Exophiala dermatiditis lipase: isolation and characterisation

Abstract

English:Lipases (EC 3.1.1.3) or acylglycerol hydrolases, which are widely distributed in nature, are enzymes that catalyse the reversible hydrolysis and synthesis of tri-, di- and monoacylglycerols. These enzymes can be used for chemical modification of lipids, processes such as hydrolysis, ester synthesis and interesterification reactions. Current research has focus sed on the determination of the 3D-structure of lipases, industrial application of lipases, immobilisation of lipases, genetic engineering, use of lipases in organic systems and the isolation of new lipases. Black yeast isolates (23) were obtained from the departmental yeast culture collection of the Department of Microbiology and Biochemistry, UOFS. These isolates were screened for lipase activity on agar plates containing four different inducers. Two isolates were chosen for further studies, namely Exophiala dermatiditis UOFS Y-2044 and Exophiala dermatiditis UOFS Y-2048. Lipase production during shake culture was determined and optimised using olive oil as inducer. Isolation of the two extracellular lipases present in the supernatant after centrifugation of the culture medium and the addition of a protease inhibitor, PMSF, included the following steps: cation exchange chromatography on Toyopearl SP-650M, anion exchange chromatography on Toyopearl Super-Q 650S, gel permeation on Toyopearl HW50F and affinity chromatography on MIMETIC Red and Yellow dye ligands for ED2044L and ED2048L, respectively. The final purification protocol for ED2044L resulted in a 58-fold purification, a specific activity of 1,73 U/mg and a final yield of 42,7%. One band with an approximate molecular mass of 23 600 was visible on SDS-PAGE. The purified ED2044L showed maximal activity under alkaline pH conditions with an optimum pH of pH8,5. The lipase had an optimum temperature of 50eC. The lipase was not thermostable at temperatures higher than 35eC, with an approximate half-live of 3,2 hours at 40eC. EDTA significantly inhibited the activity of ED2044L, indicating that this is a metalloenzyme. Calcium(II), tin(II) and copper(II) inhibited the lipase activity, whereas magnesium(II), iron(III), mercury(II), barium(II) and manganese(II) activated the lipase activity. ED2044L was affected by detergents, with CHAPS, sodium deoxycholate, Cetrimide, Triton-XlOO, Tween-80 and SDS inhibiting the lipase activity. PMSF activated the-lipase at lower concentrations and inhibited the lipase activity by approx. 30% at higher concentrations. The lipase showed interfacial activation. From the substrate specificity results, it was concluded that ED2044L prefers short chain substrates.The final purification protocol of ED2048L resulted in a 3-fold purification, a specific activity of 1,6U/mg and a final yield of 13,5%. No bands were visible on SDS-PAGE, because of the low protein concentration. The purified ED2048L showed maximal activity under alkaline pH conditions with an optimum pH of 8,5. The lipase had an optimum temperature of 65°C. The lipase was not thermostable at temperatures higher than 35°C, with an approximate half-live of >24 hours at 40°C, indicating that ED2048L was more thermostable than ED2044L. The lipase showed interfacial activation. From the substrate specificity results, it was concluded that ED2048L also prefers short chain substrates.Enough evidence to identify the two enzymes as true lipases was provided. The presence of interfacial activation with tripropionin and their activity on triacylglycerol substrates confirmed this. The aim of this study, namely to isolate and purify two lipases produced by black yeasts and compare them with each other and other lipases, was successfully completed.

