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.

Keywords

α-Pinene redox potential verbenone versatile peroxidase

Introduction

Due to their high production efficiency and simpler purification processes, it is estimated that over fifty percent of market enzymes used in various biotechnological and industrial sectors are derived from fungal sources.^1,2 In addition to several well-studied ligninolytic enzymes, ie, manganese peroxidase (MnP, EC 1.11.1.13), laccase (Lac, EC 1.10.3.2), and lignin peroxidase (LiP, EC 1.11.1.14),^3,4 versatile peroxidase (VP; EC 1.11.1.16) has been recently identified as a novel peroxidase with the catalytic characteristics of both LiP and MnP, therefore showcasing a wide substrate specificity.^5,6 Since the first VP was isolated from Pleurotus eryngii in 1996,⁷ numerous fungal VPs have been identified. Their capacity to catalyze the oxidization of aromatic compounds with high redox potential in the presence of H₂O₂ makes these enzymes highly valued in biotechnology.⁸

VP is highly valued in numerous industrial sectors for its capacity to oxidize a diverse range of substrates. For instance, VP from the fungus Lentinus squarrosulus has been shown to catalyze the conversion of lignocellulosic biomass into valuable products.⁹ Moreover, several fungal VPs have been reported as key oxidative enzymes in the degradation of environmental contaminants, ie, polycyclic aromatic hydrocarbons (PAHs), synthetic dyes, and lignin.^4,10-12 Recent studies have shown significant interest in the application of VP in the oxidation of α-pinene – a main component of plant essential oils and turpentine.^13-17 The products from the enzymatic conversion of α-pinene could be verbenol, verbenone, myrtenol, terpineol, etc, which are highly valuable compounds with various applications in, eg, insect repellent, insecticides, pharmaceuticals, and the flavors and fragrances industry.^14,17,18 White-rot fungal VPs from Ceriporia sp. ZLY-2010 and Stereum hirsutum were isolated and found to catalyze the conversion of α-pinene, primarily producing verbenone as the main metabolite. After 96 h, additional metabolites such as myrtenol, camphor, and isopinocarveol were also formed.¹⁵ Additionally, Gheorghita et al (2021) demonstrated that the utilization of a combination of versatile peroxidase and laccase significantly enhances the oxidation efficiency of α-pinene into verbenone up to 80% after a 5-h incubation time. This finding underscores the potential of using multiple ligninolytic enzymes in synergy to improve the efficiency of the biotransformation of α-pinene.¹⁴ Taken together, it is anticipated that the demand for versatile peroxidase will increase in the coming years as they have great potential as biocatalysts for various industrial processes.

Numerous fungal VPs have been studied, eg, Pleurotus eryngii, Pleurotus ostreatus, Lentinus squarrosulus, and Bjerkandera sp, etc.^6,9,19-21 Nevertheless, the VP from Candolleomyces eurysporus has not been reported yet so far. In this study, to gain further insight into the versatile peroxidase system from fungal, we purified and characterized a VP from the novel isolated fungus Candolleomyces eurysporus VAST02.52 (CeuVP) and its involvement in the biotransformation of plant α-pinene as well.

Materials and Methods

Molecular Identification and Phylogenetic Analysis

The fungus was isolated in Cúc Phương National Park (20.293500 N, 105.667528 E), Ninh Bình Province, Viet Nam and identified by analysis of the internal transcribed spacer region (ITS) amplified from the fungal genome. The Plant/Fungi DNA Isolation Kit from Norgenbiotek was used to extract genomic DNA according to the manufacturer's protocol. The ITS regions were amplified using primers of 5'-TCCGTAGGTGAACCTGCGG-3’ (ITS1) and 5'-TCCTCCGCTTATTGATATGC-3’ (ITS4). The PCR mixture (total volume of 25 µl) included 3 µl of DNA (deoxyribonucleic acid), 12 µl of 2x Master Mix from Thermo Fisher Scientific^TM (containing Taq DNA Polymerase and dNTPs), 1 µl of each primer (10 mM), and distilled water. The thermal cycling procedure began with an initial at 94 °C/5 min, followed by 35 cycles of denaturation at 95 °C/35 s, annealing at 58 °C/35 s, and extension at 72 °C/50 s. A final elongation step was performed at 72 °C/6 min. The PCR products were examined by 1.0% agarose gel electrophoresis and then directly sequenced using an ABI PRISM 3730 XL DNA Analyzer (Applied Biosystems, United States). The resulting sequences were analyzed with ChromasPro1.7.6 software and compared with the GenBank sequence database using the Basic Local Alignment Search Tool (BLAST). Construction of a phylogenetic tree with 1000 bootstrap replicates was implemented using the Maximum Likelihood (ML) method, performed by MEGA^® v11.0 software.²²

