Biomedical and Biotechnology Research Journal (Jan 2023)

Purification of laccase from the fungus Clitopilus prunulus BV18 and its application in bioconversion of tea polyphenols to bioactive theabrownins

  • Dinh Giap Vu,
  • Nhat Minh Dang,
  • Phuong Dai Nguyen Nguyen,
  • Thi Dung Ha,
  • Vinh The Mac

DOI
https://doi.org/10.4103/bbrj.bbrj_266_23
Journal volume & issue
Vol. 7, no. 4
pp. 633 – 640

Abstract

Read online

Background: Theabrownins (TBs), which are produced from tea leaves, have been shown to have biological. To extract biologically active chemicals from polyphenol structures, an oxidizing enzyme (i.e. laccase) is used to oxidize the polyphenol structure, releasing smaller and more soluble molecular compounds, including TBs. This is a biologically active compound that is beneficial to human health. Therefore, using laccase to purify is significant in enhancing the oxidation process of polyphenols from fresh tea leaves to improve the efficiency of harvesting theabrownins. Methods: to purify laccase, we use a diethylaminoethyl-cellulose (DEAE), Sephadex G-75, and Q Sepharose® columns in a three-step column chromatography procedure. In addition, TBs formed by hydrolysis were determined by high-performance liquid chromatography equipped with a refractive index detector (ultra violet-Vis 271 nm) and ICSep WA-1 Analysis column. Results: In this study, CliLac was effectively purified with a specific activity of 7.58 U/mg from strain Clitopilus prunulus BV18. Using DEAE, Sephadex G-75, and Q Sepharose® columns in a three-step column chromatography procedure, the enzyme was purified to a level of 21.4-fold purity. The enzyme with a molecular weight of 55.2 kDa demonstrated increased pH stability in the acidic range. Biochemical properties of CliLac showed that the optimum pH and temperature were 5.0 and 50°C, respectively. The activity retention was over 80% at pH 5.0 for more than 7 h of incubation. CliLac denatured at temperatures over 60°C. TBs production release increased up to 159 ppm after 72 h of incubation using a purified CliLac (10 U/gds) at 50°C. Conclusions: The study indicates that CliLac is appropriate for oxidizing the polyphenol structure and releases the TBs, which are smaller and more soluble molecular compounds.

Keywords