In order to prepare theaflavin more efficiently and economically, catechins were used as the cross-linking reagent in this study, and a polyphenol oxidase (tyrosinase) crossed-linked aggregate derived from Bacillus megaterium was prepared and used for the synthesis of theaflavin-3,3ʹ-digalate. Through optimization of enzyme cross-linking parameters and the catalytic performance before and after cross-linking, the results show that the optimal enzyme activity recovery rate of cross-linked enzyme (200 U·mL-1) could be obtained at pH 4.0, EGCG 0.5 mg·mL-1 and cross-linking time 50 min. Compared with free enzymes, cross-linked enzymes showed better catalytic performance (thermal stability, organic solvent tolerance, substrate tolerance). When the cross-linked enzyme was used to synthesize theaflavin-3,3′-digallate, the concentration of the product could reach 800 μg·mL-1, and the cross-linked enzyme could be reused for at least three batches. This method could significantly reduce the application cost of theaflavin, which has potential industrial application value.
ZHOU Jinghui
,
LIU Changwei
,
ZHANG Sheng
,
XU Gang
,
XU Wei
,
HUANG Jian′an
,
LIU Zhonghua
. Preparation of a Cross-linked Polyphenol Oxidase Aggregate Based on Catechins and Its Efficient Catalytic Synthesis of Theaflavin-3,3ʹ-digalate[J]. Journal of Tea Science, 2023
, 43(3)
: 377
-388
.
DOI: 10.13305/j.cnki.jts.2023.03.005
[1] Takemoto M, Takemoto H.Synthesis of theaflavins and their functions[J]. Molecules, 2018, 23(4): 918. doi: 10.3390/molecules23040918.
[2] He H F.Research progress on theaflavins: efficacy, formation, and preparation[J]. Food & Nutrition Research, 2017, 61(1): 1344521. doi: 10.1080/16546628.2017.1344521.
[3] Zhou H, Xia C, Yang Y, et al.The prevention role of theaflavin-3,3'-digallate in angiotensin II induced pathological cardiac hypertrophy via CaN-NFAT signal pathway[J]. Nutrients, 2022, 14(7): 1391. doi: 10.3390/nu14071391.
[4] O'Neill E J, Termini D, Albano A, et al. Anti-cancer properties of theaflavins[J]. Molecules, 2021, 26(4): 987. doi: 10.3390/molecules26040987.
[5] Pan H, Wang F, Rankin G O, et al.Inhibitory effect of black tea pigments, theaflavin-3,3'-gallate against cisplatin-resistant ovarian cancer cells by inducing apoptosis and G1 cell cycle arrest[J]. International Journal of Oncology, 2017, 51(5): 1508-1520.
[6] Ramadan G, EI-Beih N M, Talaat R M, et al. Anti-inflammatory activity of green versus black tea aqueous extract in a rat model of human rheumatoid arthritis[J]. International Journal of Rheumatic Diseases, 2017, 20(2): 203-213.
[7] Peluso I, Serafini M.Antioxidants from black and green tea: from dietary modulation of oxidative stress to pharmacological mechanisms[J]. British Journal of Pharmacology, 2017, 174(11): 1195-1208.
[8] Ge G, Yang S, Hou Z Y, et al.Theaflavin-3,3'-digallate promotes the formation of osteoblasts under inflammatory environment and increases the bone mass of ovariectomized mice[J]. Frontiers in Pharmacology, 2021, 12: 648969. doi: 10.3389/fphar.2021.648969.
[9] Luo T, Jiang J G.Anticancer effects and molecular target of theaflavins from black tea fermentation in vitro and in vivo[J]. Journal of Agricultural and Food Chemistry, 2021, 69(50): 15052-15065.
[10] Lei S, Xie M, Hu B, et al.Effective synthesis of theaflavin-3,3'-digallate with epigallocatechin-3-O-gallate and epicatechin gallate as substrates by using immobilized pear polyphenol oxidase[J]. International Journal of Biological Macromolecules, 2017, 94: 709-718.