Fungal Cultivation and Enzyme Extraction

The fungal isolate was cultured on potato dextrose agar (PDA) with the supplement antibiotics (0.004% penicillin, nystatin) at 30 °C. The fungus was then routinely propagated and preserved at −20 °C in 25% glycerol.

For versatile peroxidase production, C. eurysporus VAST02.52 was grown in liquid cultures containing the following components (per liter): glucose 25 g, yeast extract 0.5 g, veratryl alcohol 0.6 g, CuSO₄.5H₂O 15 mg, KH₂PO₄ 50 mg, (NH₄)₂SO₄ 10 mg, MgSO₄ 25 mg, CaCl₂ 10 mg, FeSO₄.7H₂O 150 mg, MnSO₄.H₂O 25 mg. After incubation at 30 °C, the culture supernatant was collected on the day of maximum enzyme activity.

Enzyme Purification

The maximum activity of versatile peroxidase was gained from fungal cultures on the seventh day. The mycelium was removed by centrifugation at 250 rpm for 15 min and filtration through GF6-filter (Whatman, UK). The supernatant was collected and subsequently concentrated by 10 kDa cut-off ultrafiltration. Protein purification was performed using fast protein liquid chromatography (FPLC) with an ÄKTA Pure system (Cytiva, U.S.). Initially, proteins were added to a DEAE cellulose column that had been equilibrated with 20 mM sodium acetate (CH₃COONa) buffer at a pH of 5.5. The flow rate was set at a steady 1 mL/min, and the elution process involved a linear gradient from 0 to 1.5 M of NaCl concentration. Subsequently, VP-active fractions were pooled and loaded to a gel filtration, Sephadex G-100 column, equilibrated with acetate buffer at a pH of 5.5. Finally, elution on HiTrap^TM Q XL was performed with a 100 mM CH₃COONa buffer (pH 6.5) starting from 0–1.0 M of NaCl at a flow rate of 0.3 mL/min. The collected portions containing VP activity were pooled and ultra-filtrated (10 kDa cut-off) in the CH₃COONa buffer, pH 6.5 and preserved at −20 °C for future studies.

The UV/Vis spectra of the purified CeuVP were recorded over the wavelength range of 300 to 600 nm in both the oxidized and reduced states of the enzyme. These measurements were conducted using a Spark microplate reader (Tecan, Männedorf, Switzerland).

Enzyme Assay

Versatile peroxidase activity was assayed by measuring the increase in A₄₂₀ in a reaction mixture of 1 mL containing 5 mM ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)), 50 mM H₂O₂, 100 mM sodium tartrate buffer, pH 3.0, and an appropriate amount of CeuVP. The enzyme assay was performed during the first one min at 25 °C and one unit of VP activity was defined as the amount of enzyme needed to oxidize 1 µmol of substrate per minute (ε₄₂₀= 36000 M⁻¹ cm⁻¹).¹⁹

Biochemical Characterization

The purified enzyme's M_W was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 25 °C using a separating gel of 12% acrylamide (Tris-HCl buffer, pH 8.8) and a stacking gel of 5% acrylamide (Tris-HCl buffer, pH 6.8). All gels were stained with Coomassie brilliant blue (R-250), followed by fixing and destaining.

The optimal pH of CeuVP was evaluated in buffer systems ranging from pH 1.0 to 7.0 using designated buffers (100 mM): sodium-tartrate (Na₂C₄H₄O₆) buffer for pH 1.0–3.0; citric acid–Na₂HPO₄ buffer for pH 4.0–5.0; sodium phosphate (Na₃PO₄) buffer for pH 6.0–7.0. To determine the optimal temperature for CeuVP, enzyme activity was gauged between 20–55 °C, as outlined in the above section of the enzyme assay. Also, the pH stability of CeuVP was evaluated by comparing its remaining activity to that under optimal conditions. The CeuVP enzyme underwent incubation at different pHs 3.0, 5.0, and 7.0, throughout 6 h with hourly measurements. Thermal stability was evaluated across a range of temperatures (20, 30, 40, 60 °C) for a duration of up to 2 h with every interval of 20 min. Additionally, the effect of various metal ions on the CeuVP activity was studied for Cu(SO₄)₂, CaCl₂, ZnCl₂, MnSO₄, FeCl₃, MgCl₂ KCl, and NaCl. The activity of CeuVP was assessed at optimal conditions and using ABTS as a substrate.

