Production, Purification And Characteristic Of Lipase From Aspergillus Niger

Nowadays, microbial lipase has become the main source of industrial application of thelipase. Lipases with special enzymatic characteristics （thermolstable, pH tolerant, etc.） arerequired for its application in food, dairy, medicine, and energy industries. In this paper, anlipase-producing microorganism from the traditional food in Tibet is screened, thecultivation conditions is optimized, the lipase is purified and its enzymatic characterization isstudied. The main research contents and results are as followings：（1）An extracellular lipase-producing strain, which is identified as Aspergillus nigerAN0512by18s rRNA sequence analysis and morphologic observation, is screened fromtraditional food in Tibet. The cultivation conditions for lipase production of AN0512areoptimized. The optimal fermentation conditions for lipase production are as followings: seedoil1%（v/v）, yeast extract powder1%（w/w）, KH2PO40.2%（w/w）, MgSO40.05%（w/w）,KCl0.05%（w/w）, inoculum size1.5%（v/v）, under which the highest enzyme activity can beachieved to30IU/mL at28℃and180r/m for96h.（2）The characterization of the crude lipase were studied, the maximal enzyme activityof the crude lipase is achieved at the condition of30℃and pH7.0; and the crude lipase isheat-resistant for it can even be retained for more than60%enzyme activity at60℃; Ca²⁺,Fe²⁺, Co²⁺, Mg²⁺and Mn²⁺activate the crude lipase.（3）With ammonium sulfate precipitation followed by ion-exchange chromatography andgel filtration, the purified lipase achieved203.6-fold purification and22.1%finally recovery.The purified enzyme was a monomeric protein of41kDa, which showed a prominent singleband on SDS-PAGE; By the analysis of MALDI-TOF-MS, the A. niger AN0512lipaseshowed high homology with esterase from A. niger CBS513.88.（4）The optimum temperature and pH for lipase activity were50℃and pH5.0,respectively. And the enzyme exhibited excellent stability for temperature and pH, retained92%activity at60℃for12h and99%activity at pH3.0for20h, respectively. Cu²⁺and Ca²⁺stimulated lipase activity whereas Fe²⁺showed strongly inhibition. Some polar organicsolvents also stimulated lipase activity. The lipase exhibited1,3-regiospecificity and showed lower Km constant（10μmol/L）which indicated grater affinity for the substrate p-NPP.