SiO_2微球与离子螯合微球的制备及应用于固定木瓜蛋白酶研究
摘要
固定化酶可以提高酶的稳定性并增加酶的使用次数，以及易于与
产物和底物分离等优点而被广泛深入的研究。但目前常用的固定化方
法在制备固定化酶时酶活损失较大，且载体常常不可重复使用或载体
的再生操作复杂，限制了固定化酶的产业化应用。本文针对目前固定
化酶方法的不足之处提出了相应对策并对新的酶固定化方法和载体
进行了探索。
本文选用木瓜蛋白酶作为固定化对象，木瓜蛋白酶是一种来源广
泛的巯醇类植物蛋白酶，在食品、轻纺、医学等领域有着广泛的应用，
木瓜蛋白酶在酶动力学和结构学上都已得到了深入的研究，因此很适
合作为评测酶固定化方法效率的模式酶。
采用正硅酸乙酯与N-(β-氨乙基)一氨丙基三乙氧基硅烷在油包
水形成的微胶囊中同步水解的方法，一步法制备了氨基化的二氧化硅
颗粒，这种颗粒大小均匀，氨基含量和颗粒大小可控，氨基含量高达
5． 6mmol／g。此颗粒经戊二醛处理后，采用共价法固定木瓜蛋白酶，
固定化最适pH6． 5，最佳给酶量为15mg／g载体，固定化酶的最适反应
温度为70℃，最适反应pH为6． 5，固定化酶热稳定性，pH耐受性，
贮存稳定性都明显高于游离酶，表明此颗粒可作为一种优良的酶固定
化载体。
本文采用了反相悬浮包埋合成磁性琼脂糖微球，采用悬浮聚合法
合成苯乙烯一甲基丙烯酸羟乙酯，经环氧氯丙烷和亚氨基二乙酸处理
使微球带上螯合基团，经吸附铜离子或镍离子后用于固定化木瓜蛋白
酶。实验的初步结果显示，磁性琼脂糖微球可用于固定化木瓜蛋白酶，
并且经洗脱液洗脱，酶可以从载体上洗脱下来，载体经再吸附金属离
子后可以再生，并可用于再次固定化木瓜蛋白酶，载体重复使用3次
后酶活仍达首次时的90％。初步实验结果表离子螯合载体可用于固定
化木瓜蛋白酶，并作为可再生载体经多次使用。
The immobilized enzymes have more stability, more use times and easier separation from substrate and product than enzymes in bulk solution. But current immobilization methods have many disadvantages, such as much enzyme activity lose after immobilization and high cost. So only a few immobilized enzymes have been used in industry widely though enzyme immobilization have been studied for about thirty years. In order to resolve these disadvantages, new enzyme carriers and immobilization methods need to be explored. In this paper we put forward some solutions and synthesized several kinds of new enzyme carriers.
We choose papain as immobilized object, and investigate performance of these carriers used as papain carrier. Papain, a thiol protease, have great industrial and medicinal potential, and is well characterized kinetically and structurally being a suitable model to compare the efficiency of various immobilization procedures and methods.
Among present enzyme carriers inorganic materials have lower cost and more stability than organic materials. But the present inorganic materials contain less active sites, and have to undergo some complicated processes before immobilizing enzyme. SiO2 particles containing amine groups were synthesized by synchronous hydrolysis of tetraethylorthosilicate(TEOS) and N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAPS) in W/O Microemulsion of TritonX-100/cyclohexane/ammonium hydroxide system. The obtained particles have a narrow diameter range, and its size can be controlled by adjusting water to surfactant molar ratio and water to TEOS and AEAPS molar ratio. Comparing with traditional porous silica beads, these panicles contain much more amine groups and its amine
group content can be easily changed when synthesizing. After treated by
glutaraldehyde, papain was immobilized on the particles by covalent method. The optimum immobilization conditions of enzyme were as follows: enzyme load was 15mg/g carrier, pH was 6.5.The pH and temperature optima were 6.5 and 70 C for immobilization enzyme. All above indicate this kind of particle can be a good enzyme immobilization carrier.
The reverse phase suspension and embedment technique were adopted to prepare magnetic agar microsphere. The dispersion medium was mixture of some organic solvents. Span-80 was used as stabilizer. The magnetic agar microsphere was coupled with epichlorohydrin, then treated with iminodiacetic acid, and magnetic metal chelate carrier was prepared. After the carrier was treated with CuCl2 or NiCl2 papain was immobilized on this carrier. Papain can bind on this carrier tightly, and papain can be washed from the carrier when papain loses activity after being used many times. The carrier can be regenerate by treated with CuCl2 or NiCl2, and can absorb papain again. The enzyme immobilized on the carrier which was used three times have 90% activity of the first time. So this microsphere can be as a good regenerable carrier. The result indicates metal chelate carrier will be a promising enzyme carrier.