Analysis of Spectroscopy

To assess temperature-induced changes in fluorescence, CeuVP samples were incubated at temperatures ranging from 25 °C to 70 °C for 2 h. The fluorescence emission spectra were measured using a fluorescence spectrometer system (FluoroMate FS-2, Scinco, Korea) at 25 °C, with excitation at 280 nm and monitoring of emission spectra between 300 and 400 nm.

To examine temperature-dependent changes in the secondary structure of CeuVP, samples (0.5 mg/mL in reaction buffer) were incubated at temperatures ranging from 25 °C to 70 °C for 2 h. The circular dichroism (CD) spectra were then measured at 25 °C using a JASCO J715 Spectropolarimeter. The resulting spectra were plotted by using GraphPad Prism 9 software.

Substrate Specificity and Kinetic Parameters

The influence of different substrates on CeuVP activity was assessed. The standard reaction mixture consisted of 5 mM of each substrate and 100 mM sodium tartrate buffer (pH 4.0). The reaction was initiated by the addition of 50 mM H₂O₂. Activities were calculated based on the molar absorbance of the reaction products, including p-aminophenol (ε₂₄₆= 15627 M⁻¹ cm⁻¹), catechol (ε₂₃₈= 6500 M⁻¹ cm⁻¹), 4-methoxyphenol (ε₂₅₃= 4990 M⁻¹ cm⁻¹), guaiacol (ε₄₅₆= 12100 M⁻¹ cm⁻¹), veratryl alcohol (VA, ε₃₁₀= 9300 M⁻¹ cm⁻¹), remazol brilliant blue R (ε₅₉₂= 6170 M⁻¹ cm⁻¹), ABTS (ε₄₂₀= 36000 M⁻¹ cm⁻¹), reactive black 5 (ε₅₉₈= 50000 M⁻¹ cm⁻¹), and MnSO₄ (Mn²⁺, ε₂₇₀= 8000 M⁻¹ cm⁻¹). The specific activity of CeuVP for substrate oxidation was determined by measuring absorbance at the corresponding wavelengths (Table 2).

The kinetic parameters of CeuVP for several substrates with concentrations from 0 to 1500 µM were investigated (Table 3). The Michaelis constant (K_M) and catalytic efficiency (k_cat/K_M) were defined by using the Lineweaver-Burk plot with different substrate concentrations.

High-Performance Liquid Chromatography (HPLC) Analysis

α-Pinene was extracted from the essential oil of Eucalyptus robusta in a highly purified state by fractional distillation (Figure S1). The experiment was performed in 5 mL of reaction mixture containing 0.3 mg/mL of α-pinene, 50 mM H₂O₂, and 4 U/mL of pure CeuVP in 250 mM sodium tartrate buffer solution at pH 3.0. The reaction mixture was incubated at room temperature for up to 7 h under shaking at 150 rpm. Afterward, the reaction mixtures were identified by using an HPLC system. The sample was loaded onto an Eclipse XDB-C18 (4.6 × 150 mm, 5 µm) column with a diode array detector (DAD). The substances were eluted with a mobile phase of acetonitrile and water (70/30, v/v) at a flow rate of 0.5 mL/min. The signals were detected at a typical wavelength of 260 nm, and the chromatogram was recorded at 20-min intervals.

Results and Discussion

Identification of Isolated Fungal Strain

Genomic DNA was extracted and amplified via PCR to identify the fungal isolates. The ITS regions were amplified with primers ITS1 and ITS4, with an annealing temperature of 58 °C. As a result, a sequence of 730 nucleotides were obtained, which has been submitted to the GenBank (NCBI) database under the accession number: PP859417. The resulting nucleotide sequence was subsequently compared to reference sequences in the GenBank database. The analysis revealed that the isolated fungal strain had a 100% identity with the Candolleomyces eurysporus NR172427 species. Consequently, the strain VAST02.52 was classified as Candolleomyces eurysporus (Psathyrellaceae, Basidiomycota), which is a newly identified fungal strain in Vietnam. Phylogenetic trees were constructed with bootstrap values supporting the branches (Figure 1).

