Molecular Docking Analysis of Soybean Peroxidase for Mitigating VOCs during Composting of Dairy Manure Mixed with Sawdust Bedding

Riuh Wardhani1 · Seunghun Lee2 · Jinho Shin2 · Yongwoo Song3 ·Abera Jabessa Fufa1 · SeongJun Park3 · Heekwon Ahn1,2,3*

1Dept. of Dairy Science, Chungnam National University, Daejeon, Korea

2Dept. of Animal Biosystems Science, Chungnam National University, Daejeon, Korea

3Dept. of Livestock Environmental Science & Technology, Chungnam National University, Daejeon, Korea

1. Introduction

Phenol, p-cresol, indole, and skatole are key odor-active VOCs in manure composting, with emissions intensifying during the active composting phase owing to rapid microbial decomposition (Jiang et al., 2023; Sanchez-Monedero et al., 2019). Soybean peroxidase (SBP) is a stable plant-derived biocatalyst that oxidizes aromatic substrates using peroxides. Studies on manure have shown that SBP-based treatments significantly reduce odorous VOCs in slurry or storage conditions (Parker et al., 2012; Maurer et al., 2017). However, composting is more dynamic and heterogeneous, with limited molecular-level evidence of the interaction between compost-derived odorants and the SBP catalytic site. Although computational docking has been used to evaluate indole-like ligands in peroxidases (Hallingback et al., 2006), it is rarely integrated with manure composting odor-mitigation studies. This study combined pilot-scale composting data with heme-site molecular docking to evaluate the SBP mitigation performance and the mechanistic plausibility of substrate-specific VOC oxidation.

2. Methods

Six pilot-scale compost piles were prepared using dairy manure solids and sawdust bedding: three untreated controls and three amended with SBP (2.24% w.b of initial compost mixture; calcium peroxide at 1.12% of SBP (w.w.)). Each pile contained 385 kg of material and was monitored for 50 days. VOC emissions were sampled using 2.25 m3 flux chambers. Gas samples were analyzed by gas chromatography for phenol, p-cresol, indole, and skatole. Molecular docking was used to analyze the interactions between SBP and VOC compounds. The soybean peroxidase crystal structure (PDB: 1FHF) was selected because of its resolved heme-containing active site relevant to aromatic substrate accessibility (Henriksen et al., 2001). Phenol, p-cresol, and indole were docked using AutoDock Vina with a heme-centered grid box, prioritizing the best heme proximal pose for each ligand (Trott and Olson, 2010; Eberhardt et al., 2021). Interaction patterns were evaluated for hydrogen bonding, aromatic stacking, and hydrophobic contacts linked to catalytically relevant binding (Hallingback et al., 2006).

3. Results and Discussion

SBP treatment reduced the total emissions of major odorous VOCs. Compared to the control, phenol, p-cresol, and indole emissions decreased by 47%, 10%, and 61%, respectively, whereas skatole was undetected during the 50-day period. Total VOC emissions decreased by 47%, with the strongest separation during the active composting stage, consistent with reports of intense odor-active VOC formation when degradable substrates and microbial activity are at their highest (Jiang et al., 2023; Sanchez-Monedero et al., 2019). The observed reduction in phenol is plausible because peroxidases facilitate the oxidation of phenolic compounds into phenoxy radicals, which subsequently undergo coupling and polymerization to form larger, less volatile products (Ryan et al., 2006; Steevensz et al., 2014). Comparable studies have documented significant reductions in phenolic odorants following treatment with SBP or other peroxidase systems, including the attenuation of p-cresol and related volatile organic compounds (VOCs). (Parker et al., 2012; Ye et al., 2009; Maurer et al., 2017). The results showed that the enzyme retained its oxidative function under aerated, heterogeneous compost conditions. Molecular docking provided structure-based evidence of substrate accessibility at the SBP heme site. Phenol, p-cresol, and indole showed favorable heme-proximal binding, with docking scores of -4.1, -4.5, and -4.5 kcal/mol and ligand-to-heme iron distances of 7.90, 8.12, and 8.10 Å, respectively. The predicted poses suggest that phenol and p-cresol are oriented for productive hydroxyl-associated electron transfer, whereas indole is stabilized by aromatic interactions near the heme edge, consistent with the general peroxidase substrate recognition behavior (Henriksen et al., 2001; Hallingback et al., 2006). The main advance of this study is the addition of docking-based molecular evidence that target compost VOCs can adopt catalytically meaningful binding modes in the SBP, unlike earlier studies that inferred oxidative deodorization from emission outcomes alone (Parker et al., 2012; Maurer et al., 2017).

4. Conclusion

This study shows that soybean peroxidase effectively reduces VOC emissions during pilot-scale dairy manure composting. Heme-site molecular docking provides a plausible mechanistic explanation for these effects. The reduction in phenol, p-cresol, and indole, along with favorable docking poses near the SBP heme cofactor, supports peroxidase treatment as a promising odor control strategy for manure systems. Docking analysis adds a mechanistic interpretation by linking emission reductions to catalytic site accessibility and ligand-specific interactions. Further research should optimize dosage, oxidant supply, and application timing while validating docking predictions with additional kinetic or reaction product measurements.

5. Acknowledgment