| Lignocellulose, as one of the most widespread natural organic polymer compound on earth, are degraded at a rather low speed, which become a rate-limiting step of the global carbon cycle. However, the production and purification process of the lignocellulosic enzyme was reported slow and less productive, which would be limited for the on-growing amount of this natural disposed energy. The reverse micelles extraction（RME）, a novel biological separation and purification technology was paid more and more attentions by researchers recently. RMs is formed by organic solvent and water combined by surfactants who has both hydrophilic and hydrophobic head in one molecule. And the built water core surrounded by the polar head of the surfactants could solubilize water-soluble enzymes as well as other polar materials, which make RME as a perfect liquid-liquid extraction technology for bioseparation. The aqueous droplets inside of the reverse micelles represent in stabilized nanometersized level, and the proteins solubilized in were shown to maintain their properties functionally and stably. Generally, this liquid-liquid extraction process consists of two fundamental steps: a forward extraction and a backward extraction; the previous one was got most of the attentions by researchers while the later one still need more work to be done since its rather important in order to acquiring high quality enzyme product.Currently, a bunch of domestic and international researches on reverse micelles are based on chemical surfactants, which have limited resources and would bring second pollutants to the environment and even worse, sometimes denaturizing enzymes. Comparatively, biosurfactant, an environment friendly organic compounds, caused gradually more interests because of its properties of biodegradable and less toxic and the RMs made of it having higher solubilization ability, more friendly environmental compatibility, and milder reaction microenvironment than the other chemical forming ones. Most of the researches about biosurfactant have been reported to be performing pretty well so far, especially the widely used rhamnolipids（RL）.In first part of the research, compared with synthetic surfactants（CTAB, AOT and Tween-80）, the properties of the aqueous core as well as the microenvironment behavior were investigated in water-in-oil microemulsions, which are formed by water and biosurfactant rhamnolipid（RL） in the solvent of isooctane/n-hexanol（1:1, v/v）. In this study, triangle phase diagram of surfactant/n-hexanol/isooctane/water was constructed to analyze the variation of phase behavior between each RMs system. And the O–H stretching vibrations of water in aqueous core was also studied by analyzing the IR spectrum over the region of 3050–3750 cm!1. The Critical Micelle Concentration（CMC） of RL solubilized in isooctane detected through the method of fluorescence was 0.055 mM"L-1; therefore RM could be constructed by RL with rather lower amount. Besides, the conductivity property of the systems was also tested, which show the optimum hydration degree（!0） as 35.1, higher than the optimum hydration degree of other RMs. The size of the aqueous core was measured as well; it was found that RL could highly increase the water solubility, resulting in larger particle diameter of RMs and supplying better reaction microenvironment for enzymolysis process.Moving to the second part of the paper, as a typical substrate of lignocellulose, guaiacol was used to detect the laccase activity in reversed micelles（RMs）. UV spectrum is analyzed to find out how varied factors affecting enzyme activity of laccase in RMs. The effect of the molar ratio of water to surfactant（!0） on enzyme hydrolytic activity was determined to be shown as a bell-shaped curve and presented a maximum at !0=19. And it has been found that while the pH was 5.2, the concentration of KCl was 0.05 mol"L-1, the best hydrolysis condition of RL system could be reached. Moreover, kinetic studies have also been taken to show that the catalytic efficiency of the laccase in RL-based RMs system was lower than in aqueous solution. Nevertheless, the RMs system obtained the highest hydrolysis rate at RL concentration of 0.055 mM/L.During the process of forward extraction of cellulase and LiP using RL-based RMs, the effects of running time, the pH value of water phase, the RL concentration as well as ionic strength and type on extraction efficiency have been studied precisely. From the hydrolysis research, we could find easily that on these conditions these two enzymes could reach their optimum activity. LiP : [RL]=50 CMC, [KCl]=40 mmol/L,pH=3.0,Running Time = 30 min,has better performance in the KCl solvent than other ionic type;Cellulase:[RL]=40 CMC,[KCl]=50 mmol/L,pH = 3.5,Running Time = 30 min,has best quality in the water core with NaCl。While for the backward extraction process, the effects of running time, the pH value, the ionic strength and type as well as the concentration and type of extra solvents on extraction efficiency were studied in the RL-based RMs for the purification of cellulase and LiP. It was found that under these conditions the extraction efficiency of the enzyme could reach the peak. LiP: [KCl]=0.5 mol/L,pH=7.0,Running Time = 30 min,Ethanol（v%） = 8%,obtaining the best quality in water core solubilized KCl; Cellulase: [KCl]=0.15 mol/L,pH=7.0,Running Time = 40 min,Ethanol（v%） = 4%, the enzyme activity reach the peak when there is NaCl existed. Through the study we could found that adding extra ethanol can improve the rate of enzyme activity recovery, extraction efficiency as well as purification fold, and also could make the process faster and smoother.In conclusion, the RL-based RMs has better quality in supplying better microenvironment for enzymatic reaction process and could act as a liquid-liquid system for enzyme extraction with two steps. The optimum conditions of the system was tested in the solvent of isooctane/n-hexanol（1:1, v/v）, which could help the system maintain better quality and give out the information for promoting this technology into a real industry level. However, there are still a lot of work needs to be done in the future if we really want to make it a blast in commercial market. |
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