Oxalate Decarboxylase Research Articles

Enzymatic and structural properties of a novel oxalate decarboxylase BsOxdC from Bacillus safensis and its potential pH-dependent catalytic mechanism.

Oxalate decarboxylase converts oxalate to formate and CO2 without requiring organic cofactors, making it biotechnologically relevant for applications in food, agriculture, and diagnostics. Its activity is highly dependent on pH; however, the pH-dependent catalytic mechanism remains poorly understood. This study identified a novel oxalate decarboxylase, BsOxdC, from Bacillus safensis and investigated its catalytic properties through heterologous expression and enzymatic assays. The purified BsOxdC efficiently degrades oxalate at an optimum temperature of 50°C and a pH of 4.0, achieving a Vmax of 8.54μmol/(min·mg). The apparent values of kcat, Km, and kcat/Km were 85.35s-1, 4.67μM, and 18.28μM/s, respectively. The predicted structure of BsOxdC features two conserved cupin barrel folds at the N-terminal and C-terminal. Additionally, the docking model of the oxalate-BsOxdC complex is more stable than those of the formate-BsOxdC or acetate-BsOxdC complexes due to its lowest binding energy. In the open conformation of BsOxdC, the carboxyl group of the catalytic residue E181, located in the active loop S180E181N182S183T184, points away from both the oxalate and the active-site Mn ion. Simulations suggest that S180 and E181 interact with the substrate via ionic bonds and/or water bridges only at low pH (4.0), not at pH8.0. Additionally, THR184 forms more molecular interactions with oxalate at pH4.0 than at pH8.0.

Decarboxylase mediated oxalic acid metabolism is important to antioxidation and detoxification rather than pathogenicity in Magnaporthe oryzae

ABSTRACT Oxalic acid (OA), an essential pathogenic factor, has been identified in several plant pathogens, and researchers are currently pursuing studies on interference with OA metabolism as a treatment for related diseases. However, the metabolic route in Magnaporthe oryzae remains unknown. In this study, we describe D-erythroascorbic acid-mediated OA synthesis and its metabolic and clearance pathways in rice blast fungus. By knocking out the D-arabino-1,4-lactone oxidase gene (Moalo1), one-third of oxalic acid remained in M. oryzae, indicating a main pathway for oxalic acid production. M. oryzae OxdC (MoOxdC) is an oxalate decarboxylase that appears to play a role in relieving oxalic acid toxicity. Loss of Mooxdc does not affect mycelial growth, conidiophore development, or appressorium formation in M. oryzae; however, the antioxidant and pathogenic abilities of the mutant were enhanced. This is owing to Mooxdc deletion upregulated a series of OA metabolic genes, including the oxalate oxidase gene (Mooxo) and Moalo1, as well as both OA transporter genes. Simultaneously, as feedback to the tricarboxylic acid (TCA) cycle, the decrease of formic acid in ΔMooxdc leads to the reduction of acetyl-CoA content, and two genes involved in the β-oxidation of fatty acids were also upregulated, which enhanced the fatty acid metabolism of the ΔMooxdc. Overall, this work reveals the role of OA in M. oryzae. We found that OA metabolism was mainly involved in the growth and development of M. oryzae, OA as a byproduct of D-erythroascorbic acid after removing H2O2, the OA-associated pathway ensures the TCA process and ATP supply.

Bidentate Substrate Binding Mode in Oxalate Decarboxylase.

