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dc.contributor.advisorLitthauer, Derek
dc.contributor.advisorPatterton, Hugh-George
dc.contributor.authorNthangeni, Mulalo Bethuel
dc.date.accessioned2018-05-08T07:46:45Z
dc.date.available2018-05-08T07:46:45Z
dc.date.issued2001-10
dc.identifier.urihttp://hdl.handle.net/11660/8246
dc.description.abstractEnglish: Lipases (EG 3.1.1.3) catalyze the hydrolysis of triacylglycerols and occur widely in nature. The lipase-catalysed reaction is reversible and a wide range of transand interesterification reactions can be catalyzed. These enzymes could be used to manufacture products which could not be obtained by conventional chemical processes, and as the advantages of the use of lipases relative to traditional chemical processes are more recognized, lipases may be expected to gain even more importance in the enzyme market. For these purposes, new lipases with a wide range of novel characteristics are required. Bacterial isolates collected mainly from alkaline dairy washes were screened for lipase production on agar plates containing Rhodamine B/olive oil, Tween BO/GaGb and tributyrin. Isolates that showed lipase production on agar plates were investigated for lipase production in liquid cultures in the presence of different carbon sources. Bacillus licheniformis and Bacillus pumilus were identified as best producers of lipase on the basis of relative activity, pH and temperature optimum using p-Nitrophenyl palmitate as the assay substrate. The production of extracellular lipase by Bacillus licheniformis in the presence of selected carbohydrates and lipidic substrates was investigated. The microorganism could not grow in mineral medium containing Tween 20, Tween BD and caproic and caprylic acids. Although the mineral medium supplemented with tributyrin, triolein, olive oil, tricaprylin, glycerol and glucose supported growth of the microorganism, no lipase production was detected; probably as a result of the lack of the lipase inducing factor. When the microorganism was grown on nutrient broth, lipase activity of about 600 units/I was achieved. This indicated the presence of the lipase producing components in the rich nutrient broth medium. Production of extracellular lipase was repressed by the addition of triacylglycerols, free fatty acids, glycerol and glucose. The repressive effects of triacylglycerols were found to be more rapid and pronounced as compared to the effects of free fatty acids and glycerol alone. The increased repressive effects of triacylglycerols could have occurred as a result of compounded effects of glycerol and free fatty acids, which are the hydrolytic products when the lipase hydrolyses the triacylglycerols. These observations suggested synergistic effect or the presence of two independent pathways by which free fatty acids and glycerol repress the production of lipase by Bacillus licheniformis. Addition of detergents Tween 20 and Tween 80 enhanced the production of lipases by the microorganism. The extracellular lipase activity increased to levels of about 2000 units/I in the presence of the detergent. The purification of the lipase from Bacillus licheniformis was attempted using a combination of ion-exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite and size exclusion chromatography. However, aggregation of lipase protein, lack of interaction, and irreversible interaction with chromatography resins resulted in only partially pure lipase preparations. The partially purified lipases showed biochemical properties similar to lipases produced by Bacillus pumilus and Bacillus subtilis. The three lipases are thermolabile, alkali tolerant and function optimally in alkaline pH conditions. The cloning and sequencing of the lipase gene from Bacillus pumilus isolate followed by amino acid analysis revealed high sequence homology suggesting similar protein folds. This led to the hypothesis that the mature lipase secreted by Bacillus Iicheniformis could have significant homology with the mature lipase secreted by the other two Bacillus species. Degenerate primers were consequently designed based on the sequences of mature lipases secreted by Bacillus pumilus and Bacillus subtilis. The primers amplified a DNA fragment of 560 bp encoding lipase activity with Bacillus Iicheniformis genomic DNA as the template. The DNA fragment encoding the mature lipase of Bacillus Iicheniformis was subcloned into the pET 