脂肪酶催化吡哆醇区域选择性酯化及其酶学特性研究
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摘要
设计了一套制备高纯度5-乙酰吡哆醇(5-AcPN)的酶催化反应体系,对产物进行了鉴定,通过对三种脂肪酶的筛选发现非水介质中Novozym 435催化吡哆醇酯化能力最强。为了改善吡哆醇溶解度并提高5-AcPN的产率,采用AOT反胶团、乙腈和离子液[bmim]PF6三种反应介质。发现离子液中的位置选择性不同于其它两种,在[bmim]PF6中吡哆醇的4-位羟基活性最高,而在乙腈和反胶团中5-位羟基活性最高。通过筛选,发现乙腈和AOT反胶团是此反应的潜在优良介质,不同的酰基供体不仅对转化率而且对区域选择率会产生重要影响。
在乙腈和AOT反胶团中,以乙酸乙烯酯为酰基供体进行Novozym 435催化吡哆醇酯化反应,系统的分析了水含量、温度、底物摩尔比以及酶载量等因素对反应的影响,确定系统的水含量是影响吡哆醇酯化的关键因素。利用响应面法对酶催化5-AcPN合成的反应条件进行了优化,建立了优化模型,并且验证了模型的有效性,确定了三套优化反应条件。结果表明,Novozym 435催化吡哆醇酯化的最大转化率可以达到99%,区域选择率为93%。利用优化条件成功的进行了放大试验,表明该技术具有潜在的工业应用价值。
以橄榄油水解为模型反应对Candida rugosa lipase (CRL)在新型CB-Span 85/Span 85/n-hexane反胶团中的活性特征进行了表征。结果表明,酶的加入导致了胶团流体力学半径的变化,Span 85和水分子之间发生了重排。发现足够的CB修饰密度对反胶团溶解水的能力和维持较高酶活具有重要意义,CB-Span 85反胶团中酶活与含水量(W0)之间的关系与AOT反胶团中不同。与水溶液中相比,CRL在反胶团中的最适pH保持不变,但其表观活性显著增加。动力学研究表明,CB-Span 85反胶团中酶催化橄榄油水解遵循米氏方程,表观米氏常数随反胶团浓度的增加而减小,说明酶与底物间的亲和力增加了。反胶团中CRL的稳定性与W0呈负相关性。
将CRL固定到磁性纳米载体上得到磁性固定化酶MIE,考察二糖对酶的保护作用。加入二糖后,MIE在干燥和储存过程中的稳定性增加了,其中海藻糖的保护作用最显著,乳糖其次,蔗糖最差,支持‘玻璃态’假说。采用两步失活动力学模型拟合了酶的失活过程,得到了失活速率常数和半衰期,结果加入海藻糖和乳糖之后,MIE的半衰期分别增长了31和23倍。
A lipase-catalyzed reaction system for the preparation of high-purity 5-O-acetylpyridoxine (5-AcPN) was designed and the products were identified. Three kinds of lipases were screened and Novozym 435 lipase (Candida antarctica lipase B) was found to be the best one to catalyze the esterification of pyridoxine (PN) in nonaqueous media. In order to improve the solubility of PN and to elevate the production of 5-AcPN, three reaction media, including AOT/n-hexane reversed micelles, acetonitrile and [bmim]PF6, were used in the reaction. It was found that the site-selectivity in [bmim]PF6 was different from that of the other two, i.e., the 4-OH of PN had the highest activity in [bmim]PF6, however, the 5-OH had the highest activity in acetonitrile and reversed micelles. Finally, acetonitrile and AOT reversed micelles were chosen as the potential media to perform the reaction. Different acyl donors affected significantly not only the degree of conversion but also the regioselectivity.
By employing vinyl acetate as acyl donor, Novozym 435-catalyzed esterification of PN was conducted in acetonitrile and AOT reversed micelles. The influence of several parameters such as water content, temperature, substrate mole ratio and enzyme loading on the reaction were analyzed systematically. The analysis confirmed that water content was the most significant factor affecting the esterification of PN. In addition, the response surface methodology (RSM) was used to optimize the reaction conditions for the enzymatic synthesis of 5-AcPN, and the best fitting models were established. The efficiency of the model was experimentally verified. Three sets of optimum reaction conditions were established. As a result, Novozym 435-catalyzed esterification of PN gave a maximum conversion of 99% and the regioselectivity of 93%. Besides, a scale-up experiment was successfully performed based on the optimized condition, indicating the industrial potentials of the technology.
The CB-Span 85/Span 85/ hexane reversed micellar system was characterized and evaluated by employing Candida rugosa lipase (CRL)-catalyzed hydrolysis of olive oil as a model reaction. The change of micellar hydrodynamic radius reflected the redistribution of Span 85 and water after enzyme addition. An adequate modification density of CB was found to be important for the reversed micelles to retain enough hydration capacity and achieve high enzyme activity. Compared with the results in AOT-based reversed micelles, CRL in this micellar system exhibited different activity behavior vs W0. The optimal pH of the encapsulated lipase kept unchanged, but the apparent activity was significantly higher than that of the enzyme in bulk solution. Kinetic studies indicated that the encapsulated lipase in the reversed micelles of CB-formulated Span 85 followed the Michaelis-Menten equation. The Michaelis constant decreased with increasing the micelles concentration, suggesting an increase of the enzyme affinity for the substrate. Stability of the lipase in the reversed micelles was negatively correlated to W0.
