Immobilization Of Cellulases On Magnetic Nanoparticles And Construction Of Genetic Transformation System For Cellulolytic Filamentous Fungi

Cellulases are enzymatic hydrolytic systems which consist of different enzymes capable of degrading cellulose into simple sugars through their synergies, and these sugars can be fermented to produce ethanol. As a result, Cellulases have great potential applications in some fields, such as agriculture, renewable energy environmental protection. The immobilized cellulase could be more stabile and reusable than free celluase, and result in lower cost in industrial application. In addition, a novel immobilization technology of celluase could bring a new idea of reasonable immobilization of muti-enzyme complex. This dissertation has focused on the study of preparation of the immobiled cellulase in polyvinyl alcohol/Fe₂O₃ nanoparticles, and the construction of transformation of Penicillium simplicissimum mediated by agrobacterium tumefaciens. The thesis mainly includes the following parts:1. The immobiled cellulase in polyvinyl alcohol/Fe₂O₃ nanoparticles were prepared by cyclic freezing–thawing process. Characters of TEM,IR and VSM suggested that the average diameters of immobilled cellulase complexs were 1μm whichcontained 10nm Fe₂O₃ naoparticle. Factors affecting activities of immobilizated cellulase were researched. About 42% activity retention of immobilized cellulase was achieved under the optimum conditions: pH 6.0, cellulase/PVA equal to 4,PVA/Fe equal to 50,11 hour for immobilization. The immobilized cellulase exhibited greater efficiency than free cellulase and retained 50% relatively activity after five cycles of reuse, which indicated that this novel method of immobilization could be propitious to reuse and improve efficiency of cellulase.2. Using bovine serum albumin (BSA) as a model protein, we established a pattern of silicone oil/Span80 microemulsion to immobilize protein and investigated the adjustment of the protein regulation of immobilizied complex by pH through ELISA, zetasizer, infrared spectroscopy. The results showed that more BSA were distributed in the exterior of complex in the high pH (pH=5,6) and more BSA were distributed in the interior of complex in the low pH(pH=3,4). The results of etasizer, infrared spectroscopy also showed the compex had homogeneous sizes and core-shell structure, moreover, the activity of BSA would be good retained by this immobilized method.3. We immobilized the cellulase (ICM) polyvinyl alcohol/Fe₂O₃ nanoparticles under the silicone oil/Span80 microemulsion system, and we also investigatied the effects of the immobilization of cellulase activity,such as types of oil phase, oil-water ratio, cellulose concentration, stirring speed, pH, cross-linking forms. The results showed that the activity of ICM was best(filter paper enzyme activity up to 7 U ? mg-1) under such condition as the oil phase of silicone oil/Span 80, the water/oil ratio of 1/25, the concentration of cellulase in aqueous at 20 mg?mL-1, stirring speed at 2000 rpm, pH 5. The analysis results of transmission electron microscopy and zetasizer showed that the immobilized enzyme formed a kind of sphere complex with the diameter of approximately 300 nm, and with the structure of aggregational Fe₂O₃ nanoparticles in their interior and, polymer in their exterior. Vibrating sample analysis showed that the saturation magnetization of immoblied enzyme is 4.87 Am2 ? kg-1, and the coercivity is 50 Oe, which suggested the complexs had a strong paramagnetic. The infrared spectroscopy images also showed that cellulase was immobilized in PVA/Fe₂O₃ system. The enzymatic charater of the immobilized enzyme showed that: although C1 activity andβ-glucosidase activity decreased in this complexs, Cx is much higher than the free enzyme activity 85 U mg^-1, indicating that this method can adjust the position of cellulase compent. Characterization results showed that the hydrolysis curve and the optimum pH value of immobilized enzyme were similar with the free enzymes, and had strong resistance to mechanical shearing.4. The crystal structure of cellulose is one of the main obstacles to degrade. Ball milling could destroy the crystal structure(MCC) but do great harm to the activity of cellulase. We combined ball milling with ICM. The analysis of retained activity showed that the specific activity of ICM was about 3.5 times of that of free cellulase after treated by the ball milling in 300rpm 6 h. The glucose analysis suggests that there are synergies between milling and immobilized enzyme for the degradation of microcrystalline cellulose. Infrared analysis showed that the ball can increase water absorption microcrystalline cellulose. XRD and ESEM analysis results suggested that ball milling loosed the crystalline surface and made amorphous structure, which was more accessible by ICM. With the enzyme hydrolysis of MCC, the fibrous structure of cellulose was broken, which is considered to be adoptable for better mechanic and chemical modification. These two reactions proceeded repeatedly and synergistically until the MCC was degraded efficiently. These results show that the combination of microemulsion immobilized potential wet milling is a new and effective method of degrading microcrystalline cellulose.5. To simplify the investigation of the cellulose degradation mechanism in filamentous fungi, a novel Agrobacterium-mediated transformation method of filamentous fungi method was established. We achieved the conversion of Penicillium simplicissimum by combining Agrobacterium tumefaciens LB4404 with plasmid pPK2. The molecular analysis showed that the hph gene was successfully transformed into Penicillium simplicissimum with a randomly single-copy insertion. The average transformation efficiency was about 50 transformants per 105 spores at the optimized conditiong: the concentration of Agrobacterium tumefacien at 250μM, the concentration of acetoyringone at 0.8 OD and the co-culture time at 48 h. The establishment of thid transformation system would contribute to the estabilishment of a more rational method of immobilized cellulase.