辣根过氧化物酶酶促体系引发乙烯基单体聚合研究
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摘要
本文以辣根过氧化物酶、乙酰丙酮(Acac)和过氧化氢组成的酶促氧化还原体系引发乙烯基单体(如丙烯酰胺AAM、甲基丙烯酸甲酯、丙烯腈、丙烯酸丁酯等)聚合展开研究；以AAM为代表单体，考察了酶促引发体系的引发和聚合机理，分析了诱导期产生原因，较详细地研究了聚合动力学及其影响因素；在上述基础上，对以胶囊固定化辣根过氧化物酶的酶促体系引发丙烯酰胺的聚合进行了探索性研究。
通过酶促体系引发各乙烯基单体的聚合研究结果表明，在低酶浓度下酶促体系能有效引发单体聚合，且水溶性单体经24小时后水溶液聚合转化率较高，而油溶性单体用乳液聚合转化率最高仅为40％。
在低酶浓度下(约0.1 mg／mL)，HRP酶促体系能进行AAM的聚合反应，经验证为HRP、Acac和H_2O_2三者协同作用，共同引发AAM聚合反应。酶促聚合产物PAAM样品M_n在1.24～4.6~*10~5DA之间，I_p在2.0～2.5之间。通过动力学实验，分析了诱导期产生机理，并通过调整Acac初始浓度使得诱导期消除，而对产物分子量无大影响。进一步比较实验和机理聚合速率方程表明，乙酰丙酮自由基(ACAC)之间会发生相互反应而消失，同时聚合增长链主要以歧化终止反应为主。
为了提高酶的使用效率，本文还设计了用海藻酸钠-壳聚糖-海藻酸钠(ACA)微胶囊法固定化辣根过氧化物酶，实验确定了钙离子、海藻酸钠等的适宜用量及溶芯时间等工艺条件，制备了以低分子量壳聚糖为壁材酶处于囊芯的具有较好弹性和机械强度的毫米级微胶囊。实验测得酶的包埋效率为51.33％，固定化酶相对游离酶活力约60％。以胶囊固定化辣根过氧化物酶酶促体系成功引发AAM的聚合。实验表明，酶以胶囊方式固定化后，体系需经较长时间后才能使AAM聚合，对钙固定化酶促引发体系的聚合场所问题，进行多方面的实验研究和分析，给出了初步的结论和解释，这是至今人们尚未涉及过的全新课题，有待深入研究。
Horseradish peroxidase (HRP) is an oxido-reductase acting on hydrogen peroxide as oxidant. With the presence of H2O2 and acetylacetone (Acac), free radical polymerization of vinyl monomers, including acrylamide (AAM), methyl metha- acrylate (MMA), acrylonitrile (AN), butyl acrylate (BA), etc, have been carried out with the initiation of HRP. The enzyme-mediated products have also been measured and characterized by IR spectra. The polymerizations of acrylamide (AAM) in water initiated by a HRP/ H2O2/Acac ternary system have been investigated. It was carried out by dilatometry to obtain the different conversion curves under different conditions. The experimental rate equation was also fitted and the further reaction mechanism.wasexplained by analysis. And what is more, Microcapsules with an alginate gel core and a polyanion-polycation membrane have been investigated to immobilize HRP enzyme for their well mechanistic property and penetratable selectivity to different molecules and AAM polymerization initiated by immobilized enzyme system has been conducted successfully.
From experiments and comparisons with various vinyl monomers mediated by HRP system, it can be learned that HRP/Acac/H2O2 ternary system is effective and potent to initiate the polymerizations of vinyl monomers. However, the conversions of different monomers are dissimilar. The water-soluble monomer has a higher conversion, while the oil-soluble substrate has a lower one, for an instance, about 40% for MMA.
AAM radical polymerizations initiated by an HRP-mediated redox system in lower HRP initial concentration (about 0.1 mg/mL) were performed with high yields in wild ways. The molecular weight of several PAAM products were measured and characterized as with Mn and DP ranging from 2.0-6.3 X 105 DA and 2.0-3.0, respectively . In the condition of fixed other concentrations, the inhibition period was dependant on [ACAC]o. The reason was qualitative interpreted caused by residual oxygen gas, but the quantitative correlations were needed to further investigate to obtain the rate constants. From the conversion curves, it was derived of polymerization rate (RP) and gained the apparent activation energy (Ea=7.65 KJ/mol). At last, we fitted the experimental rate equation and explained by analysis with
reaction mechanism.