Figure 1.

The phylogenetic tree of the fungal strain VAST02.52 was constructed using the ITS region sequence based on the Maximum Likelihood (ML) method. Coprinellus micaceus JN943115 was used as the outgroup taxon (A), the wood-rot basidiomycete Candolleomyces eurysporus (VAST02.52) (B).

Purification and Characterization of Versatile Peroxidase

After cultivating C. eurysporus VAST02.52 for 7 days, the protein with VP activity was successfully separated from polyphenolic pigments and protein impurities by a specific purification procedure. Firstly, the liquid culture underwent an effective purification process on the DEAE-cellulose column, resulting in a remarkable increase in VP-specific activity from 1.5 U/mg in the crude extract to 8.7 U/mg after passing through this weak anion exchanger (Table 1). All active fractions were pooled and used for the next separation by using a Sephadex G-100 gel filtration column, and then a specific activity up to 21.2 U/mg could be obtained. Figure 2A depicted the elution pattern of the HiTrap^TM Q XL column used in the final purification step, resulting in two separate peaks of protein (λ=280 nm). Only the second-peak exhibited VP activity (41.7 U/mg) with a yield of 20.9%, and a purification fold of 27.3. The relatively low recovery of enzyme activity (∼ 21%) observed for VP of C. eurysporus was comparable with that for a VP of edible mushroom Pleurotus eryngii,¹⁹ which was also isolated from liquid culture. A reason for the activity loss could be the interaction of the VP protein with the separation material of the purification column, eg, by an irreversible binding to the sugar moieties of the polysaccharide polymer like cellulose or sepharose. Nevertheless, a homogeneous protein with versatile peroxidase activity (designated as CeuVP) was obtained as it appeared a single band on SDS-PAGE gel. This pure enzyme was calculated for a M_W of 40 kDa (Figure 2B), which is comparable with the range of M_W reported for other fungal VPs (38-50 kDa).^9,20,23,24

Figure 2.

FPLC elution profile of the final purification step of CeuVP on HiTrap^TM Q XL column (A): absorbance at 280 nm (solid line), VP activity (black circles), and NaCl gradient (dashed line) and SDS-PAGE gel after protein purification (Lane 1: marker, lane 2: eluted fraction from HiTrap^TM Q XL column) (B). UV/Vis absorption spectra (300 to 600 nm) of CeuVP were recorded for both the oxidized (dotted line) and dithionite-reduced (solid line) states (C).

Table 1.

Summary of Versatile Peroxidase (CeuVP) Purified from Culture of Candolleomyces eurysporus.

Purification steps	Total protein (mg)	Total activity (U)	Specific activity (U mg⁻¹)	Yield (%)	Purification (fold)
Crude extract	301	460	1.5	100	1.0
DEAE Cellulose	45	390	8.7	84.8	5.7
Sephadex G-100	9.7	206	21.2	44.8	13.9
HiTrap^TM Q XL	2.3	96	41.7	20.9	27.3

Figure 2C displays the UV-visible spectrum of purified CeuVP, featuring a Soret peak at 406 nm. Upon reduction with dithionite, the Soret band shifts to 416 nm, with additional bands emerging at 522 nm and 551 nm. This spectrum closely resembles that of a heme cofactor.^25,26 Indeed, versatile peroxidases are enzymes belonging to the heme peroxidase gene family and have been reported in numerous studies.^12,27 The heme in the active center of peroxidases interacts with H₂O₂, creating highly reactive intermediates that subsequently oxidize substrates with high redox potential.²⁸

The Influcence of pH and Temperature on CeuVP Activity

The optimal pH was determined to be pH of 4.0 as the highest activity of CeuVP observed in this weakly acidic solution (Figure 3A). This optimal pH range of CeuVP could be compatible with several other VPs, including that from the Pleurotus eryngii, and Citrus sinensis.^6,19 The pH stability of CeuVP was assessed by measuring its remaining activity after being incubated at different pH levels ranging from 3.0 to 7.0 for indicated durations. As a result, this purified VP displayed improved pH stability in acidic conditions. Thus, CeuVP maintained its activity up to 75% for 6.0-h incubation at pH 3.0 and was relatively stable at pH 5.0 retaining its activity over 50%. However, it was seemingly unstable at pH 7.0, with residual activity dropping to below 40% after 6.0 h (Figure 3C). CeuVP resembled the purified VP from Lentinus squarrosulus⁹ and Bjerkandera sp.,²⁰ both of which exhibited stability in the acidic pHs. Doriv Knop et al also reported that fungal VPs usually exhibited activity under acidic pH conditions well, achieving a high redox potential.²¹

Figure 3.

