磁性纳米颗粒固定纤维素酶和产酶丝状真菌转化的研究
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摘要
纤维素酶是一组具有纤维素降解能力酶的总称,它们协同作用可以将纤维素物质水解成简单糖,进而发酵产生乙醇,从而应用于农业、再生能源以及环境保护等领域。将已商业化的分泌型木霉生产的多组分纤维素酶有效固定化,不仅可以提高酶的稳定性、重复性,降低应用成本,还能为其他大分子底物的多酶体系的合理共固定研究奠定基础。本研究主要开展磁性纳米颗粒固定化纤维素酶和丝状真菌转化系统研究,获得了以下主要研究结果:
一、以Fe_2O_3纳米颗粒和聚乙烯醇为载体物理交联固定纤维素酶方法的建立。采以Fe_2O_3纳米颗粒为载体,以聚乙烯醇为包覆物和交联物对纤维素酶进行反复冻融固定,形成了催化性质稳定的固定化复合物。采用透射电镜、红外光谱仪、振动样品磁强度计对固定化酶复合体进行了表征,结果显示:固定化酶凝胶团由大小约1μm的微凝胶团组成,微胶团内含10 nm左右的Fe_2O_3纳米颗粒。对影响固定化因素的研究发现:当pH为6,固定化时间为11 h,纤维素酶/PVA为4,PVA/Fe为50时,固定化纤维素酶效果最高。通过该方法固定后酶活回收率达42%,经过5次反应后的固定化酶相对酶活力仍保留50%以上。结果表明:基于Fe_2O_3纳米颗粒和聚乙烯醇为载体的反复冻融法可以有效固定纤维素酶。
二、以牛血清蛋白(BSA)为模式蛋白,建立了采用硅油/Span80微乳体系固定化蛋白质的方法,并探讨了pH对固定化蛋白质在复合物空间分布的影响。对制备的BSA / PVA /Fe_2O_3纳米颗粒复合物进行酶联免疫、粒径、蛋白质含量分析,结果表明:较高pH值(pH=5、6)有利于BSA分布在复合物表层,而较低pH值(pH=3、4)使BSA则更多地镶嵌在复合物内层。透射电镜、zeta电位、红外光谱分析的结果显示BSA/PVA/Fe_2O_3纳米颗粒复合物粒径分布都较为均匀,具有球型核壳状的立体结构,并较好地保持了BSA的活力。
三、利用硅油/Span80微乳体系对纤维素酶(ICM)进行了固定,考察了油相种类、油水比、纤维素酶浓度,搅拌转速,pH值,交联方式等因素对固定化纤维素酶活力的影响。结果显示:采用硅油/Span 80体系,水/油比值为1/25,水相纤维素酶浓度为20 mg·mL~(-1),搅拌转速为2000 rpm,pH值为5的条件下固定化效果最佳。滤纸酶活力可达7 U·mg~(-1)。透射电镜和粒径分析表明该固定化酶的粒径为300 nm左右,并形成Fe_2O_3纳米颗粒聚集在内,高分子物质分布在外的复合物。振动样品强度分析显示该复合物的饱和磁化强度为4.87 Am2·kg~(-1),矫顽力为50 Oe,有较强的顺磁性,红外光谱分析表明纤维素酶固定在PVA/Fe_2O_3纳米颗粒体系中。对固定化酶的性质分析表明:该固定化方法虽然C1酶活力和β-葡萄糖苷酶活力均下降,但比游离酶的Cx酶活力85 U·mg~(-1)要高很多,显示该方法能够有效调整纤维素酶组分的位置。酶学性质研究结果显示该固定化酶水解曲线、最适pH值与游离酶等性能基本相似,并具有较强的耐机械剪切能力。
四、纤维素的晶体结构是提高酶活力的主要障碍之一,球磨技术虽然能够很好地破坏结晶纤维但是对酶活力影响也比较大。实验将固定化酶与球磨联用处理纤维素。酶活力和糖化分析结果表明:与球磨(300rpm)联合处理微晶纤维素(MCC) 6 h后,微乳固定化酶的单位蛋白酶活力是游离酶活力的3.5倍,且固定化酶与球磨对于微晶纤维素的降解具有明显协同作用。红外光谱、X晶体衍射和环境扫描电镜分析结果显示:球磨能够增加微晶纤维素的吸水量;微晶纤维的有序晶体排列在处理过程中被机械球磨降低,并使更多的不定型特性的纤维素纤丝出现,而固定化酶能够迅速降解这一不定型纤丝,从而使球磨和固定化酶呈现出良好的协同降解微晶纤维素作用,表明乳固定化酶结合湿法球磨是一种新型潜在的有效降解微晶纤维素的方法。
五、为了便于探索丝状真菌纤维素降解机制,对丝状真菌的转化方法进行了探索,建立的农杆菌介导的丝状真菌转化体系。利用根癌农杆菌LB4404和质粒pPK2成功实现了简青霉菌株的转化。通过分子检测表明,hph基因成功转化至简青霉中,而且呈单拷贝。转化过程中影响遗传转化效率因素的研究结果表明在乙酰丁香酮浓度为250μM、OD值在0.8、共培养时间为48 h的条件下转化效率最高。验证结果表明在该条件下,平均转化率可达50个转化子/105孢子。该转化体系的建立为建立更合理的固定化纤维素酶模式奠定了基础。
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/Fe_2O_3 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/Fe_2O_3 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 Fe_2O_3 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/Fe_2O_3 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 Fe_2O_3 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/Fe_2O_3 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.
