Cloning, expression and enzymatic characterization of recombinant eugenol oxidase (EUGO) in Escherichia coli

Authors

  • Pham Thi My Binh VNUHCM - University of Science
  • Le Hai Yen
  • Tran Quoc Tuan
  • Nguyen Thi Hong Thuong

DOI:

https://doi.org/10.15625/1811-4989/17/3/13244

Keywords:

Eugenol oxidase, E. coli, pET-28a-EUGO, vanillin, immobilized−metal affinity chromatography

Abstract

Eugenol oxidase (EUGO), a member of the vanillyl alcohol oxidase family, catalyzes the oxidative reaction of vanillyl alcohol to vanillin. This compound is responsible for the vanilla aroma and is widely used as a flavoring agent in food, cosmetics, and pharmaceuticals. Previously, EUGO was cloned and expressed in E. coli TOP10, and purified by anion-exchange chromatography with Q-Sepharose resin but the purification factor was low. To improve the efficiency of the EUGO purification, in this study, we cloned eugo gene into pET-28a vector and expressed it in E. coli Tunetta. The SDS-PAGE analysis of protein extracts obtained from E. coli expressing EUGO under different induction conditions showed that EUGO was expressed mostly in the soluble fraction at 6 hours after induction with 0.1 mM IPTG at 25oC. EUGO was purified by immobilized−metal affinity chromatography with Ni2+-NTA agarose and the in vitro enzymatic activity was characterized. The specific activity of purified EUGO was nearly 4-fold higher than that of the crude enzyme sample. In particular, the enzyme preparation produced by the purification method based on Ni-NTA affinity in this study was 2,5-fold more pure than that produced by Q-sepharose purification method described previously.

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Published

2020-11-28

Issue

Section

Articles