大孔/介孔多级孔SiO_2的制备及其在固定化脂肪酶中的应用
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摘要
以具有三维骨架结构的环氧树脂大孔聚合物为模板,制备具有毫米级尺寸的大孔/介孔多级孔SiO_2。应用SEM、MIP、FTIR和N_2吸附-脱附法对材料孔道结构和表面性质进行表征。采用吸附法固定褶皱假丝酵母脂肪酶(CRL),研究CRL初始浓度、pH值及固定化时间对脂肪酶固定化的影响,对比研究了游离脂肪酶和固定化脂肪酶的酶学性质。结果表明,大孔/介孔SiO_2具有三维连续贯通的大孔孔道,孔壁由连续的SiO_2纳米薄膜构筑而成且表面存在丰富的介孔,比表面积为75.1m~2/g,孔隙率为92.3%;在CRL浓度为0.6mg/mL、pH值为8.0、固定化时间为10h时,固定化酶酶活达到4 825U/g。与游离脂肪酶相比,固定化脂肪酶的pH稳定性、热稳定性和储存稳定性明显提高,连续使用8次后的酶活为初始酶活的68%。利用环氧树脂大孔聚合物模板制备的大孔/介孔多级孔SiO_2在固定化酶方面具有良好的应用前景。
Millimeter-sized hierarchical macroporous/mesoporous silica was prepared by using an epoxy resin macroporous polymer with three-dimensional(3D)skeletal structure as template.Pore structures and surface properties of the as-prepared materials were characterized by SEM,MIP,FTIR and N_2adsorption-desorption.Candida rugosa lipase(CRL)was immobilized on the macroporous/mesoporous silica by adsorption method.The effects of the initial CRL concentration,pH and immobilizing time on lipase immobilization were investigated,and the properties of free and immobilized lipase were studied.The results demonstrated that the macroporous/mesoporous silica had 3Dcontinuous pass-through macroporous structure;its pore wall was constructed by continuous silica nano-film and had abundant mesopores.The specific surface area and porosity are 75.1m~2/g and 92.3%,respectively.The activity of immobilized lipase reaches 4 825U/g under conditions of initial CRL concentration 0.6mg/mL,pH of 8.0and immobilizing time of 10h.Compared with free CRL,the pH,thermal and storage stabilities of immobilized lipase were improved significantly.The immobilized lipase retained 68%residual activity after 8consecutive operations.Therefore,this hierarchical macroporous/mesoporous silica was a good potential candidate for enzyme immobilization.
Millimeter-sized hierarchical macroporous/mesoporous silica was prepared by using an epoxy resin macroporous polymer with three-dimensional(3D)skeletal structure as template.Pore structures and surface properties of the as-prepared materials were characterized by SEM,MIP,FTIR and N_2adsorption-desorption.Candida rugosa lipase(CRL)was immobilized on the macroporous/mesoporous silica by adsorption method.The effects of the initial CRL concentration,pH and immobilizing time on lipase immobilization were investigated,and the properties of free and immobilized lipase were studied.The results demonstrated that the macroporous/mesoporous silica had 3Dcontinuous pass-through macroporous structure;its pore wall was constructed by continuous silica nano-film and had abundant mesopores.The specific surface area and porosity are 75.1m~2/g and 92.3%,respectively.The activity of immobilized lipase reaches 4 825U/g under conditions of initial CRL concentration 0.6mg/mL,pH of 8.0and immobilizing time of 10h.Compared with free CRL,the pH,thermal and storage stabilities of immobilized lipase were improved significantly.The immobilized lipase retained 68%residual activity after 8consecutive operations.Therefore,this hierarchical macroporous/mesoporous silica was a good potential candidate for enzyme immobilization.
引文
1 Fernandez-Lafuente R.Lipase from thermomyces lanuginosus:Uses and prospects as an industrial biocatalyst[J].Journal of Molecular Catalysis B:Enzymatic,2010,62(3-4):197.
2 Adlercreutz P.Immobilisation and application of lipases in organic media[J].Chemical Society Reviews,2013,42(15):6406.
3 Narwal S K,Gupta R.Biodiesel production by transesterification using immobilized lipase[J].Biotechnology Letters,2013,35(4):479.
4 Moayedallaie S,Mirzaei M,Paterson J.Bread improvers:Comparison of a range of lipases with a traditional emulsifier[J].Food Chemistry,2010,122(3):495.
5 Habibi Z,Mohammadi M,Yousefi M.Enzymatic hydrolysis of racemic ibuprofen esters using rhizomucor miehei lipase immobilized on different supports[J].Process Biochemistry,2013,48(4):669.