一、以Fe_2O_3纳米颗粒和聚乙烯醇为载体物理交联固定纤维素酶方法的建立。采以Fe_2O_3纳米颗粒为载体,以聚乙烯醇为包覆物和交联物对纤维素酶进行反复冻融固定,形成了催化性质稳定的固定化复合物。采用透射电镜、红外光谱仪、振动样品磁强度计对固定化酶复合体进行了表征,结果显示:固定化酶凝胶团由大小约1μm的微凝胶团组成,微胶团内含10 nm左右的Fe_2O_3纳米颗粒。对影响固定化因素的研究发现:当pH为6,固定化时间为11 h,纤维素酶/PVA为4,PVA/Fe为50时,固定化纤维素酶效果最高。通过该方法固定后酶活回收率达42%,经过5次反应后的固定化酶相对酶活力仍保留50%以上。结果表明:基于Fe_2O_3纳米颗粒和聚乙烯醇为载体的反复冻融法可以有效固定纤维素酶。
二、以牛血清蛋白(BSA)为模式蛋白,建立了采用硅油/Span80微乳体系固定化蛋白质的方法,并探讨了pH对固定化蛋白质在复合物空间分布的影响。对制备的BSA / PVA /Fe_2O_3纳米颗粒复合物进行酶联免疫、粒径、蛋白质含量分析,结果表明:较高pH值(pH=5、6)有利于BSA分布在复合物表层,而较低pH值(pH=3、4)使BSA则更多地镶嵌在复合物内层。透射电镜、zeta电位、红外光谱分析的结果显示BSA/PVA/Fe_2O_3纳米颗粒复合物粒径分布都较为均匀,具有球型核壳状的立体结构,并较好地保持了BSA的活力。
三、利用硅油/Span80微乳体系对纤维素酶(ICM)进行了固定,考察了油相种类、油水比、纤维素酶浓度,搅拌转速,pH值,交联方式等因素对固定化纤维素酶活力的影响。结果显示:采用硅油/Span 80体系,水/油比值为1/25,水相纤维素酶浓度为20 mg·mL~(-1),搅拌转速为2000 rpm,pH值为5的条件下固定化效果最佳。滤纸酶活力可达7 U·mg~(-1)。透射电镜和粒径分析表明该固定化酶的粒径为300 nm左右,并形成Fe_2O_3纳米颗粒聚集在内,高分子物质分布在外的复合物。振动样品强度分析显示该复合物的饱和磁化强度为4.87 Am2·kg~(-1),矫顽力为50 Oe,有较强的顺磁性,红外光谱分析表明纤维素酶固定在PVA/Fe_2O_3纳米颗粒体系中。对固定化酶的性质分析表明:该固定化方法虽然C1酶活力和β-葡萄糖苷酶活力均下降,但比游离酶的Cx酶活力85 U·mg~(-1)要高很多,显示该方法能够有效调整纤维素酶组分的位置。酶学性质研究结果显示该固定化酶水解曲线、最适pH值与游离酶等性能基本相似,并具有较强的耐机械剪切能力。
四、纤维素的晶体结构是提高酶活力的主要障碍之一,球磨技术虽然能够很好地破坏结晶纤维但是对酶活力影响也比较大。实验将固定化酶与球磨联用处理纤维素。酶活力和糖化分析结果表明:与球磨(300rpm)联合处理微晶纤维素(MCC) 6 h后,微乳固定化酶的单位蛋白酶活力是游离酶活力的3.5倍,且固定化酶与球磨对于微晶纤维素的降解具有明显协同作用。红外光谱、X晶体衍射和环境扫描电镜分析结果显示:球磨能够增加微晶纤维素的吸水量;微晶纤维的有序晶体排列在处理过程中被机械球磨降低,并使更多的不定型特性的纤维素纤丝出现,而固定化酶能够迅速降解这一不定型纤丝,从而使球磨和固定化酶呈现出良好的协同降解微晶纤维素作用,表明乳固定化酶结合湿法球磨是一种新型潜在的有效降解微晶纤维素的方法。
五、为了便于探索丝状真菌纤维素降解机制,对丝状真菌的转化方法进行了探索,建立的农杆菌介导的丝状真菌转化体系。利用根癌农杆菌LB4404和质粒pPK2成功实现了简青霉菌株的转化。通过分子检测表明,hph基因成功转化至简青霉中,而且呈单拷贝。转化过程中影响遗传转化效率因素的研究结果表明在乙酰丁香酮浓度为250μM、OD值在0.8、共培养时间为48 h的条件下转化效率最高。验证结果表明在该条件下,平均转化率可达50个转化子/105孢子。该转化体系的建立为建立更合理的固定化纤维素酶模式奠定了基础。
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/Fe_2O_3 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/Fe_2O_3 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 Fe_2O_3 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/Fe_2O_3 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 Fe_2O_3 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/Fe_2O_3 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.
引文
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