Magnetic immobilized enzyme, MIE, was prepared by immobilizing CRL onto magnetic nanoparticles. The protection of MIE by disaccharides was investigated. It was shown the stability of MIE increased during drying and storage in the presence of disaccharides. Furthermore, the preservation action of trehalose was the most efficient, secondly was lactose, and sucrose was the worst one, which supported the‘glassy state theory’.‘Two-step deactivation’model was employed to fit the deactivation kinetics of MIE. Also, the deactivation rate constant and the half-life were obtained. In the presence of trehalose and lactose, the MIE half-lives increased 31- and 23-fold, respectively.
在乙腈和AOT反胶团中,以乙酸乙烯酯为酰基供体进行Novozym 435催化吡哆醇酯化反应,系统的分析了水含量、温度、底物摩尔比以及酶载量等因素对反应的影响,确定系统的水含量是影响吡哆醇酯化的关键因素。利用响应面法对酶催化5-AcPN合成的反应条件进行了优化,建立了优化模型,并且验证了模型的有效性,确定了三套优化反应条件。结果表明,Novozym 435催化吡哆醇酯化的最大转化率可以达到99%,区域选择率为93%。利用优化条件成功的进行了放大试验,表明该技术具有潜在的工业应用价值。
以橄榄油水解为模型反应对Candida rugosa lipase (CRL)在新型CB-Span 85/Span 85/n-hexane反胶团中的活性特征进行了表征。结果表明,酶的加入导致了胶团流体力学半径的变化,Span 85和水分子之间发生了重排。发现足够的CB修饰密度对反胶团溶解水的能力和维持较高酶活具有重要意义,CB-Span 85反胶团中酶活与含水量(W0)之间的关系与AOT反胶团中不同。与水溶液中相比,CRL在反胶团中的最适pH保持不变,但其表观活性显著增加。动力学研究表明,CB-Span 85反胶团中酶催化橄榄油水解遵循米氏方程,表观米氏常数随反胶团浓度的增加而减小,说明酶与底物间的亲和力增加了。反胶团中CRL的稳定性与W0呈负相关性。
将CRL固定到磁性纳米载体上得到磁性固定化酶MIE,考察二糖对酶的保护作用。加入二糖后,MIE在干燥和储存过程中的稳定性增加了,其中海藻糖的保护作用最显著,乳糖其次,蔗糖最差,支持‘玻璃态’假说。采用两步失活动力学模型拟合了酶的失活过程,得到了失活速率常数和半衰期,结果加入海藻糖和乳糖之后,MIE的半衰期分别增长了31和23倍。
A lipase-catalyzed reaction system for the preparation of high-purity 5-O-acetylpyridoxine (5-AcPN) was designed and the products were identified. Three kinds of lipases were screened and Novozym 435 lipase (Candida antarctica lipase B) was found to be the best one to catalyze the esterification of pyridoxine (PN) in nonaqueous media. In order to improve the solubility of PN and to elevate the production of 5-AcPN, three reaction media, including AOT/n-hexane reversed micelles, acetonitrile and [bmim]PF6, were used in the reaction. It was found that the site-selectivity in [bmim]PF6 was different from that of the other two, i.e., the 4-OH of PN had the highest activity in [bmim]PF6, however, the 5-OH had the highest activity in acetonitrile and reversed micelles. Finally, acetonitrile and AOT reversed micelles were chosen as the potential media to perform the reaction. Different acyl donors affected significantly not only the degree of conversion but also the regioselectivity.
By employing vinyl acetate as acyl donor, Novozym 435-catalyzed esterification of PN was conducted in acetonitrile and AOT reversed micelles. The influence of several parameters such as water content, temperature, substrate mole ratio and enzyme loading on the reaction were analyzed systematically. The analysis confirmed that water content was the most significant factor affecting the esterification of PN. In addition, the response surface methodology (RSM) was used to optimize the reaction conditions for the enzymatic synthesis of 5-AcPN, and the best fitting models were established. The efficiency of the model was experimentally verified. Three sets of optimum reaction conditions were established. As a result, Novozym 435-catalyzed esterification of PN gave a maximum conversion of 99% and the regioselectivity of 93%. Besides, a scale-up experiment was successfully performed based on the optimized condition, indicating the industrial potentials of the technology.
The CB-Span 85/Span 85/ hexane reversed micellar system was characterized and evaluated by employing Candida rugosa lipase (CRL)-catalyzed hydrolysis of olive oil as a model reaction. The change of micellar hydrodynamic radius reflected the redistribution of Span 85 and water after enzyme addition. An adequate modification density of CB was found to be important for the reversed micelles to retain enough hydration capacity and achieve high enzyme activity. Compared with the results in AOT-based reversed micelles, CRL in this micellar system exhibited different activity behavior vs W0. The optimal pH of the encapsulated lipase kept unchanged, but the apparent activity was significantly higher than that of the enzyme in bulk solution. Kinetic studies indicated that the encapsulated lipase in the reversed micelles of CB-formulated Span 85 followed the Michaelis-Menten equation. The Michaelis constant decreased with increasing the micelles concentration, suggesting an increase of the enzyme affinity for the substrate. Stability of the lipase in the reversed micelles was negatively correlated to W0.
Magnetic immobilized enzyme, MIE, was prepared by immobilizing CRL onto magnetic nanoparticles. The protection of MIE by disaccharides was investigated. It was shown the stability of MIE increased during drying and storage in the presence of disaccharides. Furthermore, the preservation action of trehalose was the most efficient, secondly was lactose, and sucrose was the worst one, which supported the‘glassy state theory’.‘Two-step deactivation’model was employed to fit the deactivation kinetics of MIE. Also, the deactivation rate constant and the half-life were obtained. In the presence of trehalose and lactose, the MIE half-lives increased 31- and 23-fold, respectively.
引文
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