Effect of the pH and temperature on the ceuVP activity. Optimum pH (A), optimum temperature (B), pH stability (C), and thermal stability (D).

The enzyme's optimal temperature was determined by evaluating its activity at a temperature range of 20–55 °C. The CeuVP exhibited a peak of 100% of its activity at 35 °C, while losing approximately 80% of maximal activity when incubated at 55 °C (Figure 3B). The optimal temperature of CeuVP differs from other VPs since almost all fungal VPs showed optimal activity at higher temperatures, ie, 50–55 °C, as found for that from Pleurotus eryngii.¹⁹ It's reported that the VP from Pleurotus ostreatus exhibited optimal activity at 55 °C.²¹ The purified enzyme exhibited significant thermal stability at moderate temperatures up to 40 °C but sharply declined its activity at higher temperatures. For instance, following a 100-min incubation at 70 °C, the enzyme retained its activity only 25% (Figure 3D).

Analysis of Spectroscopy

The effect of temperature on structural changes of CeuVP was investigated for various temperatures ranging from 25 °C to 70 °C for 2 h, and their structural integrity was evaluated by measuring fluorescence and CD spectra (Figure 4). The spectroscopic data indicated that CeuVP maintains its tertiary and secondary structure within the temperature range of 25–40 °C. However, its structure becomes denatured after 1 h of incubation at 70 °C. Overall, these data are consistent with the thermal stability results. These findings suggest that while CeuVP is robust under moderate thermal conditions, higher temperatures may compromise both its structure and function.

Figure 4.

Spectroscopic analysis of CeuVP at various temperatures. Fluorescence spectra (A), Circular dichroism (CD) spectra (B).

The Influcence of Metal Ions

The metal cations differently impacted the CeuVP at a tested concentration of 1 mM with a slight enhancement of 122% and 134%, respectively, in its activity by adding Zn²⁺ and Fe²⁺. It's not surprising since VP is a heme protein containing an iron atom in its center and is also reported as a vital metal ion for enzyme activation.²⁹ The activity was inhibited by Ca²⁺ (34%), while the addition of Cu²⁺, Mg²⁺, Na⁺, and K⁺ (102%, 98%, 96%, and 94%, respectively) did not affect CeuVP activity (Figure 5). The inhibitory effect of Ca²⁺ could be due to competitive binding at the active site or conformational changes induced by the ion. The presence of metal ions can significantly impact the efficiency of CeuVP, making it essential to understand their interaction in industrial settings. Numerous studies have highlighted the importance of controlling metal ion concentrations to optimize peroxidase activity and ensure the success of industrial processes.^29-33

Figure 5.

Effect of metal ions on the activity of CeuVP.

Substrate Specificity of CeuVP

Various selected substrates including p-aminophenol, catechol, 4-methoxyphenol, guaiacol, veratryl alcohol, remazol brilliant blue R, ABTS, reactive black 5, and Mn²⁺, respectively, were used to determine CeuVP's substrate specificity. As shown in Table 2, CeuVP exhibited 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), and 4-methoxyphenol (31.1 U/mg), while lower activity was determined for guaiacol (18.3 U/mg) and Reactive Black 5 (12.9 U/mg). Among them, ABTS, Mn²⁺, and veratryl alcohol are recognized as “classic substrates” for VP activity.^6,9,19,20,23 This suggests that CeuVP has a strong affinity for these substrates, making them ideal for studying the enzyme's kinetic properties and potential applications in biocatalysis. The relatively high activity towards catechol and p-aminophenol also indicates a versatile substrate profile, which could be beneficial for various industrial processes, including bioremediation and the synthesis of fine chemicals. Conversely, the lower activity observed with guaiacol and Reactive Black 5 may suggest a limited interaction with these substrates, highlighting the enzyme's selectivity. Understanding CeuVP's substrate specificity is crucial for optimizing its use in different biotechnological applications.

Table 2.

Specific Activity of CeuVP for the Oxidation of Selected Substrates.