Oxalate decarboxylase is an Mn- and O2-dependent enzyme in the bicupin superfamily that catalyzes the redox-neutral disproportionation of the oxalate monoanion to form carbon dioxide and formate. Its best-studied isozyme is from Bacillus subtilis where it is stress-induced under low pH conditions. Current mechanistic schemes assume a monodentate binding mode of the substrate to the N-terminal active site Mn ion to make space for a presumed O2 molecule, despite the fact that oxalate generally prefers to bind bidentate to Mn. We report on X-band 13C-electron nuclear double resonance (ENDOR) experiments on 13C-labeled oxalate bound to the active-site Mn(II) in wild-type oxalate decarboxylase at high pH, the catalytically impaired W96F mutant enzyme at low pH, and Mn(II) in aqueous solution. The ENDOR spectra of these samples are practically identical, which shows that the substrate binds bidentate (κO, κO') to the active site Mn(II) ion. Domain-based local pair natural orbital coupled cluster singles and doubles (DLPNO-CCSD) calculations of the expected 13C hyperfine coupling constants for bidentate bound oxalate predict ENDOR spectra in good agreement with the experiment, supporting bidentate bound substrate. Geometry optimization of a substrate-bound minimal active site model by density functional theory shows two possible substrate coordination geometries, bidentate and monodentate. The bidentate structure is energetically preferred by ~4.7 kcal/mol. Our results revise a long-standing hypothesis regarding substrate binding in the enzyme and suggest that dioxygen does not bind to the active site Mn ion after substrate binds. The results are in agreement with our recent mechanistic hypothesis of substrate activation via a long-range electron transfer process involving the C-terminal Mn ion.

Recombinant strains expressing oxalate decarboxylase efficiently and environmental characteristics of their life cycle assessment

Oxalate decarboxylase has garnered significant attention due to its wide-ranging applications, particularly as an effective demulsifier for oil pollution remediation. However, the accumulation of oxalate decarboxylase by strain is insufficient, and the acquisition of necessary nutrients and energy for biosynthesis also poses challenges to the environment. Therefore, it is crucial to assess the strain's capacity and environmental burden in order to guide future developments and ensure sustainable scale-up. To enhance the protein yield of oxalate decarboxylase from Bacillus mojavensis XH1 (Bacm OxdC), a specialized biodemulsifier, our study investigated the properties of Bacm OxdC and improved its expression efficacy in Escherichia coli from an environmentally friendly perspective. By employing a promoter tandem strategy and optimizing the 5′ untranslated region, recombinant strains C3 and C5 were constructed, resulting in 1.22-fold and 6.90-fold increases in Bacm OxdC expression levels compared to the initial strain C1. The bioprocess of strain C5 achieved a life cycle assessment (LCA)-based environmental impact value of 322.75, representing the lowest environmental impact observed thus far. Improvement strategies included reducing protein purification steps and electricity consumption, which can further mitigate negative environmental impacts and carbon emissions. This study offers a technical strategy for large-scale production of oxalate decarboxylase while attaining green and low-carbon objectives for biological demulsifiers.

Experimental Study on Ecological and Biogeochemical Role of Calcimycocavites in Understanding Carbon Fluxes in the Age of Global Warming and Climate Change

The present work reports the results of calcimycocavitological studies on the seashells from beaches of north Goa, India yielding distinct microfungal forms known as calcimycocavites which are important in global climate change studies. SEM studies of the calcareous shell fragments revealed the micro tunneling behavior of the fungi. Digital analysis of SEM images using Mountain Premium 7.2 software revealed the fine topography of calcimycocavites hyphae along with unidentified presumptive biomineral encrustations. The biodegradation of mycocavitogenic insoluble Calcium Oxalate by oxalotrophic members of bacterial communities may occur using bacterial Oxalate decarboxylase and formate dehydrogenase finally releasing Carbon Di Oxide and water and Calcium oxide to the local nutrient pool. Calcimycocavites may be thus involved in the biogeochemical cycling of Carbon and Calcium in the Ocean-Beach environment. The ecological, biological, and biogeochemical implications of the findings are presented concerning the possible role of calcimycocavites in Calcium and Carbon cycling by breakdown of the calcareous shells and release of inorganic and organic components in the ecosystem. The role of calcimycocavites is stressed in global climate studies.