6 Lima L N,Oliveira G C,Rojas M J,et al.Immobilization of Pseudomonas fluorescens lipase on hydrophobic supports and application in biodiesel synthesis by transesterification of vegetable oils in solvent-free systems[J].Journal of Industrial Microbiology&Biotechnology,2015,42(4):523.
7 曹林秋.载体固定化酶——原理、应用和设计[M].北京:化学工业出版社,2008:1.
8 Airestrapote A,Hoyos P,Alcántara A R,et al.Covalent immobilization of Pseudomonas stutzeri lipase on a porous polymer:An efficient biocatalyst for a scalable production of enantiopure benzoin esters under sustainable conditions[J].Organic Process Research and Development,2015,19(7):687.
9 Jia J,Yin D Z,Zhang B L,et al.Study on the immobilization of papain by macroporous resin[J].Materials Review B:Research Papers,2013,27(11):88(in Chinese).贾佳,尹德忠,张宝亮,等.大孔树脂固定木瓜蛋白酶的研究[J].材料导报:研究篇,2013,27(11):88.
10 Bernal C,Illanes A,Wilson L.Heterofunctional hydrophilic-hydrophobic porous silica as support for multipoint covalent immobilization of lipases:Application to lactulose palmitate synthesis[J].Langmuir,2014,30(12):3557.
11 Beck J S,Vartuli J C,Roth W J,et al.A new family of mesoporous molecular sieves prepared with liquid crystal templates[J].Journal of the American Chemical Society,1992,114(27):10834.
12 Zhou Z,Hartmann M.Recent progress in biocatalysis with enzymes immobilized on mesoporous hosts[J].Topics in Catalysis,2012,55(16):1081.
13 Li Y,Liu X Z,Liang Y X.Preparation of P(GMA-co-HEMA)/SiO2macroporous composite and its application in lipase i-mmobilization[J].Acta Materiae Compositae Sinica,2016,33(3):635(in Chinese).李云,刘晓贞,梁云霄.P(GMA-co-HEMA)/SiO2大孔复合材料的制备及其在固定化脂肪酶中的应用[J].复合材料学报,2016,33(3):635.
14 Bemak C,Sierra L,Mesa M.Application of hierarchical porous silica with a stable large porosity forβ-Galactosidase immobilization[J].ChemCatChem,2011,3(12):1948.
15 Miletic N,Vukocic Z,Nastasovic A,et al.Macroporous poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate)resins-versatile immobilization supports for biocatalysts[J].Journal of Molecular Catalysis B:Enzymatic,2009,56(4):196.
16 Li Y,Gao F,Wei W,et al.Pore size of macroporous polystyrene microspheres affects lipase immobilization[J].Journal of Molecular Catalysis B:Enzymatic,2010,66(1-2):182.
17 Bai Wenjing,Li Yun,Cao Dali,et al.Immobilization of pseudomonas fluorescens lipase on PDA/SiO2macroporous composite[J].Chinese Journal of Inorganic Chemistry,2016,32(11):1973(in Chinese).白文静,李云,曹大丽,等.PDA/SiO2大孔复合材料固定化荧光假单胞菌脂肪酶[J].无机化学学报,2016,32(11):1973.
18 Wang M,Qi W,Yu Q,et al.Cross-linking enzyme aggregates in the macropores of silica gel:A practical and efficient method for enzyme stabilization[J].Biochemical Engineering Journal,2010,52(2-3):168.
19 Yuan Z Y,Su B L.Insights into hierarchically meso-macroporous structured materials[J].Journal of Materials Chemistry,2006,16(7):663.
20 Li J,Li L S,Xu L.Hierarchically macro/mesoporous silica sphere:A high efficient carrier for enzyme immobilization[J].Microporous and Mesoporous Materials,2016,231:147.
21 Kilcawley K N,Wilkinson M G,Fox P F,et al.Properties of commercial microbial proteinase preparations[J].Food Biotechnology,2002,16(1):29.
22 Bradford M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(s1-2):248.
23 Chen R,Li D X.Analysis of error for pore structure of porous materials measured by MIP[J].Bulletin of The Chinese Ceramic Society,2006,25(4):198(in Chinese).陈悦,李东旭.压汞法测定材料孔结构的误差分析[J].硅酸盐通报,2006,25(4):198.
24 Yu A M,Liang Z J,Caruso F.Enzyme multilayer-modified porous membranes as biocatalysts[J].Chemistry of Materials,2005,17(1):171.