20b(+) expression vector to construct a recombinant lipase protein containing 6 histidine residues at the C-terminal. High-level expression of the lipase by Escherichia coli cells harbouring the lipase gene-containing expression vector was observed upon induction with IPTG at 30°C. A one step purification of the recombinant lipase was achieved with Ni-NTA resin. The histidine tag was removed by creating a 6X His-tag at the N-terminal of the protein followed by a rTEV protease cleavage site. The lipase protein was purified by Ni-NTA affinity chromatography followed by cleavage with rTEV protease to remove the histidine tag. The Cterminal His-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 pH 9.5-11.5 and was remarkably stable at alkaline pH values up to 20 hours. The Cterminal histidine tag was found to enhance the specific activity of the lipase in pH conditions between 10-11.5. The enzyme showed maximal activities toward p-nitrophenyl esters and triacylglycerols containing C6 and Ca fatty acyl groups. The metals that affected the lipase significantly were divalent Co, Zn and Hg which decreased the activity to less than 30% with Hg abolishing all the lipase activity. The enzyme was not inhibited by ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), or mercaptoethanol, while the classical serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) decreased the lipase activity to 40% at 1 mM concentration. The amino acid sequence of the lipase shows striking similarities to lipases from Bacillus subtilis and Bacillus pumilus. Based on the amino acid identity and biochemical characteristics, we classified Bacillus licheniformis lipase as a member of Family 1.4 lipases, together with lipolytic enzymes produced by Bacillus subtilis and Bacillus pumilus. The consensus sequence of the lipases around the nucleophilic Ser deviated from the canonical Gly-X-Ser-X-Gly. In the three Bacillus lipases, the consensus sequence was found to be Ala-His-Ser-XGly. This motif is shared with another group of larger lipases produced by thermophilic Bacillus species. Site-directed mutagenesis of the Ala and His residues near the nucleophilic Ser suggested that the lipase enzymes acquired the amino acids during evolution for optimal activity and enhanced thermal stability. Asp133, His 156 together with Ser?? were identified by site-directed mutagenesis as residues that form the catalytic triad of the lipase enzyme. This was confirmed by the three-dimensional structure model built using Bacillus subtilis Lipase A as the template. While in pursuit of cloning the promoter region of Bacillus licheniformis lipase gene, a 3.5 kb DNA fragment that showed lipolytic activity on tributyrin agar plate was obtained. Sequence analysis of the cloned DNA fragment revealed that a new carboxyl esterase gene with an open reading frame encoding a protein of 484 amino acids with estimated molecular mass of 53 kDa and a pi of 5.4 had been cloned. The cloned protein showed high amino acid identity with industrially significant enzymes belonging to the esterase family. This study has advanced the biochemical knowledge on lipases secreted by Bacillus species. Although the biochemical properties of Bacillus lipases are becoming known, the molecular mechanisms regulating the biosynthesis of the enzymes remain unknown. Future studies should therefore also attempt to elucidate the molecular mechanisms of Bacillus lipase biosynthesis. This would facilitate the bioengineering of Bacillus species to produce inducible lipase enzyme at quantities enough for application in for example, detergent and leather tanning industries. Current studies aimed at the cloning of the promoter region of the lipase from Bacillus licheniformis should be continued for the purpose of understanding the molecular regulation of the gene. The role of Bacillus lipases in biocatalytic resolution of racemic mixtures has not been fully explored. The recent availability of the three-dimensional structure of Family 1.4 lipases will enable us to understand the structural determinants of lipase properties and to construct Bacillus lipases that suit desired functions. As an example, the mature lipase produced by Bacillus pumilus (UOFS) is more than 95% identical to Bacillus lichenifomis lipase at amino acid level, but the latter lipase shows a broader alkaline optimum pH profile. The determinant for the broad alkaline pH profile has not yet been identified. The new carboxyl esterase gene cloned in this study should be over-expressed and biochemically characterized. The biotechnological applicability for the enzyme should be assessed.en_ZA
