Fermentation Technology Of Yarrowia Lipolytica Lipase Lip2 Displayed On The Cell Surface Of Saccharomyces Cerevisiae

Lipase (EC 3.1.1.3), an ester bond-hydrolase, can hydrolyze the long chain fatty acid trilgyceride to fatty acids and glycerine. It can be used in the bio-energy, food processing, and so on. People pay more attention to the exploration of bio-energy because of energy crisis in recent years. Biodiesel is a kind of ideal bio-energy. Although the chemical-enzymatic catalyzing for biodiesel preparation technics has been industrialized, it has many disadvantages, such as difficult separation, high production cost and pollution discharge. The bio-enzymatic catalyzing for biodiesel preparation method is becoming the development trend of biodiesel preparation. However, the instability, lower catalytic activity and hard to be recycled in usage of dissociative lipase, which cause high production cost, are bottlenecks for its development of industrialization. Surface displayed of lipases as a whole-cell catalyst can improve lipase's stability, catalytic activity and reusability. In this paper, with recombinant strain Saccharomyces cerevisiae EBY100/pCTLip2, optimizations of fermentation technology of surface displayed lipase Lip2 production in flask were conducted.The main work and results were as follows:1. Making a study on enzymatic properties of the surface displayed lipase. A comparison study was conducted on effect of enzyme activity of the surface displayed lipase Lip2 in different temperature, different pH and different substrate. The results show that the optimum temperature is 40°C, the optimum pH is 7.0, and the optimum substrate is C8.2. Conducting a study on fermentation parameters of surface displayed Lip2. At the single factor experiments, fermentation conditions of surface displayed Lip2 have been studied with rotating speed, liquid volume in flask, induction time, the content of GAL, the content of YNB, inoculation amount, inducing temperature, nitrogen source and so on. The optimal parameters of fermentation technology are: 40mL liquid volume in flask, 72h induction time, 20°C induction temperature, 1.5%(w/v)GAL amount, 4% inoculation amount, and best nitrogen source is YNB. 3. Based on single factorial experiments, two important factors were chosen from the seven factors related to the hydrolyzing activity of the surface displayed lipase by Plaekett-Burman method. The result shows that CAA (Casein acid hydrolysate) and GAL were two significant factors having significant effects on enzyme activity of the surface displayed lipase. Then, steepest ascent experiment design was chosen to define the optimal response region of the significant factors. Six times experiments were conducted totally. The result shows that the most large lipase production region is nearby RUN4. The optimal values for the two significant factors were determined by using response surface method. Finally, the optimal medium are 5.40 g Na2HPO4,7.45 g NaH2PO4,15.029g CAA, adding distilled water to 800ml, pH 7.0, steam sterilization under 0.1Mpa lasting for 20min, adding 100ml 13.4%(w/v) YNB solution and 100ml 13.821%(w/v) GAL solution after cooling. The optimal culture conditions are the inoculation amount equal to the 10% amount of liquid medium, initial pH value pH 7.0, rotating value 150 rpm/min, liquid volume in flask 30mL. Under these conditions, the highest hydrolyzing activity of displayed Lip2 can reach 1400.39U/g-dry cell.