25 Netto C G C M,Andrade L H,Toma H E.Enantioselective transesterification catalysis by candida antarctica,lipase immobilized on superparamagnetic nanoparticles[J].Tetrahedron Asymmetry,2009,20(19):2299.
26 Malinin A S,Rakhnyanskaya A A,Bacheva A V,et al.Activity of an enzyme immobilized on polyelectrolyte multilayers[J].Polymer Science,2011,53(1):52.
27 Liu J,Guo H,Zhou Q,et al.Highly efficient enzymatic preparation for dimethyl carbonate catalyzed by lipase from penicillium expansum,immobilized on CMC-PVA film[J].Journal of Molecular Catalysis B:Enzymatic,2013,96(12):96.
28 Wang W C,Zhou W Q,Wei W,et al.Towards a deeper understanding of the interfacial adsorption of enzyme molecules in gigaporous polymeric microspheres[J].Polymers,2016,8(4):116.
29 Wu C,Zhou G,Jiang X,et al.Active biocatalysts based on candida rugosa lipase immobilized in vesicular silica[J].Process Biochemistry,2012,47(6):953.
30 Ma H Z,Yu X W,Song C,et al.Immobilization of candida antarctica,lipase B on epoxy modified silica by sol-gel process[J].Journal of Molecular Catalysis B:Enzymatic,2016,127:76.
31 Sun H,Li Q Y,Wang X W,et al.Rapid aqueous synthesis of mesoporous carbon and their HRP immobilization performance[J].Acta Chimica Sinica,2013,71(4):619(in Chinese).孙何,李群艳,王学伟,等.介孔碳的快速水相合成及对辣根过氧化物酶的固定[J].化学学报,2013,71(4):619.
32 Yuce-Dursun B,Cigil A B,Dongez D,et al.Preparation and characterization of sol-gel hybrid coating films for covalent immobilization of lipase enzyme[J].Journal of Molecular Catalysis B:Enzymatic,2016,127:18.
2 Adlercreutz P.Immobilisation and application of lipases in organic media[J].Chemical Society Reviews,2013,42(15):6406.
3 Narwal S K,Gupta R.Biodiesel production by transesterification using immobilized lipase[J].Biotechnology Letters,2013,35(4):479.
4 Moayedallaie S,Mirzaei M,Paterson J.Bread improvers:Comparison of a range of lipases with a traditional emulsifier[J].Food Chemistry,2010,122(3):495.
5 Habibi Z,Mohammadi M,Yousefi M.Enzymatic hydrolysis of racemic ibuprofen esters using rhizomucor miehei lipase immobilized on different supports[J].Process Biochemistry,2013,48(4):669.
6 Lima L N,Oliveira G C,Rojas M J,et al.Immobilization of Pseudomonas fluorescens lipase on hydrophobic supports and application in biodiesel synthesis by transesterification of vegetable oils in solvent-free systems[J].Journal of Industrial Microbiology&Biotechnology,2015,42(4):523.
7 曹林秋.载体固定化酶——原理、应用和设计[M].北京:化学工业出版社,2008:1.
8 Airestrapote A,Hoyos P,Alcántara A R,et al.Covalent immobilization of Pseudomonas stutzeri lipase on a porous polymer:An efficient biocatalyst for a scalable production of enantiopure benzoin esters under sustainable conditions[J].Organic Process Research and Development,2015,19(7):687.
9 Jia J,Yin D Z,Zhang B L,et al.Study on the immobilization of papain by macroporous resin[J].Materials Review B:Research Papers,2013,27(11):88(in Chinese).贾佳,尹德忠,张宝亮,等.大孔树脂固定木瓜蛋白酶的研究[J].材料导报:研究篇,2013,27(11):88.
10 Bernal C,Illanes A,Wilson L.Heterofunctional hydrophilic-hydrophobic porous silica as support for multipoint covalent immobilization of lipases:Application to lactulose palmitate synthesis[J].Langmuir,2014,30(12):3557.
11 Beck J S,Vartuli J C,Roth W J,et al.A new family of mesoporous molecular sieves prepared with liquid crystal templates[J].Journal of the American Chemical Society,1992,114(27):10834.
12 Zhou Z,Hartmann M.Recent progress in biocatalysis with enzymes immobilized on mesoporous hosts[J].Topics in Catalysis,2012,55(16):1081.