In order to use the enzyme effectively, Alginate-Chitosan-Alginate (ACA) microcapsules were used to immobilize HRP. The immobilized efficiency of this method and the ralative special ratio of immobilized to free enzyme were tested as 51.33% and about 60%, respectively. AAM polymerization initiated by immobilized enzyme system has been conducted successfully.
The study of our work provides the necessary experimental basis for further investigatation.
通过酶促体系引发各乙烯基单体的聚合研究结果表明，在低酶浓度下酶促体系能有效引发单体聚合，且水溶性单体经24小时后水溶液聚合转化率较高，而油溶性单体用乳液聚合转化率最高仅为40％。
在低酶浓度下(约0.1 mg／mL)，HRP酶促体系能进行AAM的聚合反应，经验证为HRP、Acac和H_2O_2三者协同作用，共同引发AAM聚合反应。酶促聚合产物PAAM样品M_n在1.24～4.6~*10~5DA之间，I_p在2.0～2.5之间。通过动力学实验，分析了诱导期产生机理，并通过调整Acac初始浓度使得诱导期消除，而对产物分子量无大影响。进一步比较实验和机理聚合速率方程表明，乙酰丙酮自由基(ACAC)之间会发生相互反应而消失，同时聚合增长链主要以歧化终止反应为主。
为了提高酶的使用效率，本文还设计了用海藻酸钠-壳聚糖-海藻酸钠(ACA)微胶囊法固定化辣根过氧化物酶，实验确定了钙离子、海藻酸钠等的适宜用量及溶芯时间等工艺条件，制备了以低分子量壳聚糖为壁材酶处于囊芯的具有较好弹性和机械强度的毫米级微胶囊。实验测得酶的包埋效率为51.33％，固定化酶相对游离酶活力约60％。以胶囊固定化辣根过氧化物酶酶促体系成功引发AAM的聚合。实验表明，酶以胶囊方式固定化后，体系需经较长时间后才能使AAM聚合，对钙固定化酶促引发体系的聚合场所问题，进行多方面的实验研究和分析，给出了初步的结论和解释，这是至今人们尚未涉及过的全新课题，有待深入研究。
Horseradish peroxidase (HRP) is an oxido-reductase acting on hydrogen peroxide as oxidant. With the presence of H2O2 and acetylacetone (Acac), free radical polymerization of vinyl monomers, including acrylamide (AAM), methyl metha- acrylate (MMA), acrylonitrile (AN), butyl acrylate (BA), etc, have been carried out with the initiation of HRP. The enzyme-mediated products have also been measured and characterized by IR spectra. The polymerizations of acrylamide (AAM) in water initiated by a HRP/ H2O2/Acac ternary system have been investigated. It was carried out by dilatometry to obtain the different conversion curves under different conditions. The experimental rate equation was also fitted and the further reaction mechanism.wasexplained by analysis. And what is more, Microcapsules with an alginate gel core and a polyanion-polycation membrane have been investigated to immobilize HRP enzyme for their well mechanistic property and penetratable selectivity to different molecules and AAM polymerization initiated by immobilized enzyme system has been conducted successfully.
From experiments and comparisons with various vinyl monomers mediated by HRP system, it can be learned that HRP/Acac/H2O2 ternary system is effective and potent to initiate the polymerizations of vinyl monomers. However, the conversions of different monomers are dissimilar. The water-soluble monomer has a higher conversion, while the oil-soluble substrate has a lower one, for an instance, about 40% for MMA.
AAM radical polymerizations initiated by an HRP-mediated redox system in lower HRP initial concentration (about 0.1 mg/mL) were performed with high yields in wild ways. The molecular weight of several PAAM products were measured and characterized as with Mn and DP ranging from 2.0-6.3 X 105 DA and 2.0-3.0, respectively . In the condition of fixed other concentrations, the inhibition period was dependant on [ACAC]o. The reason was qualitative interpreted caused by residual oxygen gas, but the quantitative correlations were needed to further investigate to obtain the rate constants. From the conversion curves, it was derived of polymerization rate (RP) and gained the apparent activation energy (Ea=7.65 KJ/mol). At last, we fitted the experimental rate equation and explained by analysis with
reaction mechanism.
In order to use the enzyme effectively, Alginate-Chitosan-Alginate (ACA) microcapsules were used to immobilize HRP. The immobilized efficiency of this method and the ralative special ratio of immobilized to free enzyme were tested as 51.33% and about 60%, respectively. AAM polymerization initiated by immobilized enzyme system has been conducted successfully.
The study of our work provides the necessary experimental basis for further investigatation.
引文
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[2] Fahnestock S R,Yao Z,Bedzyk. Microbial Production of Spider Silk Proteins.[J]Rev.in Mol.Biotech.2000,v74,105-119.
