新型磁性高分子载体的制备、固定化脂肪酶及其催化酮洛芬手性拆分的研究
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摘要
本学位论文综述了固定化酶技术、固定化酶用磁性高分子载体材料以及脂肪酶与固定化脂肪酶用于2-芳基丙酸类化合物手性拆分的最新进展,提出了本学位论文的研究思路和选题指导思想。采用无皂种子乳液聚合和DPE可控自由基聚合法合成了两种新型磁性高分子载体材料,对合成的载体进行了表征,优化了固定化条件,研究了两种固定化酶的性质；并使用游离酶及上述两种载体材料固定化脂肪酶后用于催化酮洛芬酯类手性水解反应,研究了多种因素对于不对称水解反应的影响。
1、利用传统的化学共沉淀法合成了Fe304磁性纳米粒子,并使用油酸和γ-氨丙基三乙氧基硅烷对Fe304磁性纳米粒子进行了改性。结果表明,对磁性粒子的化学改性是成功的,且合成的磁性粒子粒径约为十几纳米,并有一定的磁响应性。
2、采用苯乙烯(St),甲基丙烯酸缩水甘油酯(GMA)和甲基丙烯酸β-羟乙酯(HEMA)作为共聚单体,利用无皂乳液聚合法合成种子乳液后将APTS-Fe3O4纳米粒子引入,得到磁性种子乳液；在此磁性种子乳液存在下,采用甲基丙烯酸丁酯(BMA)与醋酸乙烯酯(VAc)为共聚第二单体,得到磁性聚合物复合乳胶粒；对乳胶粒进一步功能化后,用共价结合法和物理吸附法对进行了脂肪酶的固定化。结果表明,合成的磁性乳胶粒具有核壳结构,且具有一定的磁响应性。且用共价结合法制得的固定化酶性质优于物理吸附法制得的固定化酶。
3、利用1,1-二苯乙烯(DPE)作为控制试剂,通过两步聚合,合成了高分子-磁性粒子复合微球。首先,以油酸改性的纳米Fe304粒子、DPE以及单体GMA、引发剂AIBN为油相,2%淀粉NaCl饱和溶液为水相,应用简单的悬浮聚合法在氮气氛围中进行第一步聚合。聚合结束后使反应体系冷却过夜,后在第一步反应体系中加入第二种单体HEMA和少量交联剂,不另加引发剂,进行第二步聚合,即得磁性高分子微球载体。用所得高分子微球进行了脂肪酶的固定化,研究了固定化酶的性质,采用SDS凝胶电泳证实了酶的连接方式。结果表明,该固定化酶在水相和有机相中均表现出了较好的催化活性,说明该载体是一种两亲型载体。
4、合成了酮洛芬的不同种酯并对其用核磁共振氢谱进行了表征。选用玫瑰假丝酵母脂肪酶作为催化剂,研究了其在水相中催化酮洛芬的不同酯水解的反应。研究了反应的各种条件,如不同底物、底物浓度、反应pH值、反应时间、有机溶剂、共溶剂DMSO的加入等对的反应的转化率及立体选择性的影响,结果表明,加入一定量DMSO,能够提高底物的转化率和产物的立体选择性。
5、选用上述合成的两种载体固定化玫瑰假丝酵母脂肪酶作为催化剂,考察了两种固定化酶对酮洛芬不同酯的选择性,对比了两种固定化酶的反应效果。结果表明,P(GMA-b-HEMA)磁性高分子微球固定化脂肪酶催化酮洛芬酯水解的效果较好,在此基础上,优化了其催化酮洛芬酯水解反应的条件,并考察了固定化酶的重复使用效果。结果表明,该固定化酶重复使用五次之后,仍能保持一定的催化活力和对底物的选择性。
This dissertation reviews the studies of enzyme immobilization technologies, novel immobilization materials of today and application of lipase and immobilized lipase on chiral separation of 2-Arylpropionic Acid. Novel magnetic polymer composites were synthesized via soap-free seeded emulsion polymerization and DPE controlled radical polymerization, respectively. After that, these novel magnetic polymer composites were used as carriers for lipase immobilization. Then free lipase and the synthesized immobilized lipases were used as catalyst to catalyze the asymmetric hydrolysis of ketoprofen ester.
1、Fe3O4 nanoparticles were synthesized by the chemical co-precipitation method, and the magnetic nanoparticles were chemically modified by oleic acid and y-aminopropyltriethoxysilane (APTS).
