Exophiala dermatiditis lipase: isolation and characterisation

[摘要] English:Lipases (EC 3.1.1.3) or acylglycerol hydrolases, which are widely distributed in nature, areenzymes 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, estersynthesis and interesterification reactions. Current research has focus sed on the determination ofthe 3D-structure of lipases, industrial application of lipases, immobilisation of lipases, geneticengineering, 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 theDepartment of Microbiology and Biochemistry, UOFS. These isolates were screened for lipaseactivity on agar plates containing four different inducers. Two isolates were chosen for furtherstudies, 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 culturemedium and the addition of a protease inhibitor, PMSF, included the following steps: cationexchange chromatography on Toyopearl SP-650M, anion exchange chromatography on ToyopearlSuper-Q 650S, gel permeation on Toyopearl HW50F and affinity chromatography on MIMETICRed and Yellow dye ligands for ED2044L and ED2048L, respectively. The final purification protocol for ED2044L resulted in a 58-fold purification, a specific activity of1,73 U/mg and a final yield of 42,7%. One band with an approximate molecular mass of 23 600was visible on SDS-PAGE. The purified ED2044L showed maximal activity under alkaline pHconditions with an optimum pH of pH8,5. The lipase had an optimum temperature of 50eC. Thelipase was not thermostable at temperatures higher than 35eC, with an approximate half-live of 3,2hours at 40eC. EDTA significantly inhibited the activity of ED2044L, indicating that this is ametalloenzyme. Calcium(II), tin(II) and copper(II) inhibited the lipase activity, whereasmagnesium(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 lowerconcentrations and inhibited the lipase activity by approx. 30% at higher concentrations. Thelipase showed interfacial activation. From the substrate specificity results, it was concluded thatED2044L prefers short chain substrates.The final purification protocol of ED2048L resulted in a 3-fold purification, a specific activity of1,6U/mg and a final yield of 13,5%. No bands were visible on SDS-PAGE, because of the lowprotein concentration. The purified ED2048L showed maximal activity under alkaline pHconditions with an optimum pH of 8,5. The lipase had an optimum temperature of 65°C. Thelipase was not thermostable at temperatures higher than 35°C, with an approximate half-live of >24hours at 40°C, indicating that ED2048L was more thermostable than ED2044L. The lipase showedinterfacial activation. From the substrate specificity results, it was concluded that ED2048L alsoprefers short chain substrates.Enough evidence to identify the two enzymes as true lipases was provided. The presence ofinterfacial 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 andcompare them with each other and other lipases, was successfully completed.

[发布机构] University of the Free State