[3] Kobayashi S,Uyama H,Kimura S.Enzymatic Polymerization.[J]Chem.Rev.2001,v101.3793-3818.
[4] 齐耿耿，孙建中，周其云，等．酶催化聚合研究进展．[J]功能高分子学报，2002，v14，n2，1-8．
[5] 刘永成，沈晶，于同隐．酶在高分子合成中的应用．[M]高分子材料科学与工程，1999，v15，n6，6-8．
[6] Zaks A,Klibanov A M. Enzymatic Catalysis at 100℃. [J]Science.v 224,1249-1251.
[7] MacDonald R T,Pulapura S K,Suirkin Y Y,et al.Enzyme-Catalyzed Ring-Opening Polymerization[J].Macromolecules,1995,v28:73-78.
[8] Kobayashi S,Shimada J,Kashiwa K,et al.Enzymatic polymerization of α-D-maltosyl fluoride utilizing α-amylase as the catalyst:a new approach for the synthesis of maltooligosaccharides[J].Macromolecules,1992,25:3237-3241.
[9] 周其凤，胡汉杰．[M]高分子化学．北京：化工出版社，2001,349-388．
[10] Park O-J,Kim D-Y,Dordick J S.Enzyme-Catalyzed Synthesis of Sugar-Containing Monomers and Linear Polymers.[J] Biotech.&Bioeng.2000,v70,208-216.
[11] Kobayashi S, Uyama H, Namekawa S, et al. Enzymatic Ring-Opening Polymerization and Copolymerization of 8-Octanolide by Lipase Catalyst.[J] Macromol.1998,v31,5655-5659.
[12] Daniel A, Abramowicz,Keese C R.Enzymatic Transesterifications of Carbonates in Water-Restricted Environment.[J] Biotech.&Bioeng.1989,v33,149-156.
[13] Linko Y-Y,Wang Z-L,Seppala J.Lipase-Catalyzed Linear Aliphatic Polyester Synthesis in Organic Solvent.[J] Enzyme Microb.Technol.1995,v17,507-511.
[14] Ikeda R, Tanaka H, Uyama H, et al. Oxidative Polymerization of 2,6-Difluorophenol to Crystalline Poly(2,6-difluoro-1,4-phenylene oxide).[J] Macromol. 2000, v33, 6648-6652.
[15] Durand A,Lalot T,Brigodiot M,et al.Enzyme-Mediated Initiation of Acrylamide Polymerization:Reaction Mechanism.[J] Polymer 2000,v41,8183-8192.
[16] Rodney R L,Stagno J L,Beckman E J,et al.Enzymatic Synthesis of Carbonate Monomers and Polycarbonates.[J] Biotech.&Bioeng.1999,v62,259-266.
[17] Svirkin Y Y, Xu J, Gross R A, et al. Enzyme-Catalyzed Stereoelective Ring-Opening Polymerization of α-Methyl-β-propiolactone. [J] Macromol.1996,v29,4591-4597.
[18] Dong H,Cao S-G,Li Z-Q,et al.Study on the Enzymatic Polymerization Mechanism of Lactone and the Strategy for Improving the Degree of Polymerization.[J] J.Polym.Sci:Part A:Polym.Chem.1999,v37,1265-1275.
[19] Akkara J A,Senecal K J,Kaplan D L.Synthesis and Characterization of Polymer Produced by Horseradish Peroxidase in Dioxane.[J] J.Polym.Sci., Polym.Chem.1991,v29,1561-1563.
[20] Dordick J S, Marletta M A, klibanov M. Polymerization of Phenols Catalyzed by Peroxidase in Nonaqueous Media. [J] Biotech.&Bioeng.1987,v30,31-36.
[21] Fukuoka T,Tonami H,Maruichi N,et al.Peroxidase-Catalyzed Oxidative Polymerization of 4,4'-Dihydroxydiphenyl Ether.Formation of α,ω-Hydroxy oligo(1,4-phenylene oxide) through an Unusual Reaction Pathway.[J] Macromol.2000,v33,9152-9155.
[22] Ikeda R, Sugihara J,Uyama H,et al.Enzymatic Oxidative Polymerization of 2,6-Dimethylphenol.[J]Macromol.1996,v29,8702-8705.
[23] Kobayashi S. Enzymatic Polymerization: A New Method of Polymer Synthesis. [J] J.Polym.Sci., Polym.Chem.1999,v37,3041-3056.
[24] Derango A R,Chiang L-C,Dowbenko R,et al.Biotechnol Tech.1992,523-526.
[25] Emery O,Lalot T,Brigodiot M,et al.Free-Radical Polymerization of Acrylamide
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