树脂固定化多酚氧化酶及其催化茶多酚形成茶黄素的研究
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摘要
酶固定化技术是20世纪60年代兴起的,在21世纪初期得到长足发展的一项高新生物技术工程。在酶固定化技术中,固定化载体对酶固定化后的酶活性高低,以及固定化酶的应用方面有着显著的影响。本实验综合固定化酶活性的高低和固定化酶的应用等因素考虑,以树脂为载体来固定化多酚氧化酶,并对固定化多酚氧化酶的性质,以及固定化多酚氧化酶催化茶多酚形成茶黄素开展了研究。
1对不同孔径,材质和极性的大孔吸附树脂和离子交换树脂载体进行了筛选,实验结果表明,在对多酚氧化酶的固定化效果方面,阳离子交换树脂载体优于大孔吸附树脂载体和阴离子交换树脂载体。在阳离子交换树脂载体中,又以树脂载体D152效果最理想。
2对树脂载体D152固定化多酚氧化酶的条件进行了优化,得到最优固定化条件:10.00g经处理的树脂载体D152,加入0.2mg/ml的多酚氧化酶溶液50ml,在4℃下,120rpm下吸附固定化2h,之后,加入0.080ml 25%戊二醛溶液, 120rpm下交联1h。
3对固定化多酚氧化酶的性质进行了研究。(1)固定化多酚氧化酶的Km值为0.113mg/ml,较SPPO增大了17.6%;(2)4℃下多酚氧化酶的半衰期由17.2天增加到52.5天,提高3倍;(3)固定化多酚氧化酶具有较好的重复使用稳定性,重复使用20次后,酶活力保留40%以上;(4)固定化多酚氧化酶的最适pH较多酚氧化酶向碱移1个单位,最适反应温度提高5℃;(5)固定化多酚氧化酶在有机溶剂中的稳定性明显优于游离态多酚氧化酶;(6)固定化酶多酚氧化酶对金属离子的抗性较游离多酚氧化酶没有明显的变化;(7)固定化酶多酚氧化酶柱具有很好的淋洗稳定性。
4将固定化多酚氧化酶应用于茶黄素的制备中,对实验主要影响因素:温度(X1),pH(X2),底物浓度(X3),反应时间(X4)和通氧量(X5),采用正交实验设计处理,对实验结果进行多项式回归方程拟合,得到多项式回归方程:Y=-0.4366+0.0186X1+0.1065X2-0.1121X3+3.6350X5-0.0002X1*X1 -0.0034X3*X3+0.0003X4*X4+0.4025X5*X5-0.0034X1*X2+0.0034X1*X3-0.0623X1*X5+0.0149X2*X3-0.0012X3*X4-0.0328X4*X5,R2=0.9998,结合多项式回归方程,对整个实验因素的最优方案进行优选,得出最佳实验处理组合:温度=64.6℃,pH=6.57,底物浓度=11.53mg/ml,反应时间=59.7min,通氧量=0.10L/min,将此组合代入回归方程,得到Y=1.03mg/ml。以以上因素水平组合,进行实验检验,得到茶黄素产量(0.90±0.06)mg/ml,验证准确率达到87.46%。
Immobilized enzyme technology is an advance bioengineering, it was rised at 1960s,and has developmented greatly 21st century. At enzyme immobilizing, immobilized carrier has significantly effection in activity of immobilization enzymed and efficiency of enzyme immobilized.This research consider comprehensively activity and application of immobilization enzyme, using resin as carrier immobilizes polyphenol oxidase, and researching on immobilization polyphenol oxidase, as well as using it catalytic oxidative tea polyphenol formation theaflavins.
1 Accord to activity and efficiency of enzyme immobilized, researched different aperture, materials and polarity of macroporous adsorption resin and ion exchange resin.Experimental results show that cation exchange resins better than macroporous adsorption resin and anion exchange resin in immobilizes polyphenol oxidase action.Among of cation exchange resins, D152 resin is the best.