2、Superparamagnetic composites based on polymer colloids were prepared by seeded emulsion. Firstly, styrene (St), glycidyl methacrylate (GMA) and 2-hydroxyethyl methacrylate (HEMA) were copolymerized by soap-free seeded emulsion polymerization, and then APTS-Fe3O4 nanoparticles were introduced into above copolymer emulsion by covalent bonding or physically absorption. Secondly, butyl methacrylate (BMA), vinyl acetate (VAc) and ethylne glycol dimethacrylate (EGDMA) were added drop wise to the the superparamagnetic seed latexes without addition of any emulsifier. The results showed that the magnetic composites of polymer colloids were successfully synthesized and had a structure with a thinner shell (around 100 nm) and a bigger cavity (around 200 nm) and also possessed a certain level of magnetic response (4.16emu/g carrier). Candida rugosa lipases (CRL) were covalently immobilized or physically absorbed on the magnetic microspheres. The immobilized lipase could be separated, recovered and reused easily and rapidly. The immobilization conditions were optimized; the properties of immobilized lipase (such as themal stability, reusability and kinetic properties) prepared by the two methods were compared. The results indicated that immobilized lipase prepared by covalent bonding held better themal stability and reusability than ICRL prepared by physically absorption.
3、OA-Fe3O4 nanoparticles were introduced into 1,1-Diphenylethylene (DPE) controlled radical polymerization system to prepare superparamagnetic microspheres for enzyme immobilization by two steps of polymerization. In the presence of DPE, glycidyl methacrylate (GMA) and 2-hydroxyethyl methacrylate (HEMA), methacryloxyethyl trimethyl ammonium chloride (MATAC) with charge were selected as copolymering monomers based on their reactive functional group and excellent biocompatibility which were suitable for immobilization of Candida rugosa lipase (CRL). SDS-PAGE analysis was also conducted to demonstrate whether CRL is covalently immobilized or only physically adsorbed. The results indicated that the polymerization was successfully carried out, and lipase was immobilized on the magnetic microspheres through ionically adsorption and covalent binding under mild conditions. The immobilized lipase exhibited high activity recovery (69.7%), better resistance to pH and temperature inactivation in aqueous phase, as well as superior reusability in nonaqueous phase. The data showed that the resulting carrier could hold an amphiphilic property.
4、The methyl ester, ethyl ester, propyl ester, isopropyl ester and butyl ester of ketoprofen were synthesized. Candida rugosa lipase was used as catalyst to catalyze the asymmetric hydrolysis reaction of ketoprofen esters. The reaction conditions (such as structure of substrate, pH, addition of DMSO and reaction time) were optimized. The obtained products were analyzed by HPLC. The results showed that the conversion% of substrate and eep value of products will increase with the addition of DMSO.
5、Candida rugosa lipase was immobilized on magnetic polymer colloids and P(GMA-b-HEMA). Then the immobilized CRL were used to catalyze asymmetric hydrolysis reaction of ketoprofen esters. The effects of the two immobilized CRL on the conversion% of substrate and eep value of products were compared. The reaction conditions (such as structure of substrate, pH, addition of DMSO and reaction time) were optimized. The reusability of immobilized CRL was also investigated.
1、利用传统的化学共沉淀法合成了Fe304磁性纳米粒子,并使用油酸和γ-氨丙基三乙氧基硅烷对Fe304磁性纳米粒子进行了改性。结果表明,对磁性粒子的化学改性是成功的,且合成的磁性粒子粒径约为十几纳米,并有一定的磁响应性。
2、采用苯乙烯(St),甲基丙烯酸缩水甘油酯(GMA)和甲基丙烯酸β-羟乙酯(HEMA)作为共聚单体,利用无皂乳液聚合法合成种子乳液后将APTS-Fe3O4纳米粒子引入,得到磁性种子乳液；在此磁性种子乳液存在下,采用甲基丙烯酸丁酯(BMA)与醋酸乙烯酯(VAc)为共聚第二单体,得到磁性聚合物复合乳胶粒；对乳胶粒进一步功能化后,用共价结合法和物理吸附法对进行了脂肪酶的固定化。结果表明,合成的磁性乳胶粒具有核壳结构,且具有一定的磁响应性。且用共价结合法制得的固定化酶性质优于物理吸附法制得的固定化酶。
3、利用1,1-二苯乙烯(DPE)作为控制试剂,通过两步聚合,合成了高分子-磁性粒子复合微球。首先,以油酸改性的纳米Fe304粒子、DPE以及单体GMA、引发剂AIBN为油相,2%淀粉NaCl饱和溶液为水相,应用简单的悬浮聚合法在氮气氛围中进行第一步聚合。聚合结束后使反应体系冷却过夜,后在第一步反应体系中加入第二种单体HEMA和少量交联剂,不另加引发剂,进行第二步聚合,即得磁性高分子微球载体。用所得高分子微球进行了脂肪酶的固定化,研究了固定化酶的性质,采用SDS凝胶电泳证实了酶的连接方式。结果表明,该固定化酶在水相和有机相中均表现出了较好的催化活性,说明该载体是一种两亲型载体。
4、合成了酮洛芬的不同种酯并对其用核磁共振氢谱进行了表征。选用玫瑰假丝酵母脂肪酶作为催化剂,研究了其在水相中催化酮洛芬的不同酯水解的反应。研究了反应的各种条件,如不同底物、底物浓度、反应pH值、反应时间、有机溶剂、共溶剂DMSO的加入等对的反应的转化率及立体选择性的影响,结果表明,加入一定量DMSO,能够提高底物的转化率和产物的立体选择性。
5、选用上述合成的两种载体固定化玫瑰假丝酵母脂肪酶作为催化剂,考察了两种固定化酶对酮洛芬不同酯的选择性,对比了两种固定化酶的反应效果。结果表明,P(GMA-b-HEMA)磁性高分子微球固定化脂肪酶催化酮洛芬酯水解的效果较好,在此基础上,优化了其催化酮洛芬酯水解反应的条件,并考察了固定化酶的重复使用效果。结果表明,该固定化酶重复使用五次之后,仍能保持一定的催化活力和对底物的选择性。
This dissertation reviews the studies of enzyme immobilization technologies, novel immobilization materials of today and application of lipase and immobilized lipase on chiral separation of 2-Arylpropionic Acid. Novel magnetic polymer composites were synthesized via soap-free seeded emulsion polymerization and DPE controlled radical polymerization, respectively. After that, these novel magnetic polymer composites were used as carriers for lipase immobilization. Then free lipase and the synthesized immobilized lipases were used as catalyst to catalyze the asymmetric hydrolysis of ketoprofen ester.