产物和底物分离等优点而被广泛深入的研究。但目前常用的固定化方
法在制备固定化酶时酶活损失较大，且载体常常不可重复使用或载体
的再生操作复杂，限制了固定化酶的产业化应用。本文针对目前固定
化酶方法的不足之处提出了相应对策并对新的酶固定化方法和载体
进行了探索。
本文选用木瓜蛋白酶作为固定化对象，木瓜蛋白酶是一种来源广
泛的巯醇类植物蛋白酶，在食品、轻纺、医学等领域有着广泛的应用，
木瓜蛋白酶在酶动力学和结构学上都已得到了深入的研究，因此很适
合作为评测酶固定化方法效率的模式酶。
采用正硅酸乙酯与N-(β-氨乙基)一氨丙基三乙氧基硅烷在油包
水形成的微胶囊中同步水解的方法，一步法制备了氨基化的二氧化硅
颗粒，这种颗粒大小均匀，氨基含量和颗粒大小可控，氨基含量高达
5． 6mmol／g。此颗粒经戊二醛处理后，采用共价法固定木瓜蛋白酶，
固定化最适pH6． 5，最佳给酶量为15mg／g载体，固定化酶的最适反应
温度为70℃，最适反应pH为6． 5，固定化酶热稳定性，pH耐受性，
贮存稳定性都明显高于游离酶，表明此颗粒可作为一种优良的酶固定
化载体。
本文采用了反相悬浮包埋合成磁性琼脂糖微球，采用悬浮聚合法
合成苯乙烯一甲基丙烯酸羟乙酯，经环氧氯丙烷和亚氨基二乙酸处理
使微球带上螯合基团，经吸附铜离子或镍离子后用于固定化木瓜蛋白
酶。实验的初步结果显示，磁性琼脂糖微球可用于固定化木瓜蛋白酶，
并且经洗脱液洗脱，酶可以从载体上洗脱下来，载体经再吸附金属离
子后可以再生，并可用于再次固定化木瓜蛋白酶，载体重复使用3次
后酶活仍达首次时的90％。初步实验结果表离子螯合载体可用于固定
化木瓜蛋白酶，并作为可再生载体经多次使用。
The immobilized enzymes have more stability, more use times and easier separation from substrate and product than enzymes in bulk solution. But current immobilization methods have many disadvantages, such as much enzyme activity lose after immobilization and high cost. So only a few immobilized enzymes have been used in industry widely though enzyme immobilization have been studied for about thirty years. In order to resolve these disadvantages, new enzyme carriers and immobilization methods need to be explored. In this paper we put forward some solutions and synthesized several kinds of new enzyme carriers.
We choose papain as immobilized object, and investigate performance of these carriers used as papain carrier. Papain, a thiol protease, have great industrial and medicinal potential, and is well characterized kinetically and structurally being a suitable model to compare the efficiency of various immobilization procedures and methods.
Among present enzyme carriers inorganic materials have lower cost and more stability than organic materials. But the present inorganic materials contain less active sites, and have to undergo some complicated processes before immobilizing enzyme. SiO2 particles containing amine groups were synthesized by synchronous hydrolysis of tetraethylorthosilicate(TEOS) and N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAPS) in W/O Microemulsion of TritonX-100/cyclohexane/ammonium hydroxide system. The obtained particles have a narrow diameter range, and its size can be controlled by adjusting water to surfactant molar ratio and water to TEOS and AEAPS molar ratio. Comparing with traditional porous silica beads, these panicles contain much more amine groups and its amine
group content can be easily changed when synthesizing. After treated by
glutaraldehyde, papain was immobilized on the particles by covalent method. The optimum immobilization conditions of enzyme were as follows: enzyme load was 15mg/g carrier, pH was 6.5.The pH and temperature optima were 6.5 and 70 C for immobilization enzyme. All above indicate this kind of particle can be a good enzyme immobilization carrier.
The reverse phase suspension and embedment technique were adopted to prepare magnetic agar microsphere. The dispersion medium was mixture of some organic solvents. Span-80 was used as stabilizer. The magnetic agar microsphere was coupled with epichlorohydrin, then treated with iminodiacetic acid, and magnetic metal chelate carrier was prepared. After the carrier was treated with CuCl2 or NiCl2 papain was immobilized on this carrier. Papain can bind on this carrier tightly, and papain can be washed from the carrier when papain loses activity after being used many times. The carrier can be regenerate by treated with CuCl2 or NiCl2, and can absorb papain again. The enzyme immobilized on the carrier which was used three times have 90% activity of the first time. So this microsphere can be as a good regenerable carrier. The result indicates metal chelate carrier will be a promising enzyme carrier.
引文
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