有序大孔二氧化硅孔壁聚合物功能化修饰及葡萄糖淀粉酶固载化
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摘要
以丙烯酸为功能单体,甘油1,3-二甘油醇酸二丙烯酸酯作为交联剂,在孔内通过自由基引发聚合,成功地制备了在孔壁表面聚合物修饰的三维有序大孔氧化硅/聚合物复合材料(3DOM Si O_2-COOH)。通过拉曼光谱、扫描电子显微镜(SEM)、BET比表面测试和片剂硬度计技术手段表征了材料孔结构特征和机械强度。结果表明,3DOM Si O_2-COOH具有均匀的相互连接的大孔结构,孔壁表面形成了一层致密的11~32 nm厚度的聚合物膜,且具有较高的机械强度高。以3DOM Si O_2-COOH材料为载体,葡萄糖淀粉酶在其内固载能均匀地分布在材料内部。固载酶和游离酶的最佳反应p H均为5,最佳反应温度为55℃,米氏常数分别为3.78和3.97 g/L。固载酶具有更高的热稳定性、p H值稳定性、储藏稳定性和重复使用稳定性。3DOM Si O_2-COOH材料可作为一种新型的固载酶载体。
This paper presented a facile method to the modification of the three-dimensionally ordered macroporous(3DOM)SiO_2hybrids with a carboxyl functionalized polymer layer on the macropore walls.Acrylic acid and glycerol 1,3-diglycerolate diacrylate were copolymerized to form the polymer layer in the macropores.Raman spectra,scanning electron microscopy(SEM)and BET measurements show that the prepared 3DOM SiO_2@polymer composites(SiO_2 -COOH)have an uniform interconnected macroporous structure and the macropore walls are covered by a nanoscaled compact polymer layer.Moreover,the 3DOM SiO_2-COOH has an improved mechanical strength.The 3DOM SiO_2 -COOH was further used as the support to immobilize glucoamylase.The results show that the immobilized enzyme homogeneously distributes in the3DOM materials.The optimum pH of immobilized and free enzymes is both at 5,and the optimal reaction temperature is at 55℃.Michaelis constants of the immobilized enzyme and free enzyme are 3.78 g/L and3.97 g/L,respectively.The immobilized enzyme presents higher thermal,pH,storage stabilities and higher reusability compared with the free enzyme.The results indicate that 3DOM SiO_2 -COOH could be a novel support for the immobilization of enzymes.
This paper presented a facile method to the modification of the three-dimensionally ordered macroporous(3DOM)SiO_2hybrids with a carboxyl functionalized polymer layer on the macropore walls.Acrylic acid and glycerol 1,3-diglycerolate diacrylate were copolymerized to form the polymer layer in the macropores.Raman spectra,scanning electron microscopy(SEM)and BET measurements show that the prepared 3DOM SiO_2@polymer composites(SiO_2 -COOH)have an uniform interconnected macroporous structure and the macropore walls are covered by a nanoscaled compact polymer layer.Moreover,the 3DOM SiO_2-COOH has an improved mechanical strength.The 3DOM SiO_2 -COOH was further used as the support to immobilize glucoamylase.The results show that the immobilized enzyme homogeneously distributes in the3DOM materials.The optimum pH of immobilized and free enzymes is both at 5,and the optimal reaction temperature is at 55℃.Michaelis constants of the immobilized enzyme and free enzyme are 3.78 g/L and3.97 g/L,respectively.The immobilized enzyme presents higher thermal,pH,storage stabilities and higher reusability compared with the free enzyme.The results indicate that 3DOM SiO_2 -COOH could be a novel support for the immobilization of enzymes.
引文
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[19]Reddy P S,Rani D J,Sulthana S. Amylases as a Tool for Industrial Application:A Review[J]. J Pure Appl Algebra,2011,5(1):167-171.
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[22]YE Shuaiguan,SUN Zuoyu,DING Yi. The Coupled Reaction of Glucose Oxidase-Per-Oxidase-o-Diansidine System for Determination of Blood-Glucose and Some Inhibitors[J]. Chem Reagent,1986,8(5):306-309(in Chinese).叶率官,孙佐宇,丁益.以葡萄糖氧化酶-过氧化物酶-邻联二茴香胺偶联反应动力学方法(比色)测定血中葡萄糖及某些抑制剂[J].化学试剂,1986,8(5):306-309.
[23]Nair D P,Podgorski M,Chatani S,et al. The Thiol-Michael Addition Click Reaction:A Powerful and Widely Used Tool in Materials Chemistry[J]. Chem Mater,2014,26(1):724-744.
