非水介质中漆酶催化酚类化合物特性及产物抗氧化活性研究
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摘要
现代酶学研究发现,在非水介质中酶可能表现出较水介质中更高的催化活性并且具有全新的性质,有利于制备新型功能型化合物。本研究以白腐菌漆酶为试材,以咖啡酸和阿魏酸为模式底物,于10种有机溶剂的非水反应介质中进行体外酶促氧化试验。研究了不同非水介质对酶促氧化反应的影响规律,分析氧化产物的理化性质、稳定性及其抗氧化活性,进行了氧化产物的组分分析和质谱鉴定。得出如下结果:
(1)以咖啡酸和阿魏酸为底物,漆酶催化的最佳反应条件为:pH值分别5.15和4.85;温度均30℃;底物浓度分别8mmol/L和5mmol/L;酶浓度分别为0.05U/mL和0.025U/mL。
(2)以10种有机溶剂的不同混水比例二相介质反应得出,低体积分数的亲水溶剂和高体积分数的疏水溶剂对酶促反应具有促进的作用。漆酶催化咖啡酸的最适反应介质为40%甲醇,92%正己烷;催化咖啡酸的最适反应介质为40%甲醇,92%的异辛烷。
(3)咖啡酸和阿魏酸氧化产物的最大吸收波长分别为366nm和372nm。咖啡酸氧化产物具有较强的热稳定性,对光不敏感;阿魏酸氧化产物热稳定性差,对光敏感,应低温避光保存。
(4)用DPPH,FRAP和还原力测定法测定咖啡酸和阿魏酸及其氧化产物的抗氧化活性,结果表明:DPPH和FRAP法的测定结果基本一致,酚类底物在疏水介质中生成的氧化产物抗氧化活性相对较高,咖啡酸在92%的正己烷,阿魏酸在92%异辛烷中的产物活性最高。最大清除率可达到85.4%,IC_(50)最小值0.049mmol/L;还原力测定结果为酚类底物的抗氧化能力远远大于氧化产物。另外,咖啡酸及其氧化产物的抗氧化活性明显强于阿魏酸。
(5)对氧化产物进行HPLC及HPLC/MS分析,可知咖啡酸氧化生成4种产物,主要推测为咖啡酸二聚体(C_(18)H_(14)O_8),四聚体(C_(36)H_(28)O_(16))以及无法推测出结构的多聚体。阿魏酸氧化生成2种氧化产物,主要推测为阿魏酸二聚体(C_(20)H_(18)O_8)和三聚体(C_(30)H_(26)O_(12))。抗氧化活性高的产物中以低聚物为主。
Modern enzymology reveals that. enzyme in non-aqueous medium show very high catalytic activity and has new properties, such as products in non-aqueous media with strong thermal stability, storage stability and three-dimensional structure of selectivity , etc. For the synthesis of functional polymers, the non-aqueous media is conducive to the formation of phenolic oligomers with more stable and enhanced oxidation ability than the monomer, which can help to improve the laccase stability in practical application and catalytic preparation of new functional compounds.
In the study, 10 kinds of organic solvents with different water content were employed as reaction media to tests the activity of laccase catalyzed oxidation of phenolic compounds.. Stability and antioxidant activity, of end products were determinrd and, composition as well as structure were identified by HPLC,HPLC / MS analysis..Main results are as following:
(1) For model substrates of caffeic acid and ferulic acid, optimum reaction condition for Trametes versicolor laccase were determined as: pH5.15 and pH4.85, temperatures 30℃, substrate concentration of 8mmol/L and 5mmol/L, enzyme concentration of 0.05U/mL and 0.025U/mL,respectively.
(2) Different organic solvents with different fraction of water showed different role for laccase catalyzation. Enzyme activity were enhanced in either low volume fraction of hydrophilic solvent or high volume fraction of hydrophobic solvents. The most suitable reaction mediumof the laccase-catalyzed oxidations for caffeic acid and ferulic acid are 40% methanol, 92% hexane and 40% methanol, 92% isooctane.
(3) The maximum absorption wavelength of laccase-catalyzed end-product for caffeic acid and ferulic acid were 388,408 nm. Oxidation products of caffeic acid are stable in the light relatively and have a stronger heat resistance; Oxidation products of ferulic acid are poor thermal stability and sensitivity to light, and should be stored in cool and dark condition.
(4) Antioxidant activities of each substrate and their end product were valuated by through DPPH, FRAP and total reducing ability method. The results show that DPPH and FRAP method was consistent. The oxidation phenolic products generated in the hydrophobic medium owned relatively strong antioxidant activities, the maximum clearance rate could reach 85.4% and minimum IC_(50) is 0.049mmol / L, and reducing ability of phenolic substrate is much greater than the oxidation products. The antioxidant activity of caffeic acid and its Oxidation products was significantly stronger than that of ferulic acid, and it may be related to the number of phenolic hydroxyl groups and the degree of polymerization of a relationship.