Afrikaans: Lipases (EC 3.1.l.3) of asielhidrolases, wat wyd verspreid in die natuur voorkom, is ensieme wat die omkeerbare hidrolase en sintese van tri-, di- of monoasielgliserole kataliseer. Lipases kan egter ook gebruik word vir die chemiese modifisering van lipiede asook prosesse soos hidrolise, ester sintese en interesterifikasie. Huidige navorsing behels die bepaling van die 3D-struktuur van lipases, industriële toepassing van lipases, gebruike van lipases in organiese sisteme, asook die volgehoue isolasie van nuwe lipases.Swart gis isolate (23) is verkry van die departementele gis versameling van die Departement van Mikrobiologie en Biochemie, UOVS. Die isolate is getoets vir lipase produksie deur dit uit te streep op agar plaatjies met vier verskillende induseerders. Twee isolate is gekies vir verdere studies, naamlik Exophiala dermatiditis UOFS Y-2044 en Exophiala dermatiditis UOFS Y-2048. Lipase produksie is gedurende kultivering bepaal en geoptimiseer deur olyfolie as induseerder te gebruik. Die isolasie van die twee esktrasellulêre lipases teenwoordig in die bovloeistof na sentrifugering van die kultuur medium en die byvoeging van a protease inhibeerder, PMSF, het die volgende stappe ingesluit: katioon-uitruilingschromatografie m.b.v. Toyopearl SP-650M, anioonuitruilingschromatografie m.b.v. Toyopearl Super-Q 650S, gelfiltrasie m.b.v. Toyopearl HW50F en affiniteits chromatografie m.b.v. MIMETIC Red en Yellow kleur ligande vir onderskeidelik ED2044L en ED2048L. Die resultaat van die finale suiwering van ED2044L was 'n 58-voudige suiwering met 'n spesifieke aktiwiteit van 1,73U/mg en 'n finale opbrengs van 42,7%. Een band met 'n relatiewe molekulêre massa van 23 600 was sigbaar op die SDS-PAGE gel. Die suiwer ED2044L het maksimale aktiwiteit onder alkaliese toestande getoon met 'n optimum pH van 8,5. Die lipase het 'n optimum temperatuur van 50°C gehad. Die lipase is nie termostabiel by temperature bokant 35°C en het 'n relatiewe half-leeftyd van 3,2 ure by 40°C getoon. EDTA het 'n noemenswaardige inhibering effek op die lipase gehad, wat impliseer dat die ensiem miskiem 'n metalo-ensiem is. Die lipase aktiwiteit is geïnhibeer deur kalsium(I1), tin(II) en koper(II). Magnesium(II), yster(III), kwik(II), barium(I1) en mangaan(I1) het gelei tot die aktivering van die lipase se aktiwiteit. Die effek van die wasmiddels CHAPS, natrium deoksikolaat, Cetrimide, Triton-X-lOO, Tween-80 en SDS het gelei tot die inhibering van ED2044L aktiwiteit. PMSF het die lipase aktiwiteit geaktiveer by laer konsentrasies en daarna by hoër konsentrasies sowat 30% geïnhibeer. Die lipase het skeidingsvlak aktivering getoon. Van die substraat spesifisiteit studies, kan dit afgelei word dat die lipase kort ketting substrate verkies. Die resultaat van die finale suiwering van ED2048L was 'n 3-voudige suiwering met 'n spesifieke aktiwiteit van 1,6U/mg en 'n finale opbrengs van 13,5%. Geen bande was sigbaar op die SDSPAGE gel nie, heel waarskynlik toe te skryf aan die baie lae proteïen konsentrasie van die lipase. Die suiwer ED2048L het maksimale aktiwiteit onder alkaliese toestande getoon met 'n optimum pH van 8,5. Die lipase het 'n optimum temperatuur van 65°C gehad. Die lipase is nie termostabiel by temperature bokant 35°C en het 'n relatiewe half-leeftyd van >24 ure by 40°C getoon, wat impliseer dat ED2048L meer termostabiel is as ED2044L. Die lipase het skeidingsvlak aktivering getoon. Van die substraat spesifisiteit studies, kan dit afgelei word dat die lipase kort ketting substrate verkies. Voldoende bewyse om die twee ensieme as egte lipases te identifiseer is gevind. Die voorkoms van skeidingsvlak aktivering met tripropionien en die aktiwiteit van die ensieme met twee triasielgliserol substrate was 'n bevestiging hiervan. Die doel van die projek, naamlik die isolasie en suiwering van twee lipases wat deur swart giste geproduseer is, en die vergelyking van die twee lipases met mekaar en ander lipases, is suksesvol afgehandel.