Purification Of Enzymes By Reversed Micellar Extraction And Degradation Of Polycyclic Aromatic Hydrocarbons In Reversed Micelles

The existence of solid waste in the environment makes it more urgent to get more and more lignocellulase.Reversed micellar extraction is a novel method in the environment engineering.It is a hot topic of extracting enzymes and degrading pollutants by employing reversed micelles.And it is also a challenge to make this method work,especially when the efficiency of the synthetic surfactant based reversed micellar extraction and degradation of solid waste is low,and the research of biosurfactant based reversed micellar extraction is limited.The biosurfactant based reversed micelle technology promotes the development of environmental pollutant management.In this dissertation,we first investigated the function of the biosurfactant based reversed micelles,then extracted and purified the enzymes by the reversed micelles and biosurfactant precipitation.We conducted mixed reversed micellar extraction and obtained the optimum operating factors in both methods.Meanwhile,experiments on synchronous extraction of similar enzymes in the crude broth were carried out.Electron spin resonance spectroscopy was applied to test the micro-parameters of reversed micelles when enzymes immobilized.On the basis of the present research on the reversed micelles,the degradation of environment pollutants like polycyclic aromatic hydrocarbons were conducted and the parameters were optimized.The major work of this dissertation is shown as following:First,we constructed the biosurfactant based reversed micelles and investigated the solubilization of water in the reversed micelles.Then we compared the results with synthetic surfactant based reversed micelles.The critical micellar concentration of each system were determined and the results showed that the demand of rhamnolipid was lower.We can see from the particle size experiment that there were different sizes of reversed micelles,and the percentage of bigger particles was about70% and the smaller one possessed 30%,which demonstrated that most of the reversed micelles were assembled together,only small percentage of them distributed randomly.Generally speaking,the characters of biosurfactant based reversed micelles had the superiority towards the synthetic ones.In the second section,base on the construction of reversed micelle,reversed micellar extraction of laccase was conducted to purify laccase.Meanwhile,the comparison of synthetic surfactant based reversed micellar extraction was made.Fromthe comparison,the biosurfactant based reversed micellar extraction showed both better activity recovery and protein recovery.Also,the possibility of reuse of reversed micelles was proved.The third part of the dissertation describes another method of purifying enzymes using biosurfactant.Biosurfactant precipitation was used to purify cellulase.Similarly,the biosurfactant showed the superiority when compared with the synthetic ones.The optimum parameters also were found out during the process.Base on the signal surfactant reversed micellar extraction,the fourth part of this work shows the mixed reversed micellar extraction of enzyme.Biosurfactant and synthetic surfactant were mixed at different ratios.The experiment also found out the best parameters during the extraction process.The research of the mixed reversed micellar extraction can provide guidance for the industry in extraction and lower the price.The fifth part of the dissertation focuses on the synchronous extraction of enzymes which have similar function but different structures.The biosurfactant based reversed micelles showed better extraction efficiency,which also proved the possibility of synchronous extraction of different enzymes.The sixth section shows the electron spin resonance spectroscopy analysis on reversed micelles.The enzyme was immobilized in the water core of the reversed micelles.The functional parameters such as hyperfine splitting constant and rotation correlation time can be obtained by using this technique,which can infer the information about the movement of the probe in the reversed micelles.The ultracentrifugation analysis and Cryo-SEM further verified the distribution and size of the reversed micelles.The Fourier transform infrared spectroscopy study showed the conformation change of enzyme which trapped into the reversed micelles.Based on all the work above,the final part of the work describes the degradation of environment pollutant inside reversed micelles.We inspected the effects of several factors on the degradation efficiency,such as reaction time,surfactant concentration and laccase concentration.This work provided a new way in catalysis of polycyclic aromatic hydrocarbons,and promoted the specific methods of applying reversed micellar system in environmental engineering field.