dc.description.abstractAfrikaans: Lipases (E.C. 3.1.1.3), wat wyd in die natuur voorkom, kataliseer die hidrolise van triasielgliserole. Die lipase-gekataliseerde reaksie is omkeerbaar en 'n wye verskeidenheid trans- en interesterifiseringreaksies is moontlik. Die ensieme kan die sintese van produkte wat nie gewoonweg deur konvensionele chemiese metodes gemaak kan word, nie moontlik maak, en daar kan verwag word dat, soos die toepaslikheid van ltpases meer bekend word, hulle steeds belangriker in die ensiem mark sal word. Daarom sal nuwe lipases met nuwe eienskappe benodig word. Bakteriese isolate wat hoofsaaklik van alkaliese melkery afloopwater versamel is, is op Rhodamien B/olyfolie-, Tween 80/CaC12- en tributirien-bevattende agarplate getoets. Isolate wat lipase vervaardig het, is in skudkulture vir lipase-produksie in die teenwoordigheid van verskillende koolstofbronne getoets. Bacillus licheniformis en Bacillus pumilus is as die beste lipase-produseerders geïdentifiseer met p-nitrofeniel palmitaat as toetssubstraat en deur relatiewe aktiwiteit, optimum pH en temperatuur as maatstaf te gebruik. Lipase produksie deur Bacillus licheniformis is ondersoek in die teenwoordigheid van verskillende koolhidrate en lipiedsubstrate. Die mikro-organisme kon nie groei in 'n mineraalmedium wat met Tween 20, Tween 80, kaproësuur of kaprillensuur gesupplementeer was nie. Dieselfde medium met tributirien, trioleïen, olyfolie, gliserol of glukose gesupplementeer is, kon groei ondersteun maar geen lipase aktiwiteit kon gemeet word nie, waarskynlik weens die gebrek aan 'n induksiefaktor. Dit het daarop gedui dat daar in 'n ryk voedingsop medium waarskynlik 'n lipase induseerder teenwoordig was. Die byvoeging van triasielgliserole, vrye vetsure, gliserol of glukose het die produksie van die ekstrasellulêre lipase onderdruk. Die onderdrukkende effek van triasielgliserole was vinniger en sterker as die van vetsure of gliserol afsonderlik, moontlik as gevolg van die gesamentlike effek van die twee verbindings wat beide die produkte van hidrolise is. Die waarnemings het gedui op 'n sinergistiese effek van moontlik twee afsonderlike bane waar deur vrye vetsure en gliserol die produksie van Bacillus licheniformis lipase onderdruk. Byvoeging van Tween 20 of Tween 80 het die produksie van lipases deur die organisme versterk en 'n toename tot so veel as 2000 eenhede/ml veroorsaak. Suiwering van die lipase is deur middel van 'n kombinasie van ioonuitruilings-, hidrofobiese interaksie-, hidroksielapatiet- en uitsluitingschromatografie was onsuksesvol. Aggregasie, 'n gebrek aan interaksie met die kolommedia of onomkeerbare binding van die lipase-proteïen aan die chromatografiemedium het die suiwering bemoeilik en slegs 'n gedeeltelik-gesuiwerde proteïen kon verkry word. Die lipasepreparaat het dieselfde eienskappe as dié van Bacillus pumi!is en Bacillus subti!is getoon. AI drie ensieme was termies labiel, kon alkali tolereer en het optimaal in alkaliese medium gefunksioneer. Die klonering en nukleotiedvolgordebepaling van die lipase geen van 'n Bacillus pumi!is isolaat het daarop gedui dat hierdie lipase en die een van Bacillus subti!is dieselfde struktuur kon hê. Dit kon daarop dui dat die lipase van Bacillus !icheniformis beduidende homologie met die ensiem van die ander twee bacilli kon toon. Degenererende voorlopers is, met die nukleotiedopeenvolging van die Bacillus pumi!is en Bacillus subti!is as basis, ontwerp. 'n DNA fragment van 560 basispare wat vir lipase aktiwiteit kodeer en met die genomiese DNA as templaat, is met die gebruik van die voorlopers geamplifiseer. Die DNA fragment wat vir die lipase kodeer is in 'n pET 20b(+) uitdrukkingsvektor gesubkloneer om 'n lipase konstruk te verkry met 6 histidienresidue aan die C-terminale van die proteïen toegevoeg. Hoë uitdrukkingsvlakke van die lipase is verkry met Escherichia coli selle wat die vektor bevat na induksie met IPTG by 30°C. 