Substrates	Wavelength (nm)	Specific activity (U/mg)
p-Aminophenol	246	68.9
Cathecol	238	77.8
4-Methoxyphenol	253	31.1
Guaiacol	456	18.3
Veratryl alcohol	310	112.7
Remazol Brilliant Blue R (RBBR)	592	56.2
ABTS	420	231.6
Reactive Black 5	598	12.9
MnSO₄	270	205.8

Previous studies have shown that VP enzymes possess the catalytic activities of both MnP and LiP, enabling them to oxidize Mn²⁺ like MnP and high-redox potential non-phenolic compounds like LiP.^20,34,35 In this study, CeuVP was able to oxidize all tested substrates, including phenolic and non-phenolic aromatic compounds, as well as Mn²⁺. Therefore, it can be classified into a new group of versatile peroxidases.

Enzyme Kinetics

The kinetic constants of purified CeuVP were thoroughly investigated using H₂O₂, ABTS, VA and Mn²⁺ (MnSO₄) as substrates and the reaction rates exhibited variations corresponding to the substrate concentrations. The determination of Michaelis constants (K_M= 8.4 to 1300 µM) and catalytic efficiencies (k_cat/K_M values = 0.41 to 3.59 µM⁻¹. s⁻¹) could be implemented for all the substrates that were tested. In the reaction, the co-substrate ABTS was oxidized by CeuVP at varying concentrations of H₂O₂, resulting in a K_M value of 8.4 µM and a kcat value of 30.2 s⁻¹. When Mn²⁺ was used as a co-substrate, the K_M value slightly increased to 12.5 µM, and the k_cat value slightly decreased to 22.9 s⁻¹. Consequently, the catalytic efficiency (k_cat/K_M) of H₂O₂ as a substrate (k_cat/K_M -ABTS) was found to be 1.96-fold higher than that of Mn²⁺ (k_cat/K_M – Mn²⁺). In contrast, when VA was used as a co-substrate, the K_M value dramatically increased to 35.5 µM, and the k_cat value decreased to 19.4 s⁻¹. As a result, the catalytic efficiency of ABTS was found to be 6.52-fold higher than that of VA (Table 3).

Table 3.

Kinetic Constants for CeuVP Oxidation of Selected Substrates.

Substrate	K_M, µM	k_cat, s⁻¹	k_cat/K_M, µM⁻¹.s⁻¹
H₂O₂^a	8.4 ± 0.91	30.2 ± 0.07	3.59
H₂O₂^b	35.5 ± 1.13	19.4 ± 0.04	0.55
H₂O₂^c	12.5 ± 0.83	22.9 ± 0.05	1.83
ABTS	50.2 ± 2.01	103.6 ± 0.08	2.06
VA	1300 ± 1.52	537 ± 0.03	0.41
Mn²⁺	121.9 ± 1.06	85.3 ± 0.07	0.70

^a)

ABTS was oxidized at different concentrations of H₂O₂

^b)

VA was oxidized at different concentrations of H₂O₂

^c)

Mn²⁺ was oxidized at different concentrations of H₂O₂

When oxidizing different concentrations of ABTS, VA and Mn²⁺ in the presence of H₂O₂ as a co-substrate, CeuVP exhibited K_M values of 50.2, 1300, and 121.9 µM, respectively. Additionally, the k_cat/K_M for ABTS as a substrate is 5.02 and 2.94-fold higher than that for VA and Mn²⁺, respectively. In summary, the kinetic study results indicate that CeuVP prefers substrates in descending order ie, ABTS > Mn²⁺> VA.

It has been known that VP oxidizes numerous substrates, making it suitable for a wide range of applications. Among these, ABTS has been the most extensively studied as the primary substrate for VP. The kinetic data revealed a substantial difference in the K_M value for ABTS between CeuVP and other fungal VPs. Specifically, the K_M value of CeuVP (50.2 µM) was lower (reversely higher affinity) than that of the VPs from the white-rot fungus Pleurotus eryngii (203.09 µM),¹⁹ and Pleurotus sapidus (524 µM).¹⁰ Moreover, the k_cat/K_M value (2.06 s⁻¹. µM⁻¹) of CeuVP was 41.2 and 187.3-fold higher than that of VPs from Phanerocheate chrysosporium (0.05 s⁻¹. µM⁻¹) and Citrus sinensis (0.011 s⁻¹. µM⁻¹), respectively.⁶