Performance of Philippine Native Pig Fed with Ensiled Pongapong

Native animals are regarded as an essential component of most agricultural production systems in rural settings. Amorphophallus campanulatus can be used as food and animal feed, however its application is limited due to its high oxalate concentration and low crude protein level. Fermentation with oxalolytic bacteria, such as Bacillus subtilis, which produces the oxalate decarboxylase enzyme, has been utilized to boost the nutritional value of pongapong. This study aims to determine the growth performance of native pig fed with fermented pongapong as feed supplement. Specifically, to determine the effect of fermented/ensiled pongapong on the body weight-gain in weight, growth increment, feed conversion efficiency and feed conversion ratio on Philippine native pigs; determine the best level of silage pongapong to growth of Philippine Native pigs; determine the nutrient composition of fermented Amorphophalus campanulatus; and determine the economics of fermented pongapong as feed supplement for native pigs in one production cycle. The Complete Randomized Design (CRD) was utilized in the study. The treatments were follows:T1=Formulated feeds ,T2=75% Formulated feeds + 25% silage AC ,T3=50% Formulated feeds 50% silage AC, T4=25% Formulated feeds + 75% silage AC, T5= Pure ensiled AC. Based on the analysis of variance shows insignificant among the treatments as to the initial weight, feed conversion efficiency and growth rate. Also, there are significant differences in the body weight, gain weight and feed conversion ration while it shows highly significant feed consumption throughout the duration of the study. Thus, 75% formulated feeds and 25% fermented pongapong and 50% Formulated feeds + 50% fermented pongapong results to best growth. It demonstrates that feeding native pigs with formulated feed including fermented pongapong was employed as an alternative diet that is potentially cost effective and useful in native pig production in the province. This information could help growers reduce feed costs.

Transcriptomic and biochemical analysis of the mechanism of sodium gluconate promoting the degradation of benzo [a] pyrene by Bacillus subtilis MSC4

Benzo[a]pyrene (B[a]P) is a carcinogenic environmental pollutant widely present in the environment and can enter the human body through the food chain. It is therefore essential to treat and remediate the B[a]P-contaminated environment. Microbial remediation of B[a]P-contaminated environments is considered to be one of the most effective strategies, and the addition of biostimulants is a feasible method to further improve the effectiveness of microbial remediation. In this study, we used Bacillus subtilis MSC4 to screen for the stimulation of sodium gluconate, which promoted B[a]P degradation. Based on biochemical and transcriptomic analyses, Sodium gluconate was found to significantly increase the biomass of MSC4 and the expression of most genes involved in B[a]P degradation. Activities of central carbon metabolism, fatty acid β-oxidation and oxidative phosphorylation were all promoted. The significant increase in acid-induced oxalate decarboxylase expression indicates a decrease in intracellular pH, which promoted the synthesis of acetoin and lactate. Genes involved in the nitrogen cycle, especially nitrification and denitrification, were significantly up-regulated, contributing to B[a]P degradation. Genes involved in the synthesis of enzyme cofactors, including thiamine, molybdenum cofactors, NAD and heme, were up-regulated, which contributes to increasing enzyme activity in metabolic pathways. Up-regulation of genes in flagella assembly, chemotaxis, and lipopeptide synthesis is beneficial for the dissolution and uptake of B[a]P. Genes related to the sugar transport system were upregulated, which facilitates the transport and absorption of monosaccharides and oligosaccharides by MSC4. This study provides a theoretical basis for the further application of sodium gluconate in the treatment of PAH-contaminated sites.

The CsPbs2-interacting protein oxalate decarboxylase CsOxdC3 modulates morphosporogenesis, virulence, and fungicide resistance in Colletotrichum siamense

The HOG MAPK pathway mediates diverse cellular and physiological processes, including osmoregulation and fungicide sensitivity, in phytopathogenic fungi. However, the molecular mechanisms underlying HOG MAPK pathway-associated stress homeostasis and pathophysiological developmental events are poorly understood. Here, we demonstrated that the oxalate decarboxylase CsOxdC3 in Colletotrichum siamense interacts with the protein kinase kinase CsPbs2, a component of the HOG MAPK pathway. The expression of the CsOxdC3 gene was significantly suppressed in response to phenylpyrrole and tebuconazole fungicide treatments, while that of CsPbs2 was upregulated by phenylpyrrole and not affected by tebuconazole. We showed that targeted gene deletion of CsOxdC3 suppressed mycelial growth, reduced conidial length, and triggered a marginal reduction in the sporulation characteristics of the ΔCsOxdC3 strains. Interestingly, the ΔCsOxdC3 strain was significantly sensitive to fungicides, including phenylpyrrole and tebuconazole, while the CsPbs2-defective strain was sensitive to tebuconazole but resistant to phenylpyrrole. Additionally, infection assessment revealed a significant reduction in the virulence of the ΔCsOxdC3 strains when inoculated on the leaves of rubber tree (Hevea brasiliensis). From these observations, we inferred that CsOxdC3 crucially modulates HOG MAPK pathway-dependent processes, including morphogenesis, stress homeostasis, fungicide resistance, and virulence, in C. siamense by facilitating direct physical interactions with CsPbs2. This study provides insights into the molecular regulators of the HOG MAPK pathway and underscores the potential of deploying OxdCs as potent targets for developing fungicides.

