Screening And Isolation Of A High-yield Thermostable Lipase Producing Strain And Research On The Production, Purification And Properties Of The Lipase

Lipases belong to a common class of enzymes which can hydrolyze triglycerides at the lipid-water interface and are mainly obtained from animals, plants and microorganisms. Many industrial lipase-calalyzed processes are mostly performed at high temperature, which requires the lipases to be thermostable and with having exceptionally high optimum reaction temperature. Considering that, the isolation of strains producing thermal stable lipases from the extreme hot environment could be an ideal way to achieve the desirebale thermostable properties. In the current study, all the isolates of steamer cushions were characterized for their ability to produce lipases. Only four strains produced lipolytic enzymes which were further screened to produce heat resistant lipases, only one strain belonging to the fungus, Aureobasidium pullulans, produced thermostable lipases and was named as S3. Following this screening, the optimum culture medium was developed for this strain based on single factor method. The novel lipase was purified and characterized for its stability towards pH, temperature, metallic ions and organic solvents.Among the four lipase producing strains, the two strains S3and S4produced extracellular lipases and the temperature-bearing characteristics of the lipases produced by them were studied. For S3and S4, the lipase activities of the fermentation supernatant were10.61U/mL and14.43U/mL, respectively. The optimum temperature of the lipase prodecued by the strain S3was50℃with no activity loss at50℃for5h. The lipase from strain S4showed maximum activity at60℃with no activity loss at40℃for5h, but only42.19%activity was maintained at50℃for5h.The strain S3was selected as the target strain due to the better thermostable properties and lower viscosity of the fermentation broth that could make the separation and purification of the lipase more easily.The fermentation conditions and culture medium components have been optimized by single factor experiment. The optimal composition of the culture medium (w/v) was2%yeast extract,2%tryptone,1%glucose,1%maltose,0.15%Tween20and4.5%corn oil. The optimal fermentation inoculum is1.5%,with a total fermentation volume of30mL (250mL Erlenmeyer flasks). The best enzyme producing condition was found96h at30℃,200r/min. The lipase yield of18.28U/mL was obtained at the optimal conditions, which is2.16-fold that of the original fermentation.The extracellular lipase from the strain S3was purified by centrifugation, filtration, ultrafiltration and DEAE-Sepharose Fast Flow anion exchange chromatography. The lipase was finally purified to18.16-fold with a7.26%lipase activity and the specific activity of the purified lipase was17.74U/mg pro. The purified lipase had a single band with a relative molecular weight of39.5kDa on SDS-PAGE gel. The lipase was found to be maintaining a high activity at10℃to40℃and was also stable in an alkaline range (pH7.0-10.0), with the optimum temperature and pH being40℃and7.0, respectively. The lipase was stable in30%organic solvents such as hexane, n-propanol, isopropanol and DMSO. Meanwhile, it displayed a preferable stability in the presence of10mM of K+, Mg2+, Ca2+, Ba2+and Fe2+ions, and Triton X-100, SDS, EDTA, DTT. Furthermore, the lipase activity was remarkably improved by emulsifier Tween20and Tween80. Besides, it could also tolerate high concentration of NaCl.