[24]Gomez J L,Bodalo A,Gomez E,et al. Immobilization of Peroxidases on Glass Beads:An Improved Alternative for Phenol Removal[J]. Enzyme Microb Technol,2006,39(5):1016-1022.
[25]GUO Ming,YAN Bingyu,WANG Chunpeng,et al. Preparation of Cellulose-Based Immobilized Enzyme Matrix and the Exploration of Immobilized Laccase Performance[J]. Sci Silvae Sin,2013,49(11):122-128(in Chinese).郭明,燕冰宇,王春鹏.纤维素基质固载酶材料制备及固定化漆酶性能[J].林业科学,2013,49(11):122-128.
[26]Volokitina M V,Korzhikov-Vlakh V A,Tennikova T B,et al. Macroporous Monoliths for Biodegradation Study of Polymer Particles Considered as Drug Delivery Systems[J]. J Pharm Biomed Anal,2017,145:169-177.
[27]Gashtasbi F,Ahmadian G,Noghabi K A. New Insights into the Effectiveness of Alpha-Amylase Enzyme Presentation on the Bacillus Subtilis Spore Surface by Adsorption and Covalent Immobilization[J]. Enzyme Microb Technol,2014,64/65:17-23.
[28]Gupta K,Jana A K,Kumar S,et al. Solid State Fermentation with Recovery of Amyloglucosidase from Extract by Direct Immobilization in Cross Linked Enzyme Aggregate for Starch Hydrolysis[J]. Biocatal Agric Biotechnol,2015,4(4):486-492.
[29]WANG Feng,LIANG Liman,GU Zhengbiao,et al. Immobilization of Amylase on PS-TEPA and PS-PEG Resins[J].Chinese J Appl Chem,2008,25(11):1259-1265(in Chinese).王峰.梁力曼,顾正彪,等. PS-TEPA和PS-g-PEG树脂固载淀粉酶[J].应用化学,2008,25(11):1259-1265.
[2]Yamada R,Suzuki Y,Yasuda M,et al. Immobilization of Proteins on Synthetic Resins Using Super-critical Carbon Dioxide[J]. J Supercrit Fluids,2016,107:566-570.
[3]Huttner S,Gomes M Z D V,Iancu L,et al. Immobilisation on Mesoporous Silica and Solvent Rinsing Improve the Transesterification Abilities of Feruloyl Esterases from Myceliophthora Thermophila[J]. Bioresour Technol,2017,239:57-65.
[4]Vasconcellos A D,Paula A S,Filho R A L,et al. Synergistic Effect in the Catalytic Activity of Lipase Rhizomucor Miehei Immobilized on Zeolites for the Production of Biodiesel[J]. Micropor Mesopor Mater,2012,163:343-355.
[5]Shahrestani H,Taheri-kafrani A,Soozanipour A,et al. Enzymatic Clarification of Fruit Juices Using Xylanase Immobilized on 1,3,5-Triazine-Functionalized Silica-Encapsulated Magnetic Nanoparticles[J]. Biochem Eng J,2016,109:51-58.
[6]Velev O D,Jede T A,Lobo R F,et al. Porous Silica via Colloidal Crystallization[J]. Nature,1997,389(6650):447-448.
[7]Pandya P H,Jasra R V,Newalkar B L,et al. Studies on the Activity and Stability of Immobilized Alpha-Amylase in Ordered Mesoporous Silicas[J]. Micropor Mesopor Mater,2005,77(1):67-77.
[8]Yan W,Zheng X H,Min Z. Study of Immobilization of Laccase on Mesoporous Molecular Sieve MCM-41[J]. J Chem Eng Chinese Univ,2008,22(1):83-87.
[9]Schroden R C,Al-daous M,Blanford C F,et al. Optical Properties of Inverse Opal Photonic Crystals[J]. Chem Mater,2002,14(8):3305-3315.
[10]Yang J,Chen Z L,Wang H,et al. Highly Ordered Three-Dimensional Ti O2@C Nanotube Arrays as Freestanding Electrode for Sodium-Ion Battery[J]. Mater Lett,2017,207:149-152.
[11]Li X,Liu Y,Deng J,et al. Enhanced Catalytic Performance for Methane Combustion of 3DOM Co Fe2O4by Co-Loading MnOxand Pd-Pt Alloy Nanoparticles[J]. App Surf Sci,2017,403:590-600.
[12]Ye P,Wan R B,Wang X P. Quantitative Enzyme Immobilization:Control of the Carboxyl Group Density on Support Surface[J]. J Mol Catal B:Enzym,2009,61(3/4):296-302.