Recent Advances of Oxalate Decarboxylase: Biochemical Characteristics, Catalysis Mechanisms, and Gene Expression and Regulation.

Oxalate decarboxylase (OXDC) is a typical Mn2+/Mn3+ dependent metal enzyme and splits oxalate to formate and CO2 without any organic cofactors. Fungi and bacteria are the main organisms expressing the OXDC gene, but with a significantly different mechanism of gene expression and regulation. Many articles reported its potential applications in the clinical treatment of hyperoxaluria, low-oxalate food processing, degradation of oxalate salt deposits, oxalate acid diagnostics, biocontrol, biodemulsifier, and electrochemical oxidation. However, some questions still remain to be clarified about the role of substrate binding and/or protein environment in modulating the redox properties of enzyme-bound Mn(II)/Mn(III), the nature of dioxygen involved in the catalytic mechanism, and how OXDC acquires Mn(II) /Mn(III). This review mainly summarizes its biochemical and structure characteristics, gene expression and regulation, and catalysis mechanism. We also deep-mined oxalate decarboxylase gene data from National Center for Biotechnology Information to give some insights to explore new OXDC with diverse biochemical properties.

Mechanism of oxalate decarboxylase Oxd_S12 from Bacillus velezensis BvZ45-1 in defence against cotton verticillium wilt.

Verticillium wilt, a soilborne vascular disease caused by Verticillium dahliae, strongly affects cotton yield and quality. In this study, an isolated rhizosphere bacterium, designated Bacillus velezensis BvZ45-1, exhibited >46% biocontrol efficacy against cotton verticillium wilt under greenhouse and field conditions. Moreover, through crude protein extraction and mass spectrometry analyses, we found many antifungal compounds present in the crude protein extract of BvZ45-1. The purified oxalate decarboxylase Odx_S12 from BvZ45-1 inhibited the growth of V. dahliae Vd080 by reducing the spore yield, causing mycelia to rupture, spore morphology changes, cell membrane rupture, and cell death. Subsequently, overexpression of Odx_S12 in Arabidopsis significantly improved plant resistance to V. dahliae. Through studies of the resistance mechanism of Odx_S12, V. dahliae was shown to produce oxalic acid (OA), which has a toxic effect on Arabidopsis leaves. Odx_S12 overexpression reduced Arabidopsis OA content, enhanced tolerance to OA, and improved resistance to verticillium wilt. Transcriptomics and quantitative real-time PCR analysis revealed that Odx_S12 promoted a reactive oxygen species burst and a salicylic acid- and abscisic acid-mediated defence response in Arabidopsis. In summary, this study not only identified B. velezensis BvZ45-1 as an efficient biological control agent, but also identified the resistance gene Odx_S12 as a candidate for cotton breeding against verticillium wilt.

Investigation of non-classical secretion of oxalate decarboxylase in Bacillus mojavensis XH1 mediated by exopeptide YydF: Mechanism and application

Non-classical secretory proteins are widely found in bacteria and have been extensively studied due to their important physiological roles. However, the relevant non-classical secretory mechanisms remain unclear. In this study, we found that oxalate decarboxylase (Bacm OxDC) from Bacillus mojavensis XH1 belongs to non-classical secretory proteins. Its N-terminus showed high hydrophilicity, which was different from the conventional signal peptide. The truncation test revealed that the deletion of the N-terminus affects the structure resulting in its inability to cross the cell membrane. Further studies verified that the exported peptide YydF played an important role in the secretion process of Bacm OxDC. Experimental results on the secretion mechanism indicated that Bacm OxDC bound to the exported peptide YydF and they are translocated to the cell membrane together, after which Bacm OxDC caused cell membrane relaxation for transmembrane secretion. Thereafter, three recombinant proteins were successfully secreted with certain enzymatic activity by fusing Bacm OxDC as a guide protein with various target proteins. To the best of our knowledge, this was the first time that non-classical secretion mechanism in bacteria has been analyzed. The novel discovery may provide a reference and broaden the horizons of the secretion pathway and expression regulation of proteins.