2 Researched the optimum action conditions of D152 resin carrier immobilized polyphenol oxidase,obtained the optimum action conditions:10.00g pretreatmented D152 resin,adding in 50ml 0.2mg/ml polyphenol oxidase solution, 120 rotates per minute adsorptive-immobilized 2 hours at 4℃,then adding in 0.080ml 25% glutaraldehyde solution crosslinking action 1 hour at 4℃120 rotates per minute.
3 Researched the properties of immobiliztion polyphenol oxidase.(1)The Km of immobilization polyphenol oxidase is 0.113mg/ml, it has increased 17.6% compare with SPPO;(2)The half-life of polyphenol oxidase from 17.2 days increased to 52.5 days,increasing to 300 precent; (3)Immobilized polyphenol oxidase has well stability of reuse,reusing 20 times,then activity retentives 40%; (4)The optimum pH of polyphenol oxidase moves to alkali one unit, The optimum temperature increases 5℃;(5)The stability in organic solvent of immobilizatio n polyphenol oxidase better than solution polyphenol oxidase;(6)The resistance of metal ions hasn’t significantly change about immobilization polyphenol oxidase and solution polyphenol oxidase;(7)The immobilization polyphenol oxidase column has well stability.
4 Applied immobilization polyphenol oxidase to preparing theaflavins.Using orthogonal test treatment considering the main effect factors of IPPO catalytics TP forming TFs as temperature(X1),pH(X2), contents of substrate(X3),action time(X4) and speed of adding oxygen(X5).Using the experiment results anlysis obtaining polynomial regression equation: Y=-0.4366+0.0186X1+0.1065X2-0.1121X3 +3.6350X5-0.0002X1*X1-0.0034X3*X3+0.0003X4*X4+0.4025X5*X5-0.0034X1*X2+0.0034X1*X3-0.0623X1*X5+0.0149X2*X3-0.0012X3*X4-0.0328X4*X5,R2=0.9998.By rhe regression equation, obtaining the optimum experiment conditions as X1=64.6℃, X2=6.57, X3=11.53mg/ml, X4=59.7min, X5=0.10L/min,the theoretical value of Y is 1.03mg/ml. Design experiment as the optimum conditions,getting the observation value of Y is (0.90±0.06)mg/ml,,the accuracy of authentication is 87.46%.
1对不同孔径,材质和极性的大孔吸附树脂和离子交换树脂载体进行了筛选,实验结果表明,在对多酚氧化酶的固定化效果方面,阳离子交换树脂载体优于大孔吸附树脂载体和阴离子交换树脂载体。在阳离子交换树脂载体中,又以树脂载体D152效果最理想。
2对树脂载体D152固定化多酚氧化酶的条件进行了优化,得到最优固定化条件:10.00g经处理的树脂载体D152,加入0.2mg/ml的多酚氧化酶溶液50ml,在4℃下,120rpm下吸附固定化2h,之后,加入0.080ml 25%戊二醛溶液, 120rpm下交联1h。
3对固定化多酚氧化酶的性质进行了研究。(1)固定化多酚氧化酶的Km值为0.113mg/ml,较SPPO增大了17.6%;(2)4℃下多酚氧化酶的半衰期由17.2天增加到52.5天,提高3倍;(3)固定化多酚氧化酶具有较好的重复使用稳定性,重复使用20次后,酶活力保留40%以上;(4)固定化多酚氧化酶的最适pH较多酚氧化酶向碱移1个单位,最适反应温度提高5℃;(5)固定化多酚氧化酶在有机溶剂中的稳定性明显优于游离态多酚氧化酶;(6)固定化酶多酚氧化酶对金属离子的抗性较游离多酚氧化酶没有明显的变化;(7)固定化酶多酚氧化酶柱具有很好的淋洗稳定性。
4将固定化多酚氧化酶应用于茶黄素的制备中,对实验主要影响因素:温度(X1),pH(X2),底物浓度(X3),反应时间(X4)和通氧量(X5),采用正交实验设计处理,对实验结果进行多项式回归方程拟合,得到多项式回归方程:Y=-0.4366+0.0186X1+0.1065X2-0.1121X3+3.6350X5-0.0002X1*X1 -0.0034X3*X3+0.0003X4*X4+0.4025X5*X5-0.0034X1*X2+0.0034X1*X3-0.0623X1*X5+0.0149X2*X3-0.0012X3*X4-0.0328X4*X5,R2=0.9998,结合多项式回归方程,对整个实验因素的最优方案进行优选,得出最佳实验处理组合:温度=64.6℃,pH=6.57,底物浓度=11.53mg/ml,反应时间=59.7min,通氧量=0.10L/min,将此组合代入回归方程,得到Y=1.03mg/ml。以以上因素水平组合,进行实验检验,得到茶黄素产量(0.90±0.06)mg/ml,验证准确率达到87.46%。