[13]Gornowich D B,Blanchard G J. Enhancement of Enzyme Activity by Confinement in an Inverse Opal Structure[J]. J Phys Chem C,2012,116(22):12165-12171.
[14]Jiang Y,Wang Y,Wang H,et al. Facile Immobilization of Enzyme on Three Dimensionally Ordered Macroporous Silica via a Biomimetic Coating[J]. New J Chem,2015,39(2):978-984.
[15]Wu Q,Liao J,Yin Q,et al. Synthesis, Characterization and Catalytic Activity of Ordered Macroporous Silicas Functionalized with Organosulfur Groups[J]. Mater Res Bull,2008,43(5):1209-1217.
[16]Lowe A B. Thiol-Yne Click'/Coupling Chemistry and Recent Applications in Polymer and Materials Synthesis and Modification[J]. Polymer,2014,55(22):5517-5549.
[17]Heggli M,Tirelli N,Zisch A,et al. Michael-Type Addition as a Tool for Surface functionalization[J]. Bioconjug Chem,2003,14(5):967-973.
[18]Berkel K Y,Hawker C J. Tailored Composite Polymer-Metal Nanoparticles by Miniemulsion Polymerization and Thiol-ene Functionalization[J]. J Polym Sci Polym Chem,2010,48(7):1594-1606.
[19]Reddy P S,Rani D J,Sulthana S. Amylases as a Tool for Industrial Application:A Review[J]. J Pure Appl Algebra,2011,5(1):167-171.
[20] Amirbandeh M,Taheri-kafrani A. Immobilization of Glucoamylase on Triazine-Functionalized Fe3O4/Graphene Oxide Nanocomposite:Improved Stability and Reusability[J]. Int J Biol Macromol,2016,93:1183-1191.
[21]Isobe N,Lee D S,Kwon Y J,et al. Immobilization of Protein on Cellulose Hydrogel[J]. Cellulose,2011,18(5):1251-1256.
[22]YE Shuaiguan,SUN Zuoyu,DING Yi. The Coupled Reaction of Glucose Oxidase-Per-Oxidase-o-Diansidine System for Determination of Blood-Glucose and Some Inhibitors[J]. Chem Reagent,1986,8(5):306-309(in Chinese).叶率官,孙佐宇,丁益.以葡萄糖氧化酶-过氧化物酶-邻联二茴香胺偶联反应动力学方法(比色)测定血中葡萄糖及某些抑制剂[J].化学试剂,1986,8(5):306-309.
[23]Nair D P,Podgorski M,Chatani S,et al. The Thiol-Michael Addition Click Reaction:A Powerful and Widely Used Tool in Materials Chemistry[J]. Chem Mater,2014,26(1):724-744.
[24]Gomez J L,Bodalo A,Gomez E,et al. Immobilization of Peroxidases on Glass Beads:An Improved Alternative for Phenol Removal[J]. Enzyme Microb Technol,2006,39(5):1016-1022.
[25]GUO Ming,YAN Bingyu,WANG Chunpeng,et al. Preparation of Cellulose-Based Immobilized Enzyme Matrix and the Exploration of Immobilized Laccase Performance[J]. Sci Silvae Sin,2013,49(11):122-128(in Chinese).郭明,燕冰宇,王春鹏.纤维素基质固载酶材料制备及固定化漆酶性能[J].林业科学,2013,49(11):122-128.
[26]Volokitina M V,Korzhikov-Vlakh V A,Tennikova T B,et al. Macroporous Monoliths for Biodegradation Study of Polymer Particles Considered as Drug Delivery Systems[J]. J Pharm Biomed Anal,2017,145:169-177.
[27]Gashtasbi F,Ahmadian G,Noghabi K A. New Insights into the Effectiveness of Alpha-Amylase Enzyme Presentation on the Bacillus Subtilis Spore Surface by Adsorption and Covalent Immobilization[J]. Enzyme Microb Technol,2014,64/65:17-23.
[28]Gupta K,Jana A K,Kumar S,et al. Solid State Fermentation with Recovery of Amyloglucosidase from Extract by Direct Immobilization in Cross Linked Enzyme Aggregate for Starch Hydrolysis[J]. Biocatal Agric Biotechnol,2015,4(4):486-492.
[29]WANG Feng,LIANG Liman,GU Zhengbiao,et al. Immobilization of Amylase on PS-TEPA and PS-PEG Resins[J].Chinese J Appl Chem,2008,25(11):1259-1265(in Chinese).王峰.梁力曼,顾正彪,等. PS-TEPA和PS-g-PEG树脂固载淀粉酶[J].应用化学,2008,25(11):1259-1265.