Anoxalate decarboxylase-like cupin domain containing protein is involved in imparting acid stress tolerance in Bacillus amyloliquefaciens MBNC.

We report here the structural and functional properties of an oxalate decarboxylase (OxDC)-like cupin domain-containing protein of Bacillus amyloliquefaciens MBNC and its role in imparting tolerance to acid stress conditions. Quantitative real-time PCR (qPCR) analysis revealed 32-fold and 20-fold upregulation of the target gene [(OxDC')cupin] under acetic acid stress and hydrochloric acid stress, respectively, indicating its association with the acid stress response. Bacterial cells with targeted inactivation of the (OxDC')cupin gene using the pMUTIN4 vector system showed decreased growth and survival rate in acidic pH, with drastically reduced exopolysaccharide production. In Silico protein-protein interaction studies revealed seven genes (viz. glmS, nagA, nagB, tuaF, tuaF, gcvT, and ykgA) related to cell wall biosynthesis and biofilm production to interact with OxDC-like cupin domain containing protein. While all these seven genes were upregulated in B. amyloliquefaciens MBNC after 6h of exposure to pH 4.5, the mutant cells containing the inactivated (OxDC')cupingene displayed significantly lower expression (RQ: 0.001-0.02) (compared to the wild-type cells) in both neutral and acidic pH. Our results indicate that the OxDC-like cupin domain containing protein is necessary for cell wall biosynthesis and biofilm production in Bacillus amyloliquefaciens MBNC for survival in acid-stress conditions.

Zn2+ regulates human oxalate metabolism by manipulating oxalate decarboxylase to treat calcium oxalate stones

A high concentration of oxalate is associated with an increased risk of kidney calcium oxalate (CaOx) stones, and the degradation of exogenous oxalate mostly depends on oxalate-degrading enzymes from the intestinal microbiome. We found that zinc gluconate supplement to patients with CaOx kidney stones could significantly improve the abundance of oxalate metabolizing bacteria in humans through clinical experiments on patients also subjected to antibiotic treatment. The analysis of clinical samples revealed that an imbalance of Lactobacillus and oxalate decarboxylase (OxDC) was involved in the formation of CaOx kidney stones. Then, we identified that Zn2+ could be used as an external factor to improve the activity of OxDC and promote Lactobacillus in the intestinal flora, and this treatment achieved a therapeutic effect on rats with stones aggravated by antibiotics. Finally, by analyzing the three-dimensional structure of OxDC and completing in vitro experiments, we propose a model of the Zn2+-induced reduction of CaOx kidney stone symptoms in rats by increasing the metabolism of oxalate through the positive effects of Zn2+ on Lactobacillus and OxDC.

Potential of oxalate decarboxylase of Pseudomonas sp. OXDC12 in degradation of oxalate content of vegetables

Potential of oxalate decarboxylase of Pseudomonas sp. OXDC12 in degradation of oxalate content of vegetables

MALDI mass spectrometry-based identification of antifungal molecules from endophytic Bacillus strains with biocontrol potential of Lasiodiplodia theobromae, a grapevine trunk pathogen in Peru

