Immobilization Of Mushroom Tyrosinase And Its Application In The Synthesis Of Polyphenol Compounds

Polyphenol compounds have received extensive attention in development of pharmaceuticals and cosmetic products as well as health and functional foods,mainly due to the recognition of their antioxidant properties such as being anti-inflammation,anti-tumor,anti-cardiovascular and anti-aging.Organic-inorganic hybrid nanomaterials are new nanoscaled materials self-assembled from metal ions(such as cupper)and organic components(such as proteins).They have multi-layered structure and large specific surface area,and can be used as supports or carriers for enzyme immobilization with many advantages.Enzymes can also be immobilized by forming cross-linked enzyme aggregates(CLEAs),which combine purification and immobilization into a single operation.This immobilization technique has been applied to many fields.Tyrosinase is a copper-containing oxidoreductase responsible for the catalysis of the strict ortho-hydroxylation of monophenols to o-diphenols and their subsequent oxidation to o-quinones.There have been some studies on tyrosinase CLEAs and their applications(mostly from our laboratory),but the use of organic-inorganic hybrid nanomaterials(i.e.,the nanoflower immobilization technology)to immobilize tyrosinase has not been reported so far.In this paper,for the first time the nanoflower immobilization technology was utilized to immobilize mushroom tyrosinase in the form of tyrosinase-Cu₃(PO₄)₂hybrid composites(TCHCs).TCHCs were successfully used as catalyst for the synthesis of catechol-functionalized polyphenol products such as L-3,4-dihydroxyphenylalanine(L-DOPA)and piceatannol.In order to enhance their operational stability,TCHCs were further entrapped into alginate gels,which were demonstrated to exhibit excellent recyclability and storage stability.Furthermore,tyrosinase CLEAs were also used as catalysts for synthesis of 3'-hydroxypterostilbene.The major contents are given below:1.Preparation and characterization of TCHCsTCHCs were prepared by using crude extract from fresh mushrooms and the preparation conditions were optimized through response surface methodology(RSM).Energy dispersive X-ray diffraction spectroscopy confirmed that the enzyme was successfully immobilized on copper phosphate.Scanning electron microscopy(SEM)showed that the immobilized enzyme were spherical beads with a porous and fluffy surface.The highly porous structure is beneficial,enabling substrate molecules to easily get access to the enzyme for the reaction to proceed.TCHC retained high activity and stability even under extreme conditions.2.TCHC-catalyzed synthesis of polyphenol compoundsTCHCs was used as a catalyst for the synthesis of L-DOPA and piceatannol.Formation of the products was confirmed by both HPLC and TLC.The impacts of reaction conditions,such as enzyme dosage,reaction temperature,pH,concentrations of L-ascorbic acid and ethylenediaminetetraacetic acid(EDTA),were investigated.The optimal reaction conditions for the two synthetic reactions were determined.Under the optimal reaction conditions,L-DOPA and piceatannol were produced with excellent productivities(254.4 mg/L/h and 1923.4 mg/L/h,respectively),which are the highest as compared to the data reported in literature.Our experiments have thus demonstrated that TCHCs can selectively and efficiently catalyze the synthesis of polyphenol compounds.3.Enhanced stability of TCHCs via entrapment into Cu alginate gelsIn order to enhance their operational stability,TCHCs were encapsulated into Cu alginate gels.The TCHC-alginate beads maintained their original activity for over 1month when stored at 30? and retained 78.6%of the original activity after reaction for 10 cycles.The porous structure of TCHCs were also retained well as shown by SEM.This clearly demonstrates that entrapment into Cu alginate gels can significantly enhance the robustness and operability of TCHCs.4.CLEA-catalyzed synthesis of 3'-hydroxypterostilbeneEnzymatic synthesis of 3'-hydroxypterostilbene was explored,with tyrosinase immobilized as cross-linked enzyme aggregates(CLEA)as the catalyst.RSM was employed to optimize the reaction conditions.Finally,a high yield of 77.9%was obtained,which is also obviously much higher than the one obtained by chemical synthesis.This is the first study of biocatalytic production of 3?-hydroxypterostilbene,successfully demonstrating the feasibility of utilizing tyrosinase CLEAs as catalyst for ortho-hydroxylation to produce 3?-hydroxypterostilbene.In summary,mushroom tyrosinase immobilized by using the above two technologies presented excellent catalytic properties and can be used as efficient catalysts for the synthesis of catechol-functionalized polyphenol products.Compared to the CLEA methodology,the nanoflower immobilization technology shows the advantages of higher enzyme activity and simpler preparation conditions.The operability of TCHCs can be significantly enhanced by encapsulating them into copper alginate gels.