| High effective enzymes have been drawing increasing attention in the field of biotechnological industries.Lipases,one group of important industrial enzymes,are widely used in bio-energy,medicine,chemicals and food industry.However,most lipases are sensitive to environment,easy to be inactivated,high cost and low catalytic efficiency.These drawbacks seriously hamper their wide applications in industry.To solve the problems mentioned above,in this dissertation a first attempt was conducted to give special insights into the conformation and unique catalytic mechanism of lipases in order to find novel strategy for immobilization.Lipases are a group of interfacial hydrolytic enzymes whose active centers are covered by an ?-helix,called ‘lid structure'.Lipases will convert into active state with their lids open when exposed to an oil-water interface.Emulsion and hydrophobic carrier can also activate lipases because they provide similar effect to oil-water interface.Therefore,on the basis of this special activation mechanism,a series of immobilization technologies,such as spherzyme,NKA hydrophobic macroporous resin immobilization and PEI microcapsule,were employed to improve catalytic performance of lipases.And the immobilized lipases were further used in biodiesel production to test their application property.The main research and results are summarized below:1.Through analyzing the conformation of ROL(Rhizopus oryzae lipase)and BCL(Burkholderia cepacia lipase)molecules,we found that hydrophilic amino acid residues tended to distribute in the side away from the active center,while hydrophobic amino acid residues tended to distribute around the active center.Moreover,?-NH2,which would be used to crosslink with glutaraldehyde,was far away from the active site.So,choosing free ?-NH2 amino acid residues as binding sites had very slight influence on enzyme structure.After immobilization,the activity recovery rates of BCL spherzymes and ROL spherzymes were 204.7% and 179.2%,respectively.To further optimize spherzymes,EDA was added as a modifying reagent,and the activity recovery rate of BCL was improved 1.82 times.Then,5% BSA was used as a protective reagent during immobilization,and the activity recovery rate was also enhaced 2.24 times.Enzymological properties testing revealed that the properties of the spherezymes,including pH stability,thermostability and organic solvent tolerance,were simultaneously improved in some way.2.To enhance the mechanical property of the immobilized lipases,NKA was used as carrier.The optimal parameters were pH 5,enzyme loading 40 mg/300 mg-carriers,obsorption time 10 min and temperature 12°C.Under the conditions,the enzyme activity recovery rate of ROL attained 3,062%.For BCL,the best immobilization parameters were pH value 7,enzyme loading 400 mg/500 mg-carriers,adsorption time 10 min and temperature 37°C,under which,the enzyme activity recovery rate reached 2,203%.Furthermore,non-aqueous modifications,including bio-imprinting,lecithin coating and freeze-dried protection,were conducted.Each one could enhance lipases properties.Then,three kinds of the modified methods were combined and the activity of the immobilized enzyme was 350,298 U/g.Compared with the original immobilized enzyme,the modified immobilized enzyme increased 2.09 times.In subsequent enzymological properties characterization,the properties of NKA immobilized lipases,including pH stability,thermostability and organic solvent tolerance were markedly improved.3.To make full use of interface activation,lipases were immobilized in polyethyleneimine(PEI)microcapsules and further modified with oxidized multiwall carbon nanotubes(MWCNTs).The optimum immobilization parameters for BCL were pH value 8.5,linking reagent amount 0.9 mM and reaction time 3 min.The activity recovery ratio was 2,215%.The best immobilization parameters for ROL were pH value 8.0,linking reagent amount 0.9 mM and reaction time 3 min.The activity recovery ratio reached 3,598%.Consequently,the properties of microcapsules were characterized.The permeability of microcapsules was examined,and revealed that anionic dye could permeate into the microcapsuels while zwitterionic dye only occured on the surface of the microcapsules.FITC-dextran(isosulfocyanide)4k and 10 k were used as probe to detect the profile of microcapsules.Illustrated by confocal laser scanning microscope(CLSM),it was found that microcapsules,whose barrier properties against molecules with diameter >4.6 nm,possessed a semipermeable and porous membrane structure.Then,the lipases distribution in the microcapsuels was explored.The results showed that lipases preferred to mainly locate in the inner side of the wall and partly in the center of the microcapsules with high protein concentration;however,lipases only located in the inner side of the wall with low protein concentration.The oxidized multiwall carbon nanotubes(MWCNTs)were further added to modify microcapsules,resulting in even higher activity,especially the activity of ROL increased to 5,254%.The nanocomposites were examined by scanning electron microscope(SEM)and zeta-potential analyzer.The results indicated the superior catalytic performances were attributed to the augmented interface and decreased positive charge(from +43.6 mV to 17.5 mV).Enzymological properties characterization demonstrated that the property of microcapsules,including pH stability,thermostability and organic solvent tolerance were significantly improved.4.The NKA immobilized lipases and PEI microcapsules were then used as catalysts for biodiesel production.NKA-ROL was used with other lipase to synergistically catalyze biodiesel preparation.The previous study indicated that synergistic strategy could not only cut down production cost of biodiesel,but also improve the performance of lipases catalysis.The preliminary results showed that the biodiesel yield of combined lipases was 30% higher than that of single ROL in 30 h.Then,the synergy strategy was systematically examined and the optimized conditions were the ratio of mixed lipases(NKA-ROL: Novozym 435)3:1,enzyme loading 6%,initial alcohol oil ratio 1:2,water content 4% and reaction temperature 45°C.Under the optimal conditions,the biodiesel yield was as high as 98.31% and the reaction duration was successfully shortened from 60 h to 21 h.When PEI-ROL was used for biodiesel production(enzyme amount 25%,water content 4%,reaction temperature 45°C,alcohol/oil molar ratio 5:1),the biodiesel yield was up to 95.91%.5.Carbon nanotube modified microcapsules were also used for biodiesel synthesis.The biodiesel yield of the modified BCL and ROL microcapsules increased to 97.15% and 97.38%,respectively.The reusability of the modified microcapsules was also improved.For BCL,the modified microcapsules kept 89.32% conversion yield after ten batches of usage,while original microcapsules only kept 73.66% conversion yield.For ROL,the modified microcapsules kept 76.94% conversion yield after ten batches of usage,while original microcapsules only kept 42.98% conversion yield. |
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