Study On Laccase Catalyzed Oxidation Of Phenolic Compounds In Non-Aqueous Media And Anti-Oxidation Activity Of Its End Products

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 IC₅₀ 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 (C₁₈H₁₄O₈), tetramer (C₃₆H₂₈O₁₆) and not enterpreted polymer. Ferulic acid generated 2 oxidation products, dimmer (C₂₀H₁₈O₈) and trimer (C₃₀H₂₆O₁₂). 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.