[11] Cao L, Van Rantwijk F, Sheldon R A.Cross-linked enzyme aggregates: a simple and effective method for the immobilization of penicillin acylase[J]. Organic Letters, 2000, 2(10): 1361-1364.
[12] Blanco-Llamero C, García-García P, Señoráns F J.Cross-linked enzyme aggregates and their application in enzymatic pretreatment of microalgae: comparison between CLEAs and combi-CLEAs[J]. Frontiers in Bioengineering and Biotechnology, 2021, 9: 794672. doi: 10.3389/fbioe.2021.794672.
[13] Chen N, Chang B, Shi N, et al.Cross-linked enzyme aggregates immobilization: preparation, characterization, and applications[J]. Critical Reviews in Biotechnology, 2022: 1-15. doi: 10.1080/07388551.2022.2038073.
[14] Ba S, Haroune L, Cruz-morató C, et al. Synthesis and characterization of combined cross-linked laccase and tyrosinase aggregates transforming acetaminophen as a model phenolic compound in wastewaters[J]. The Science of the Total Environment, 2014, 487: 748-755.
[15] Diaz-Vidal T, Armenta-Perez V P, Rosales-Rivera L C, et al. Cross-linked enzyme aggregates of recombinant Candida antarctica lipase B for the efficient synthesis of olvanil, a nonpungent capsaicin analogue[J]. Biotechnology Progress, 2019, 35(4): e2807. doi: 10.1002/btpr.2807.
[16] Muley A B, Awasthi S, Bhalerao P P, et al.Preparation of cross-linked enzyme aggregates of lipase from aspergillus niger: process optimization, characterization, stability, and application for epoxidation of lemongrass oil[J]. Bioprocess and Biosystems Engineering, 2021, 44(7): 1383-1404.
[17] Xu D Y, Yang Y, Yang Z.Activity and stability of cross-linked tyrosinase aggregates in aqueous and nonaqueous media[J]. Journal of Biotechnology, 2011, 152(1/2): 30-36.
[18] Yang Z, Chen J Y, Xu D Y.Use of cross-linked tyrosinase aggregates as catalyst for synthesis of L-DOPA[J]. Biochemical Engineering Journal, 2012, 63: 88-94.
[19] Quan T H, Benjakul S, Sae-Leaw T, et al.Protein-polyphenol conjugates: antioxidant property, functionalities and their applications[J]. Trends in Food Science & Technology, 2019, 91: 507-517.
[20] Frazier R A, Deaville E R, Green R J, et al.Interactions of tea tannins and condensed tannins with proteins[J]. Journal of Pharmaceutical and Biomedical Analysis, 2010, 51(2): 490-495.
[21] Czubinski J, Dwiecki K.A review of methods used for investigation of protein-phenolic compound interactions[J]. International Journal of Food Science & Technology, 2017, 52(3): 573-585.
[22] Liu H, Han G, Zhang H, et al.Improving the physical and oxidative stability of emulsions based on the interfacial electrostatic effects between porcine bone protein hydrolysates and porcine bone protein hydrolysate-rutin conjugates[J]. Food Hydrocolloids, 2019, 94: 418-427. doi: 10.1016/j.foodhyd.2019.03.037.
[23] Zhou J, Liu C, Zhao S, et al.Improved yield of theaflavin-3,3'-digallate from Bacillus megaterium tyrosinase via directed evolution[J]. Food Chemistry, 2022, 375: 131848. doi: 10.1016/j.foodchem.2021.131848.
[24] Goldfeder M, Egozy M, Shuster Ben-Yosef V, et al. Changes in tyrosinase specificity by ionic liquids and sodium dodecyl sulfate[J]. Applied Microbiology and Biotechnology, 2013, 97(5): 1953-1961.
[25] Schoevaart R, Wolbers M W, Golubovic M, et al.Preparation, optimization, and structures of cross-linked enzyme aggregates (CLEAs)[J]. Biotechnology and Bioengineering, 2004, 87(6): 754-762.
浙公网安备 33019902000101号 
