Exophiala dermatiditis lipase: isolation and characterisation

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.