13 Li Y,Liu X Z,Liang Y X.Preparation of P(GMA-co-HEMA)/SiO2macroporous composite and its application in lipase i-mmobilization[J].Acta Materiae Compositae Sinica,2016,33(3):635(in Chinese).李云,刘晓贞,梁云霄.P(GMA-co-HEMA)/SiO2大孔复合材料的制备及其在固定化脂肪酶中的应用[J].复合材料学报,2016,33(3):635.
14 Bemak C,Sierra L,Mesa M.Application of hierarchical porous silica with a stable large porosity forβ-Galactosidase immobilization[J].ChemCatChem,2011,3(12):1948.
15 Miletic N,Vukocic Z,Nastasovic A,et al.Macroporous poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate)resins-versatile immobilization supports for biocatalysts[J].Journal of Molecular Catalysis B:Enzymatic,2009,56(4):196.
16 Li Y,Gao F,Wei W,et al.Pore size of macroporous polystyrene microspheres affects lipase immobilization[J].Journal of Molecular Catalysis B:Enzymatic,2010,66(1-2):182.
17 Bai Wenjing,Li Yun,Cao Dali,et al.Immobilization of pseudomonas fluorescens lipase on PDA/SiO2macroporous composite[J].Chinese Journal of Inorganic Chemistry,2016,32(11):1973(in Chinese).白文静,李云,曹大丽,等.PDA/SiO2大孔复合材料固定化荧光假单胞菌脂肪酶[J].无机化学学报,2016,32(11):1973.
18 Wang M,Qi W,Yu Q,et al.Cross-linking enzyme aggregates in the macropores of silica gel:A practical and efficient method for enzyme stabilization[J].Biochemical Engineering Journal,2010,52(2-3):168.
19 Yuan Z Y,Su B L.Insights into hierarchically meso-macroporous structured materials[J].Journal of Materials Chemistry,2006,16(7):663.
20 Li J,Li L S,Xu L.Hierarchically macro/mesoporous silica sphere:A high efficient carrier for enzyme immobilization[J].Microporous and Mesoporous Materials,2016,231:147.
21 Kilcawley K N,Wilkinson M G,Fox P F,et al.Properties of commercial microbial proteinase preparations[J].Food Biotechnology,2002,16(1):29.
22 Bradford M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(s1-2):248.
23 Chen R,Li D X.Analysis of error for pore structure of porous materials measured by MIP[J].Bulletin of The Chinese Ceramic Society,2006,25(4):198(in Chinese).陈悦,李东旭.压汞法测定材料孔结构的误差分析[J].硅酸盐通报,2006,25(4):198.
24 Yu A M,Liang Z J,Caruso F.Enzyme multilayer-modified porous membranes as biocatalysts[J].Chemistry of Materials,2005,17(1):171.
25 Netto C G C M,Andrade L H,Toma H E.Enantioselective transesterification catalysis by candida antarctica,lipase immobilized on superparamagnetic nanoparticles[J].Tetrahedron Asymmetry,2009,20(19):2299.
26 Malinin A S,Rakhnyanskaya A A,Bacheva A V,et al.Activity of an enzyme immobilized on polyelectrolyte multilayers[J].Polymer Science,2011,53(1):52.
27 Liu J,Guo H,Zhou Q,et al.Highly efficient enzymatic preparation for dimethyl carbonate catalyzed by lipase from penicillium expansum,immobilized on CMC-PVA film[J].Journal of Molecular Catalysis B:Enzymatic,2013,96(12):96.
28 Wang W C,Zhou W Q,Wei W,et al.Towards a deeper understanding of the interfacial adsorption of enzyme molecules in gigaporous polymeric microspheres[J].Polymers,2016,8(4):116.
29 Wu C,Zhou G,Jiang X,et al.Active biocatalysts based on candida rugosa lipase immobilized in vesicular silica[J].Process Biochemistry,2012,47(6):953.
30 Ma H Z,Yu X W,Song C,et al.Immobilization of candida antarctica,lipase B on epoxy modified silica by sol-gel process[J].Journal of Molecular Catalysis B:Enzymatic,2016,127:76.
31 Sun H,Li Q Y,Wang X W,et al.Rapid aqueous synthesis of mesoporous carbon and their HRP immobilization performance[J].Acta Chimica Sinica,2013,71(4):619(in Chinese).孙何,李群艳,王学伟,等.介孔碳的快速水相合成及对辣根过氧化物酶的固定[J].化学学报,2013,71(4):619.
32 Yuce-Dursun B,Cigil A B,Dongez D,et al.Preparation and characterization of sol-gel hybrid coating films for covalent immobilization of lipase enzyme[J].Journal of Molecular Catalysis B:Enzymatic,2016,127:18.