Isolation Screening And Fermentation Conditions Of Lipase Producing Strains And Studies On Enzymatic Characteristics

Lipases of triacylglycerol hydro lases （EC3.1.1.3） are a class ofenzymes, termed as carboxyl esterases, which catalyze the hydro lys is andsynthes is to form esters fro m the glycerol a nd the long cha in fatty acids at theoil–water interface, a phenomenon k nown as interfac ial activation. Lipasesconstitute one o f the most important groups o f ind ustria l enzymes due to the iruniq ue ability of activities in both aqueous and non-aqueous solvent systems.Because of these advantages, they are wide ly used in severa l areas ofmed ic ina l, biotechnolo gica l, detergent, dairy, food, textile, surfactant,pharmaceutica l and hydro lys is of fats and oil processing. Using lipasecatalytic catering waste oil biod iesel, can not only solve the increasinglyserious problem of waste oil, more can produce green energy-biodiese l. Soscreening and research microbia l lipase either from the economic interests, orfro m environme nta l effect into cons ideration, a ll ha ve very importantsignifica nce.In order to obtain the lipase substrate specific ity of waste cooking oil, theseven stra ins was isolated fro m the oily waste soil, a fter two round pla ntscreen, whic h can growth with olive oil and produce yellow circ le in the plant.Through ferme ntation and enzymatic activity determinatio n, CJLU-31wasselected to the further work. According to the morpholo gical, phys io lo gical,bioche mica l identification a nd ITS sequence ana lys is, this stra in be longed toAspergillus and named as Aspergillus oryzae CJLU-31.The fermentation cond itio ns of Aspergillus oryzae CJLU-31wasinvestigated in shake flask, through an Optimizatio n design was obtained bysingle factor and ortho gona l test. The optimized fermentation mediumcontained （g/l）: beef e xtract30, olive o il3.75, polyving akoho l-1240.125%,（NH4）2SO41.0, MgSO47H2O1.0, K2HPO41.0. While the ferme ntationcondition was optimized, which was pH7.0,32°C, for48h, and150r/min and2%（V/V） of the inoc ulum s ize. Under these conditio ns, the lipase activity can reached460.7U/mg, whic h was1.92folds of that before optimizatio n.After40%-60%ammonium sulfate precipitatio n and DEAE-Sepharose FF,Sephadex G-100two-step chromatography, a25.1-fold purity of lipase withspecifc activity of11552.2U/mg protein and molecular mass of27kDa wasobtained.The purifed lipase showed maximum activity and stability at pH4.0and40°C. The lipase retained high activity over ranges of pH（2.0-5.0） andtemperature （30-50°C）, and was enhanced by low concentratio n meta l ions （1mM）, such as K⁺, Li⁺, Mg²⁺, Zn²⁺, Mn²⁺and Ca²⁺, while inhibited signifcantlyby Fe²⁺, Fe³⁺ and Cu²⁺. The enzyme in10%（v/v） weak polar organic solve nt（ether） can keep99%of the initia l lipase activity,20%（v/v） glycerin canincrease the activity and the enzyme to10%（v/v） short cha in a lcoho l hascertain to lerance; This enzyme has good substrate specific ity of waste cookingoil;0.1%polyving akohol-124can be as surfactant to activate its activity. TheKm and Vma x of the lipase acting on olive oil were0.11mM and0.41mM/mim, respective ly. Heating and cooling refold-treatment can activate itsactivity.Aspergillus oryzae CJLU-31lipase is the acid lipase whic h has goodtemperature stability and substrate specializatio n of waste cooking o il. Thesefeatures provided the lipase from Aspergillus oryzae CJLU-31as a potentia lapplication for lipase-catalyzed synthesis o f fatty acid methyl esters （biodiesel）us ing waste cooking o il.