| Pichia anomala Kurtzman 1003 is an yeast which can excrete lipase to the culture medium.A suitable culture medium which is good for lipase production and purification was obtained.The lipase was purified by ammonium sulphate precipitation,Sephacryl S-100 and DEAE Sepharose Fast Flow chromatography.The molecular weight of the lipase was estimated to be 54.9kDa according to the SDS-PAGE.The optimum temperature and pH of the lipase are 45℃and pH5.5,respectively.The lipase can hydrolyze many esters including fatty acid esters of C4-C18 carbon chain,olive oil, soybean oil,peanut oil,but shows higher activities on short chain fatty acid(＜C12) esters.1.Medium optimizing and lipase purifying for P.anomala 1003After testing several culture media,an optimum medium was obtained which is suitable for the cell growth,lipase yield and purification of lipase.The formula is as below:bean cake 6.0%,NH4NO3 0.6%,KH2PO4 0.3%,MgSO4.7H2O 0.25%,olive oil emulsification 0.1%,pH 5.8.P.anomala 1003 was growing in above medium at 28℃for 36 hours.Extracellular lipase,which is excreted to the medium by the strain,was purified by ammonium sulphate precipitation,Sephacryl S-100 and DEAE Sepharose Fast Flow chromatography, ultrafiltration.The lipase was purified and concentrated from the culture supernatant,with the fold of 206,yield of 6.09%.2.Characterization of purified lipase from P.anomala 1003The molecular weight of the lipase was 54.9kDa estimated according to the transport ratio by SDS-PAGE.In the range of pH4.5-6.5,30-50℃,the enzyme shows higher activities on ester hydrolyzation,the optimum pH and temperature are pH5.5 and 45℃,respectively.The lipase is stable under 50℃or ranges of pH4.0-7.5.The activities was promoted by the metal ions of Ca2+,Mg2+,and inhibited by most of the heavy metal ion,especially Cu2+, Fe3+and EDTA.Several detergents such as Triton X-100,Tween 80,Sodium Desoxycholate also can promote the activity of P.anomala 1003 lipase,while SDS acted as an inhibition factor.The results of substrate specificity assay showed that:P.anomala 1003 lipase can hydrolyze all the testing esters,but shows higher activities on short chain fatty acid(＜C12)esters.3.Sequencing of P.anomala 1003 lipase by MALDI-TOF-MSThe amino acid sequence of P.anomala 1003 lipase remained unknown and four small peptides sequence of the enzyme were obtained by MALDI-TOF-MS.The amino acid sequence of the peptides were:-V-Y-A-V-F-R-;-V-Y-Q(or K)-E-T-D-I(or L)-V-P-R-;-I(or L)-V-W-G-G-D-I(or L)-P-I(or L)-P-S-Y-S-R-;-I(or L)-Q(or K)-Y-G-E-V-T-I(or L)-I(or L)-T-Y-A-S-I(or L)-R-.These sequences provide useful information for PCR primer design and gene clone. |
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