Lasiodiplodia theobromae, a grapevine trunk pathogen, is becoming a significant threat to vineyards worldwide. In Peru, it is responsible for Botryosphaeria dieback in many grapevine-growing areas and it has spread rapidly due to its high transmissibility; hence, control measures are urgent. It is known that some endophytic bacteria are strong inhibitors of phytopathogens because they produce a wide range of antimicrobial molecules. However, studies of antimicrobial features from endophytic bacteria are limited to traditional confrontation methods. In this study, a MALDI mass spectrometry-based approach was performed to identify and characterize the antifungal molecules from Bacillus velezensis M1 and Bacillus amyloliquefaciens M2 grapevine endophytic strains. Solid medium antagonism assays were performed confronting B. velezensis M1 - L. theobromae and B. amyloliquefaciens M2 - L. theobromae for antifungal lipopeptides identification. By a MALDI TOF MS it was possible identify mass spectra for fengycin, iturin and surfactin protoned isoforms. Masses spectrums for mycobacillin and mycosubtilin were also identified. Using MALDI Imaging MS we were able to visualize and relate lipopeptides mass spectra of fengycin (1463.9 m/z) and mycobacillin (1529.6 m/z) in the interaction zone during confrontations. The presence of lipopeptides-synthesis genes was confirmed by PCR. Liquid medium antagonism assays were performed for a proteomic analysis during the confrontation of B. velezensis M1 - L. theobromae. Different peptide sequences corresponding to many antifungal proteins and enzymes were identified by MALDI TOF MS/MS. Oxalate decarboxylase bacisubin and flagellin, reported as antifungal proteins, were identified at 99 % identity through peptide mapping. MALDI mass spectrometry-based identification of antifungal molecules would allow the early selection of endophytic bacteria with antifungal features. This omics tool could lead to measures for prevention of grapevine diseases and other economically important crops in Peru.

Selective incorporation of 5-hydroxytryptophan blocks long range electron transfer in oxalate decarboxylase.

Oxalate decarboxylase from Bacillus subtilis is a binuclear Mn-dependent acid stress response enzyme that converts the mono-anion of oxalic acid into formate and carbon dioxide in a redox neutral unimolecular disproportionation reaction. A π-stacked tryptophan dimer, W96 and W274, at the interface between two monomer subunits facilitates long-range electron transfer between the two Mn ions and plays an important role in the catalytic mechanism. Substitution of W96 with the unnatural amino acid 5-hydroxytryptophan leads to a persistent EPR signal which can be traced back to the neutral radical of 5-hydroxytryptophan with its hydroxyl proton removed. 5-Hydroxytryptophan acts as a hole sink preventing the formation of Mn(III) at the N-terminal active site and strongly suppresses enzymatic activity. The lower boundary of the standard reduction potential for the active site Mn(II)/Mn(III) couple can therefore be estimated as 740 mV against the normal hydrogen electrode at pH4, the pH of maximum catalytic efficiency. Our results support the catalytic importance of long-range electron transfer in oxalate decarboxylase while at the same time highlighting the utility of unnatural amino acid incorporation and specifically the use of 5-hydroxytryptophan as an energetic sink for hole hopping to probe electron transfer in redox proteins.

Cloning and Molecular Characterization of CmOxdc3 Coding for Oxalate Decarboxylase in the Mycoparasite Coniothyrium minitans.

Coniothyrium minitans (Cm) is a mycoparasitic fungus of Sclerotinia sclerotiorum (Ss), the causal agent of Sclerotinia stem rot of oilseed rape. Ss can produce oxalic acid (OA) as a phytotoxin, whereas Cm can degrade OA, thereby nullifying the toxic effect of OA. Two oxalate decarboxylase (OxDC)-coding genes, CmOxdc1 and CmOxdc2, were cloned, and only CmOxdc1 was found to be partially responsible for OA degradation, implying that other OA-degrading genes may exist in Cm. This study cloned a novel OxDC gene (CmOxdc3) in Cm and its OA-degrading function was characterized by disruption and complementation of CmOxdc3. Sequence analysis indicated that, unlike CmOxdc1, CmOxdc3 does not have the signal peptide sequence, implying that CmOxDC3 may have no secretory capability. Quantitative RT-PCR showed that CmOxdc3 was up-regulated in the presence of OA, malonic acid and hydrochloric acid. Deletion of CmOxdc3 resulted in reduced capability to parasitize sclerotia of Ss. The polypeptide (CmOxDC3) encoded by CmOxdc3 was localized in cytoplasm and gathered in vacuoles in response to the extracellular OA. Taken together, our results demonstrated that CmOxdc3 is a novel gene responsible for OA degradation, which may work in a synergistic manner with CmOxdc1.