1、Fe3O4 nanoparticles were synthesized by the chemical co-precipitation method, and the magnetic nanoparticles were chemically modified by oleic acid and y-aminopropyltriethoxysilane (APTS).
2、Superparamagnetic composites based on polymer colloids were prepared by seeded emulsion. Firstly, styrene (St), glycidyl methacrylate (GMA) and 2-hydroxyethyl methacrylate (HEMA) were copolymerized by soap-free seeded emulsion polymerization, and then APTS-Fe3O4 nanoparticles were introduced into above copolymer emulsion by covalent bonding or physically absorption. Secondly, butyl methacrylate (BMA), vinyl acetate (VAc) and ethylne glycol dimethacrylate (EGDMA) were added drop wise to the the superparamagnetic seed latexes without addition of any emulsifier. The results showed that the magnetic composites of polymer colloids were successfully synthesized and had a structure with a thinner shell (around 100 nm) and a bigger cavity (around 200 nm) and also possessed a certain level of magnetic response (4.16emu/g carrier). Candida rugosa lipases (CRL) were covalently immobilized or physically absorbed on the magnetic microspheres. The immobilized lipase could be separated, recovered and reused easily and rapidly. The immobilization conditions were optimized; the properties of immobilized lipase (such as themal stability, reusability and kinetic properties) prepared by the two methods were compared. The results indicated that immobilized lipase prepared by covalent bonding held better themal stability and reusability than ICRL prepared by physically absorption.
3、OA-Fe3O4 nanoparticles were introduced into 1,1-Diphenylethylene (DPE) controlled radical polymerization system to prepare superparamagnetic microspheres for enzyme immobilization by two steps of polymerization. In the presence of DPE, glycidyl methacrylate (GMA) and 2-hydroxyethyl methacrylate (HEMA), methacryloxyethyl trimethyl ammonium chloride (MATAC) with charge were selected as copolymering monomers based on their reactive functional group and excellent biocompatibility which were suitable for immobilization of Candida rugosa lipase (CRL). SDS-PAGE analysis was also conducted to demonstrate whether CRL is covalently immobilized or only physically adsorbed. The results indicated that the polymerization was successfully carried out, and lipase was immobilized on the magnetic microspheres through ionically adsorption and covalent binding under mild conditions. The immobilized lipase exhibited high activity recovery (69.7%), better resistance to pH and temperature inactivation in aqueous phase, as well as superior reusability in nonaqueous phase. The data showed that the resulting carrier could hold an amphiphilic property.
4、The methyl ester, ethyl ester, propyl ester, isopropyl ester and butyl ester of ketoprofen were synthesized. Candida rugosa lipase was used as catalyst to catalyze the asymmetric hydrolysis reaction of ketoprofen esters. The reaction conditions (such as structure of substrate, pH, addition of DMSO and reaction time) were optimized. The obtained products were analyzed by HPLC. The results showed that the conversion% of substrate and eep value of products will increase with the addition of DMSO.
5、Candida rugosa lipase was immobilized on magnetic polymer colloids and P(GMA-b-HEMA). Then the immobilized CRL were used to catalyze asymmetric hydrolysis reaction of ketoprofen esters. The effects of the two immobilized CRL on the conversion% of substrate and eep value of products were compared. The reaction conditions (such as structure of substrate, pH, addition of DMSO and reaction time) were optimized. The reusability of immobilized CRL was also investigated.
引文
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