Biotransformation of α-Pinene

The oxidation of aromatic compounds by the peroxidase has been reported by previous observations, for instance, their capability to degrade the α-pinene to potential products, ie, verbenol, verbenone, myrtenol, terpineol….^14-17 In the present study, incubation of plant derived α-pinene with CeuVP in the presence of H₂O₂ resulted in the liberation of verbenone as the sole metabolite, which was detected by HPLC analysis using an Eclipse XDB-C18 column (Figure 6). The biotransformation products of α-pinene can vary depending on the enzymes from different fungal sources. Indeed, Gheorghita et al demonstrated that versatile peroxidase from the white-rot fungus Pleurotus eryngii primarily oxidized α-pinene to verbenol, while laccase extracted from Trametes sp. predominantly converted it to verbenone.¹⁴ Furthermore, Su-Yeon Lee et al demonstrated that a complex of manganese-dependent peroxidase and laccase enzymes from two white rot fungi, Ceriporia sp. ZLY-2010 and Stereum hirsutum, was used for the biotransformation of α-pinene, resulting in the major products α-terpineol and verbenone, respectively. Additionally, minor products included myrtenol, camphor, and isopinocarveol.¹⁵ These studies underscore the significant influence that different fungal enzymes can have on the biotransformation pathways of α-pinene. For instance, the selective oxidation to verbenol or verbenone demonstrates how enzyme choice can direct the outcome of the biotransformation. This specificity is crucial for industrial applications where targeted production of certain compounds is desired. Moving forward, a deeper understanding of the mechanisms behind these enzymatic reactions could lead to the development of more efficient biocatalysts, expanding the range of valuable products derived from α-pinene. Furthermore, exploring the use of these fungal enzymes in combination or under varying conditions may reveal new possibilities for optimizing product yields and diversifying the array of biotransformation products. In this study, the oxidative α-pinene assisted by CeuVP to verbenone as the principal reaction product after seven hours of incubation. This property of CeuVP shows potential for application in various industries, however, to improve the transformation efficiency of α-pinene, VP should catalyze in combination with other oxidative enzymes, eg, laccase, which are also high redox potential enzymes.³⁵

Figure 6.

HPLC elution profile of α-pinene conversion of catalyzed by CeuVP.

Conclusions

The anion exchange and gel filtration column chromatography techniques were used to purify the VP enzyme from C. eurysporus VAST02.52, achieving a 27.3-fold increase in purity. The molecular weight of CeuVP was determined by SDS-PAGE analysis to be 40 kDa. The biochemical properties of CeuVP showed the pH optimum and stability within the acidic pH range. The optimum temperature for CeuVP was 35 °C, with activity maintained at moderate temperatures. The versatile peroxidase from C. eurysporus VAST02.52 exhibits high specific activity with ABTS as the primary substrate and is capable of oxidizing all substrates tested. The study also suggests that CeuVP is effective in the bioconversion of α-pinene into verbenone, a promising compound with numerous applications in the flavors and fragrances industry. For further studies, molecular work and recombinant expression should be considered.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X241290677 - Supplemental material for The Wood-rot Basidiomycete Candolleomyces eurysporus VAST02.52 Produces a Versatile Peroxidase that Catalyses the Biotransformation of α-Pinene to Release Verbenone

Supplemental material, sj-docx-1-npx-10.1177_1934578X241290677 for The Wood-rot Basidiomycete Candolleomyces eurysporus VAST02.52 Produces a Versatile Peroxidase that Catalyses the Biotransformation of α-Pinene to Release Verbenone by Do Huu Nghi, Pham Thi Hong Minh, Nguyen Thi Hong Van and Dang Thu Quynh in Natural Product Communications

Footnotes

Acknowledgements

The authors thank Institute of Chemistry (VAST) for measuring the CD spectra.

Author Contributions

D.H.N. designed the research. D.T.Q., P.T.H.M., N.T.H.V., and D.H.N. performed the research. D.H.N. and D.T.Q. wrote the paper.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval

Ethical Approval is not applicable for this article.

Funding

This research is funded by Vietnam Academy of Science and Technology (VAST) under grant number VAST02.02/23-24.

Informed Consent

There are no human subjects in this article and informed consent is not applicable.

ORCID iDs

Nguyen Thi Hong Van

Dang Thu Quynh

Supplemental Material

Supplemental material for this article is available online.

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