Oxalic acid degradation in wood-rotting fungi. Searching for a new source of oxalate oxidase

Oxalate oxidase (EC 1.2.3.4) is an oxalate-decomposing enzyme predominantly found in plants but also described in basidiomycete fungi. In this study, we investigated 23 fungi to determine their capability of oxalic acid degradation. After analyzing their secretomes for the products of the oxalic acid-degrading enzyme activity, three groups were distinguished among the fungi studied. The first group comprised nine fungi classified as oxalate oxidase producers, as their secretome pattern revealed an increase in the hydrogen peroxide concentration, no formic acid, and a reduction in the oxalic acid content. The second group of fungi comprised eight fungi described as oxalate decarboxylase producers characterized by an increase in the formic acid level associated with a decrease in the oxalate content in their secretomes. In the secretomes of the third group of six fungi, no increase in formic acid or hydrogen peroxide contents was observed but a decline in the oxalate level was found. The intracellular activity of OXO in the mycelia of Schizophyllum commune, Trametes hirsuta, Gloeophyllum trabeum, Abortiporus biennis, Cerrena unicolor, Ceriosporopsis mediosetigera, Trametes sanguinea, Ceriporiopsis subvermispora, and Laetiporus sulphureus was confirmed by a spectrophotometric assay.

Degradation of oxalic acid by Trichoderma afroharzianum and its correlation with cell wall degrading enzymes in antagonizing Botrytis cinerea.

Oxalic acid (OA) is one of the pathogenic factors of Botrytis cinerea. Trichoderma afroharzianum exerts both antagonistic and oxalate-degrading effects on B. cinerea. This study aimed to investigate the relationship between the elimination of OA by T. afroharzianum and its antagonistic effects on B. cinerea. Reversed-phase high performance liquid chromatogram (RP-HPLC) analysis showed that T. afroharzianum LTR-2 eliminated 10- or 20-mmol/L OA within 120 h, with the degradation being particularly efficient at the concentration of 20 mmol/L. RNA-seq analysis showed that the oxalate decarboxylase (OXDC) gene Toxdc, β-1,3-exoglucanase gene Tglu and aspartic protease gene Tpro of LTR-2 were significantly upregulated after treatment with 20-mmol/L OA. RT-qPCR analysis showed that under the conditions of confrontation, Toxdc and three cell wall degrading enzyme (CWDE) genes were upregulated before physical contact with B. cinerea. In addition, RT-qPCR analysis showed that OA synthesis in B. cinerea was not significantly affected by LTR-2. The results revealed a correlation between OA degradation and mycoparasitism in T. afroharzianum when antagonising B. cinerea at the transcriptional level. The relationship between OA degradation by T. afroharzianum and its effects against B. cinerea provide a new perspective on the antagonism of T. afroharzianum against B. cinerea. In addition, this study provides theoretical data for the scientific application of T. afroharzianum in the field of biocontrol.

Dodecenylsuccinic anhydride-modified oxalate decarboxylase loaded with magnetic nano-Fe3O4@SiO2 for demulsification of oil-in-water emulsions

The inability to demulsify oil-in-water emulsions via green and efficient processes is a challenging problem in many industrial processes. As a novel biodemulsifier, protein demulsifiers display excellent dispersibility and stability, but their demulsification mechanisms are not clear, which severely restricts their large-scale production and application. In this study, the demulsification mechanism of the high-efficiency protein biodemulsifier oxalate decarboxylase (Bacm OxdC), which is secreted by the Bacillus mojavensis XH1 strain, for an oil-in-water emulsion was analyzed. The results showed that Bacm OxdC was spontaneously adsorbed at the oil-water interface and turned its hydrophobic amino acids outward to increase its hydrophobicity and break the emulsified system. Furthermore, it effectively reduced the oil-water interfacial tension and interfacial film strength, thereby reducing the oil-water interfacial energy and finally enabling demulsification. To further improve the demulsification efficiency and reusability, Fe3O4@SiO2@OxdC-DDSA was prepared. This method provided a magnetic response for Bacm OxdC and enabled efficient demulsification. The demulsification rate of Fe3O4@SiO2@OxdC-DDSA reached 98.1% at 24 h, which was 30.7% higher than that of the original Bacm OxdC. After three cycles, the demulsification rate still reached 89.3%, proving it has excellent recyclability. This work is the first study on the demulsification mechanism of protein biodemulsifiers and provides useful insights into the demulsification mechanism of biodemulsifiers for oil-in-water emulsions. In addition, a promising high-efficiency modification technique for protein biodemulsifiers was proposed, which provided information for the development of biodemulsifiers for oil-water separation.