The Wood-rot Basidiomycete Candolleomyces eurysporus VAST02.52 Produces a Versatile Peroxidase that Catalyses the Biotransformation of α-Pinene to Release Verbenone

Abstract

Background: Versatile peroxidase (VP; EC 1.11.1.16) has recently emerged as a novel peroxidase highly valued in biotechnology due to its exceptional ability to oxidize a diverse range of substrates into value-added products. Objectives: To gain insight into the versatile peroxidase system from fungi, this study was designed to purify and characterize a CeuVP from the newly isolated fungus Candolleomyces eurysporus and to investigate its involvement in the biotransformation of plant α-pinene. Materials and methods: The fungal isolate was identified by analysis of the internal transcribed spacer region. VP-active protein was purified from liquid culture of C. eurysporus VAST02.52 by using fast protein liquid chromatography. The catalytic properties and stability of enzyme were studied for various substrates, different pH- and temperatures. Moreover, its involvement in the biotransformation of plant α-pinene was assessed by HPLC chromatography. Results: CeuVP was successfully purified with 27.3-fold purity and a 20.9% yield by using three steps of anion exchange and size exclusion chromatography. The molecular weight of CeuVP was determined to be 40 kDa by SDS-PAGE electrophoresis. This pure enzyme exhibited optimal activity at pH 4.0 and 35 °C. CeuVP showed the highest specific activity of 231.6 U/mg against ABTS, followed by Mn2+ (205.8 U/mg), veratryl alcohol (112.7 U/mg), cathecol (77.8 U/mg), p-aminophenol (68.9 U/mg), RBBR (56.2 U/mg), 4-methoxyphenol (31.1 U/mg), guaiacol (18.3 U/mg), and Reactive Black 5 (12.9 U/mg). Moreover, CeuVP catalyzed the oxidation of α-pinene derived from the essential oil of Eucalyptus robusta to release verbenone as the sole metabolite after a 7 h incubation. Conclusions: Versatile peroxidase (CeuVP) from C. eurysporus VAST02.52 exhibits the highest specific activity against ABTS while also catalyzing oxidation of various substrates tested. Moreover, this study indicated that CeuVP could be effective in the bioconversion of α-pinene into verbenone, a promising compound with numerous applications in the flavors and fragrances industry.