Immobilized enzyme technology is an advance bioengineering, it was rised at 1960s,and has developmented greatly 21st century. At enzyme immobilizing, immobilized carrier has significantly effection in activity of immobilization enzymed and efficiency of enzyme immobilized.This research consider comprehensively activity and application of immobilization enzyme, using resin as carrier immobilizes polyphenol oxidase, and researching on immobilization polyphenol oxidase, as well as using it catalytic oxidative tea polyphenol formation theaflavins.
1 Accord to activity and efficiency of enzyme immobilized, researched different aperture, materials and polarity of macroporous adsorption resin and ion exchange resin.Experimental results show that cation exchange resins better than macroporous adsorption resin and anion exchange resin in immobilizes polyphenol oxidase action.Among of cation exchange resins, D152 resin is the best.
2 Researched the optimum action conditions of D152 resin carrier immobilized polyphenol oxidase,obtained the optimum action conditions:10.00g pretreatmented D152 resin,adding in 50ml 0.2mg/ml polyphenol oxidase solution, 120 rotates per minute adsorptive-immobilized 2 hours at 4℃,then adding in 0.080ml 25% glutaraldehyde solution crosslinking action 1 hour at 4℃120 rotates per minute.
3 Researched the properties of immobiliztion polyphenol oxidase.(1)The Km of immobilization polyphenol oxidase is 0.113mg/ml, it has increased 17.6% compare with SPPO;(2)The half-life of polyphenol oxidase from 17.2 days increased to 52.5 days,increasing to 300 precent; (3)Immobilized polyphenol oxidase has well stability of reuse,reusing 20 times,then activity retentives 40%; (4)The optimum pH of polyphenol oxidase moves to alkali one unit, The optimum temperature increases 5℃;(5)The stability in organic solvent of immobilizatio n polyphenol oxidase better than solution polyphenol oxidase;(6)The resistance of metal ions hasn’t significantly change about immobilization polyphenol oxidase and solution polyphenol oxidase;(7)The immobilization polyphenol oxidase column has well stability.
4 Applied immobilization polyphenol oxidase to preparing theaflavins.Using orthogonal test treatment considering the main effect factors of IPPO catalytics TP forming TFs as temperature(X1),pH(X2), contents of substrate(X3),action time(X4) and speed of adding oxygen(X5).Using the experiment results anlysis obtaining polynomial regression equation: Y=-0.4366+0.0186X1+0.1065X2-0.1121X3 +3.6350X5-0.0002X1*X1-0.0034X3*X3+0.0003X4*X4+0.4025X5*X5-0.0034X1*X2+0.0034X1*X3-0.0623X1*X5+0.0149X2*X3-0.0012X3*X4-0.0328X4*X5,R2=0.9998.By rhe regression equation, obtaining the optimum experiment conditions as X1=64.6℃, X2=6.57, X3=11.53mg/ml, X4=59.7min, X5=0.10L/min,the theoretical value of Y is 1.03mg/ml. Design experiment as the optimum conditions,getting the observation value of Y is (0.90±0.06)mg/ml,,the accuracy of authentication is 87.46%.
引文
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