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.