.4 hadbeen cloned. The cloned protein showed high amino acid identity with industriallysignificant enzymes belonging to the esterase family.This study has advanced the biochemical knowledge on lipases secreted byBacillus species. Although the biochemical properties of Bacillus lipases arebecoming known, the molecular mechanisms regulating the biosynthesis of theenzymes remain unknown. Future studies should therefore also attempt toelucidate the molecular mechanisms of Bacillus lipase biosynthesis. This wouldfacilitate the bioengineering of Bacillus species to produce inducible lipaseenzyme at quantities enough for application in for example, detergent and leathertanning industries. Current studies aimed at the cloning of the promoter region ofthe lipase from Bacillus licheniformis should be continued for the purpose ofunderstanding the molecular regulation of the gene.The role of Bacillus lipases in biocatalytic resolution of racemic mixtures has notbeen fully explored. The recent availability of the three-dimensional structure ofFamily 1.4 lipases will enable us to understand the structural determinants oflipase properties and to construct Bacillus lipases that suit desired functions. Asan example, the mature lipase produced by Bacillus pumilus (UOFS) is morethan 95% identical to Bacillus lichenifomis lipase at amino acid level, but thelatter lipase shows a broader alkaline optimum pH profile. The determinant forthe broad alkaline pH profile has not yet been identified.The new carboxyl esterase gene cloned in this study should be over-expressedand biochemically characterized. The biotechnological applicability for theenzyme should be assessed.

[发布机构] University of the Free State