'n Enkel stap suiweringsprosedure deur chromatografie op Ni-NTA chromatografiemedium het gevolg. Die histidien merker kon verwyder word deur 'n 6X histidien volgorde aan die N-terminale punt van die proteïen te heg en dit na proteïenuitdrukking met rTEV protease te verwyder, Suiwering van die volledig geprosesseerde ensiem was daarna op die Ni-NTA kolom moontlik. Die ensiem met die C-terminale histidien merker sowel as die volledig geprosesseerde ensiem is daarna gekarakteriseer. Die gesuiwerde ensiem het 'n spesifieke aktiwiteit van 130 eenhede/mg getoon met p-nitrofeniel palmitaat as substraat. Die ensiem was maksimaal aktief by pH van 9.5-11 en was besonder stabiele by alkaliese pH tot by pH 12. Die Cterminale histidien merker het die lipase aktiwiteit by pH 10-11.5 versterk. Die ensiem was maksimaal aktief met p-nitrofeniel esters of triasielgliserole met C6 of Ca asielgroepe. Divalente Co, Zn, en Hg het die grootste effek op die aktiwiteit gehad; eersgenoemde twee het die aktiwiteit tot minder as 30% laat afneem terwyl Hg die ensiem totaal gedeaktiveer het. Die ensiem was nie deur EDTA, ditiotreïtol of merkaptoetanol geïnhibeer nie maar die klassieke serien proteaseinhibeerder, feniel-metiel-sulfonielfluoried (PMSF) (1mM) het die lipaseaktiwiteit tot minder as 40% van die kontrole laat afneem. Die aminosuuropeenvolging van die lipase het besondere ooreenkomste met lipases van Bacillus subtilis en Bacillus pumilis getoon. Met aminosuuropeenvolging en biologiese eienskappe as maatstaf is die Bacillus licheniformis lipase as 'n lid van die Familie 1.4 lipase geklassifiseer, saam met die lipases van Bacillus subtilis en Bacillus pumilis. Die konsensus-opeenvolging in die omgewing van die nukleofiliese serien het van die gewone Gly-X-Ser-X-Gly afgewyk. In die Bacillus lipases is die opeenvolging Ala-His-Ser-X-Gly, 'n motief wat ook in die groep groter lipases van die termofiele Bacillus spesies. Setelgerigte mutagenese van die Ala en His residue na-aan die nukleofiliese Ser het aangedui dat die lipase-ensieme dié aminosure verkry het, waarskynlik tydens evolusie vir optimale aktiwiteit en verhoogde termiese stabiliteit. Asp133, His156 en Ser?? is as die katalitiese drietal geïdentifiseer deur setelgerigte mutagenese. Dit is bevestig deur "n driedimensionele model van die ensiem te bou met die Bacillus subtilis lipase A as templaat. In "n poging om die promotorgebied van die Bacillus licheniformis te kloneer, is "n 3.5 kb DNA fragment verkry wat ltpolltiese aktiwiteit op tributirien agarplate getoon het. Nukleotied opeenvolgingsanalise van die gekloneerde fragment het "n nuwe karboksiel esterasegeen opgelewer met "n oop leesraam wat kodeer vir "n proteïen met 484 aminosure, "n geskatte molekulêre massa van 53 kDa en "n pi van 5.4. Hierdie studie het beduidend bygedra tot die beskikbare inligting aangaande die lipases van die Bacillus spesies. Alhoewel meer omtrent die molekulêre eienskappe van die Bacillus lipases bekend word, is daar min bekend oor die molekulêre meganismes wat die uitskeiding van die ensieme reguleer. Toekomstige navorsing behoort daarop gerig te wees om hierdie kwessie op te klaar. Dit sou die biogeniëring van Bacillus spesies fasiliteer en moontlik verhoogde uitskeidingsvlakke van die ensiem moontlik maak vir toepassing in die leer- of wasmiddelbedryf. Huidige studies wat gerig is om die promotorgedeelte van die lipase geen van Bacillus licheniformis te kloneer behoort daarom voortgesit te word. Die toepassing van Bacillus lipases in die biokatalitiese skeiding van rasemiese mengsels is nog nie volledig ondersoek nie. Die onlangse publikasie van die driedimensionele struktuur van 'n Familie 1.4 lipase maak nou meer sinvolle konstruksie van pasgemaakte Bacillus lipases moontlik. As voorbeeld kan genoem word dat die aminosuuropeenvolging van die volledig geprosesseerde lipase van Bacillus pumilis (UOFS) meer as 95% ooreenkoms toon met die ensiem van Bacillus licheniformis maar laasgenoemde toon 'n wyer optimale pH profiel. Die rede vir die verskillende pH profiel is nog onbekend. Die nuwe karboksiel esterasegeen wat gekloneer is behoort in groot maat uitgedruk te word en biochemies gekarakteriseer te word. Die biotegnologiese toepaslikheid van die ensiem kan dan ook bepaal word.en_ZA
dc.description.sponsorshipNational Research Foundation (NRF)en_ZA
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.subjectBacillus (Bacteria)en_ZA
dc.subjectLipaseen_ZA
dc.subjectEnzymes -- Analysisen_ZA
dc.subjectThesis (Ph.D. (Microbiology and Biochemistry))--University of the Free State, 2001en_ZA
dc.titleMolecular and kinetic properties of recombinant Bacillus lipaseen_ZA
dc.typeThesisen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA


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