| Cellulase is a kind of compound-enzyme which can hydrolyze cellulose and can be widely used in the food,fermentation,environmental protection,textile, energy,such as feed industry.Aqueous enzymatic extraction of oil has a lot of incomparable advantages than traditional squeeze method and extracting method that it has high oil quality,mild conditions and it can reduce the energy consumption and reduce chemical reagent usage,which has been already got more and more attention of scholars both at home and abroad.And cellulase is the most important enzymes reported in many literature.At present the production level of cellulase and the enzymatic activity are very low and stability of enzyme activity is inferior;technological research of Aqueous enzymatic extraction of oil is still not perfect that the problems of the best conditions of enzyme solution,solvent residual,emulsion breaking in emulsion system and so on need further researching.In addition,there are a few reports of aqueous enzymatic extraction of cornus Wilsoniana fruit oil.So it has positive and realistic significance to expand correlative research to increase cellulase activity and to go into aqueous enzymatic extraction of cornus Wilsoniana fruit oil.In this thesis the Penicillium sp. preserved in our lab was chosen as starting strain,and the conditions of shaking-flask fermentation were optimized and processes of fermentation in a5L bioreactor was discussed,then the purification,and batch fermentation kinetics,lipase isolation and purification,lipase immobilization and its catalyzing esterification were studied in this paper.The results were as follows:Monofactorial experiments and response surface methodology were applied to optimize and got the optimal culture medium formula (W/V):bran2.955%,medicine medium3.25%,microcrystalline cellulose3.75%,ammonium sulfate1.5%,calcium chloride0.03%;the optimal conditions to produce;The optimal enzyme production conditions:speed220r/min,temperatrure30℃,pH5.5,fermentation time132h. Three groups experiment were done under these optimal conditions and the cellulose activity were7109.22U/mL which has been improved greatly compared to the initial enzyme activity of6620U/mL. The fermentation process of the Penicillium sp. was magnified in a5L fermenter and based on the data gotten from its batch fermentation in the5L fermenter,and combined mathematical reasoning,kinetic models of cell growth,synthesis of lipase and substrate consumption were established. These were as follows:(1) The kinetic model of cell growth(2) The kinetic model of synthesis of cellulose(3) The kinetic model of substrate consumptionThese established models could describe well the dynamic process of synthesis of cellulase by the batch fermentation of the Penicillium sp.,and it could give some help on the further scale-up. The isolation and purification of cellulase was studied byhyperfiltration,ammoniu-m sulfate salting-out and lyophilization.The results showed that the purification protocol resulted in2.23-fold purified naringinase with a final yield of88.1%,and the specific activity of cellulase reached5.848x104U/g from the initial2.628X104U/g.Aqueous enzymatic extraction of cornus Wilsoniana fruit oil was studied.Based on processes for oil extraction of single and composite,Monofactorial experiments and Orthogonal experiments were applied to optimize and got the optimal processes for oil extraction:crushing degree80mesh,solid-liquid ratio1:5,microwave power750W and time4min,pH5.0, temperature45℃,enzymatic hydrolysis time2.0h,then at the original temperature add pectinase0.75%,neutral protease0.75%,pH7.0,enzymatic hydrolysis time4.5h.Three groups experiment were done under these optimal conditions and the average oil extraction efficiency yield could reach84.22%,which has been improved greatly compared to the initial oil extraction efficiency yield of23.56%.The optimization of process were real and effective, whic h can be further used in industrialized mass production. |
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