Immobilization Of Burkholderia Cepacia Lipase Based On New Materials And New Strategies And Its Applications

High effective lipases have been widely applied in energy industry,medicine industry,fine chemical synthesis and food industry,etc.Immobillization can solve the problems of free lipase.Usually,the traditional immobilization is to use one kind of immobilization strategy in one immobilization carrier.Meanwhile,for such method,most of the weight of the immobilized lipase is the immobilization material.Moreover,there are few researches on combining immobilization strategies and carrier structures together.In order to solve the aforementioned problems,based on the analysis of molecular conformation of Burkholderia cepacia lipase,three strategies were employed in this study:The synergistic action of ionic exchange and covalent bonding on the activity of the immobilized lipase was studied.Melamine-glutaraldehyde hyperbranched compound was first linked to the surface of magnetic nanoparticles,and the new carrier greatly increased the target protein loading;the oxidized CNTs were inserted into the core of the nanoflower and the new hybrid nanostructure with enhanced structure were successfully prepared for the first time.The prepared immobilized lipases were then applied to catalyze different reactions.Good catalytic properties were respectively confirmed.The main researches and results are summarized below:1.The Burkholderia cepacia lipase was taken as target enzyme in this study.After bioinformatic analysis for structures and amino acids distribution of BCL molecule,three kinds of immobilized lipase were designed to increase protein content of the immobilized lipase and enhance enzyme activity.They are BCL-GEAMNP,which combined ion exchange and covalent bonding,BCL-GTAMNP,which coupled oriented immobilization strategy with melamine-glutaraldehyde dendrimer modification,and oxidized carbon nanotube modified phosphate-protein hybrid nanoflowers.2.To study BCL-GEAMNP,the properties of magnetic nanoparticles and BCL-GEAMNP were characterized by different methods and the success of carrier synthesis and immobilization were finally confirmed.After optimization for immobilization conditions,the activity recovery of the transesterification reached 147.4%.The prepared BCL-GEAMNP was then used to produce biodiesel.tert-Butanol was selected as the reaction medium due to its solubility for all of the substrates and products.Different reaction conditions were optimized and under the optimum ones,the biodiesel yield could reach 90.2%in 9 h,and 96.8%in 12 h.The result of reusability shows BCL-GEAMNP has better operational stability and higher catalytic efficiency tha n BCL-GAMNP which just used covalent bonding as the immobilization strategy.3.The properties of carriers modified with different polymers and immobilized lipases were characterized by different methods and one-step synthesis of melamine-glutaraldehyde dendrimer-like polymer were selected as the most appropriate strategy.The success of carrier synthesis and immobilization were confirmed at the same time.The single factor experiment showed that the immobilization efficiency,specific activity of esterification,and activity recovery could reach 84.2%,76675.6 U/g protein and 5815.1%respectively under the optimum immobilization conditions.The obtained BCL-GTAMNP was then used in the resolution reaction between 1-phenylethanol and vinyl acetate.After selection for different reaction media and optimization for different reaction conditions,the best coversion and ee_s values could reach 45.8%and 84.3%.After vinyl acetate treatment to the immobilized lipases for 1 h,the ee_s values could reach 98.8%in only 20min.4.Seven kinds of hybrid nanomaterials were studied.FSEM and FT-IR spectra confirmed the successful syntheses of the immobilized lipase on these nanomaterials.The esterification activities of the immobilized lipases with CNT are higher than the others.All immobilized lipases were used to catalyze the resolution reaction between1-phenylethanol and vinyl acetate.The ee_s values of the reactions were increased obviously.ThereactionconditionsofCNT/BCL,Ca₃?PO₄?₂/CNT/BCL,Fe₃?PO₄?₂/CNT/BCL,and Cu₃?PO₄?₂/CNT/BCL were further optimized.Among them,Cu₃?PO₄?₂/CNT/BCL showed the best performance,and its conversion and ee_s value could reach 49.6%and 98.3%.After 8 cycles of reuse for all immobilized lipases,the conversions and ee_s values had nearly no loss.The enhanced activity of the immobilized lipase using two-step immobilization strategy indicated that the insertion of carbon nanotubes inside the hybrid nanostructures could provide new adsorption sites for BCL molecules.Thus,the combined immobilization strategy of adsorption and precipitation crystallization can enhance the activities of the immobilized lipases.5.To further explore the application of the immobilized lipases in nonaqueous media,BCL-GAMNP,BCL-GEAMNP,BCL-GTAMNP,and Cu₃?PO₄?₂/CNT/BCL were used to produce 2-phenylpropyl acetate.The esterification activities of the immobilized lipases were higher than the activity of BCL powder.The BCL-GTAMNP and Cu₃?PO₄?₂/CNT/BCL had even better performance and were further applied to catalyze the transesterification between hydratropyl alcohol and vinyl acetate.The reaction conditions were optimized.The conversion could reach 99.9%for BCL-GTAMNP in 20 min.Simultaneously,the conversion could reach 99.2%for Cu₃?PO₄?₂/CNT/BCL within only10 min.The four immobilized lipases were recycled for 8 batches and their activities had nearly no loss.BCL-GTAMNP and Cu₃?PO₄?₂/CNT/BCL had better performance.The results proved that the immobilized lipases could also be applied in the synthesis of2-phenylpropyl acetate.The biocatalysis showed considerable advantages over the chemical catalysts.In conclusion,after bioinformatic analysis for BCL molecule,three immobilized strategies were employed to prepare three different novel immobilized lipases.They all exhibited more stable structures,higher enzyme activities,better tolerance,and better performance in biodiesel production,chiral resolution and 2-phenylpropyl acetate synthesis.The immobilized lipases all showed good potential in industrial application.