(5) Results of HPLC and HPLC/MS anslysis thowed that caffeic acid generated 4 oxidation products, three of them were identifies as dimer (C_(18)H_(14)O_8), tetramer (C_(36)H_(28)O_(16)) and not enterpreted polymer. Ferulic acid generated 2 oxidation products, dimmer (C_(20)H_(18)O_8) and trimer (C_(30)H_(26)O_(12)). Different reaction media has an important influence on the degree of polymerization and formation of products. 92% n-hexane and isooctane is conducive to oxidation of caffeic acid and ferulic acid in the formation of highly reactive oligomers.
(1)以咖啡酸和阿魏酸为底物,漆酶催化的最佳反应条件为:pH值分别5.15和4.85;温度均30℃;底物浓度分别8mmol/L和5mmol/L;酶浓度分别为0.05U/mL和0.025U/mL。
(2)以10种有机溶剂的不同混水比例二相介质反应得出,低体积分数的亲水溶剂和高体积分数的疏水溶剂对酶促反应具有促进的作用。漆酶催化咖啡酸的最适反应介质为40%甲醇,92%正己烷;催化咖啡酸的最适反应介质为40%甲醇,92%的异辛烷。
(3)咖啡酸和阿魏酸氧化产物的最大吸收波长分别为366nm和372nm。咖啡酸氧化产物具有较强的热稳定性,对光不敏感;阿魏酸氧化产物热稳定性差,对光敏感,应低温避光保存。
(4)用DPPH,FRAP和还原力测定法测定咖啡酸和阿魏酸及其氧化产物的抗氧化活性,结果表明:DPPH和FRAP法的测定结果基本一致,酚类底物在疏水介质中生成的氧化产物抗氧化活性相对较高,咖啡酸在92%的正己烷,阿魏酸在92%异辛烷中的产物活性最高。最大清除率可达到85.4%,IC_(50)最小值0.049mmol/L;还原力测定结果为酚类底物的抗氧化能力远远大于氧化产物。另外,咖啡酸及其氧化产物的抗氧化活性明显强于阿魏酸。
(5)对氧化产物进行HPLC及HPLC/MS分析,可知咖啡酸氧化生成4种产物,主要推测为咖啡酸二聚体(C_(18)H_(14)O_8),四聚体(C_(36)H_(28)O_(16))以及无法推测出结构的多聚体。阿魏酸氧化生成2种氧化产物,主要推测为阿魏酸二聚体(C_(20)H_(18)O_8)和三聚体(C_(30)H_(26)O_(12))。抗氧化活性高的产物中以低聚物为主。
Modern enzymology reveals that. enzyme in non-aqueous medium show very high catalytic activity and has new properties, such as products in non-aqueous media with strong thermal stability, storage stability and three-dimensional structure of selectivity , etc. For the synthesis of functional polymers, the non-aqueous media is conducive to the formation of phenolic oligomers with more stable and enhanced oxidation ability than the monomer, which can help to improve the laccase stability in practical application and catalytic preparation of new functional compounds.
In the study, 10 kinds of organic solvents with different water content were employed as reaction media to tests the activity of laccase catalyzed oxidation of phenolic compounds.. Stability and antioxidant activity, of end products were determinrd and, composition as well as structure were identified by HPLC,HPLC / MS analysis..Main results are as following:
(1) For model substrates of caffeic acid and ferulic acid, optimum reaction condition for Trametes versicolor laccase were determined as: pH5.15 and pH4.85, temperatures 30℃, substrate concentration of 8mmol/L and 5mmol/L, enzyme concentration of 0.05U/mL and 0.025U/mL,respectively.
(2) Different organic solvents with different fraction of water showed different role for laccase catalyzation. Enzyme activity were enhanced in either low volume fraction of hydrophilic solvent or high volume fraction of hydrophobic solvents. The most suitable reaction mediumof the laccase-catalyzed oxidations for caffeic acid and ferulic acid are 40% methanol, 92% hexane and 40% methanol, 92% isooctane.
(3) The maximum absorption wavelength of laccase-catalyzed end-product for caffeic acid and ferulic acid were 388,408 nm. Oxidation products of caffeic acid are stable in the light relatively and have a stronger heat resistance; Oxidation products of ferulic acid are poor thermal stability and sensitivity to light, and should be stored in cool and dark condition.
(4) Antioxidant activities of each substrate and their end product were valuated by through DPPH, FRAP and total reducing ability method. The results show that DPPH and FRAP method was consistent. The oxidation phenolic products generated in the hydrophobic medium owned relatively strong antioxidant activities, the maximum clearance rate could reach 85.4% and minimum IC_(50) is 0.049mmol / L, and reducing ability of phenolic substrate is much greater than the oxidation products. The antioxidant activity of caffeic acid and its Oxidation products was significantly stronger than that of ferulic acid, and it may be related to the number of phenolic hydroxyl groups and the degree of polymerization of a relationship.
(5) Results of HPLC and HPLC/MS anslysis thowed that caffeic acid generated 4 oxidation products, three of them were identifies as dimer (C_(18)H_(14)O_8), tetramer (C_(36)H_(28)O_(16)) and not enterpreted polymer. Ferulic acid generated 2 oxidation products, dimmer (C_(20)H_(18)O_8) and trimer (C_(30)H_(26)O_(12)). Different reaction media has an important influence on the degree of polymerization and formation of products. 92% n-hexane and isooctane is conducive to oxidation of caffeic acid and ferulic acid in the formation of highly reactive oligomers.
引文
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