Identification of fungal enzymes involving 3-phenoxybenzoic acid degradation by using enzymes inhibitors and inducers

Graphical abstract


A R T I C L E I N F O Method name: Identification of fungal enzymes involving 3-phenoxybenzoic acid degradation by using enzymes inhibitors and inducers
Keywords: 3-phenoxybenzoic acid, Degrading-enzymes, Enzyme inhibitor, Enzyme inducer  Table   Subject Area: Environmental Science More specific subject area: Biodegradation Method name: Identification of fungal enzymes involving 3-phenoxybenzoic acid degradation by using enzymes inhibitors and inducers Name and reference of original method: The measurement for 3-phenoxybenzoic acid and its intermediate metabolites in medium by high-performance liquid chromatography (HPLC) was based on the methods reported by   [1] and Zhao et al. (2016) [2]. The inducible or non-inducible nature of degrading-enzymes was measured as the protocol reported by Fang et al. (2015) [3]. The activity of Cytochrome P450 (CYP450) was measured using previously described by Uno et al. (2017) [4]; the activity of lignin peroxidase (LiP), laccase, and manganese peroxidase (MnP) was determined according to Guo

Introduction
Chemical pesticides are important components of modern global agricultural systems, leading to a substantial improvement in crop yields by controlling insects [1]. Nevertheless, some negative influence including disastrous contamination of ecosystems (i.e., dust, soils, air, sediments, and water) and adulterating human foods have been appeared due to the extensive application of chemical pesticides [1]. Since the partial or total ban on organochlorine and organophosphorus pesticides, pyrethroids have been widely used and account for 25 % of the global pesticides market [2]. Long term and excessive spraying have become common methods for pyrethroids application. The pyrethroid residues in the environment and food as a result of these spraying methods has attracted more attention because of their potential to contaminate the food supply chain and threaten human health [3]. As an intermediate metabolite of pyrethroids, 3-phenoxybenzoic acid (3-PBA) is more toxic than its parent compounds and has been detected in milk, soil, and human urine [3][4][5]. 3-PBA can be metabolized to less potent metabolites through microbial degradation. It has been predicted that oxidases were the main enzymes involving 3-PBA degradation by microorganisms [4][5][6], and the degrading-enzymes needed further investigation.
Some contaminants containing similar chemical structures with 3-PBA and its intermediate metabolites could be degraded by oxidases such as laccase, MnP, LiP, and dioxygenase [7,8], and many fungi including Aspergillus oryzae have these enzyme genes [9]. Therefore, it could be reasoned that these oxidases might also participate the degradation.

Method
This MethodsX is presented to researchers as a step by step procedure to study the co-metabolic enzymes and pathways of 3-PBA degradation by fungi, as shown in Fig. 1. Generally, the metabolites of 3-PBA degradation could be identified by gas chromatography-mass spectrometry [10,11]. The strain Aspergillus oryzae M-4 that is able to degrade 3-PBA via co-metabolism was obtained from the soy sauce koji. The intermediate metabolites of 3-PBA degradation include 3-hydroxy-5-phenoxy benzoic acid, phenol, gallic acid, and catechol [12,13]. The concentrations of 3-PBA and its intermediate metabolites can be detected by high-performance liquid chromatography (HPLC) following the  [14].
The same volumes of filter liquor and cell debris solutions were added into phosphate buffer solution containing 0.048 mM each of 3-PBA, and intermediate metabolites and the degradation of the compounds was measured. The reaction was conducted in different duration (24, 48, 72 h) at 30 C and 180 rpm. Then, the reaction was terminated by adding equal volumes of acetonitrile. The concentrations of 3-PBA or its potential metabolites were measured, and degradation was calculated. The results indicated that degradation of 3-PBA and its potential metabolites by the filter liquor and cell debris solutions at 72 h was not significantly higher than that at 48 h. The location of enzymes involving 3-PBA and intermediate metabolites were understood.
Based on the chemical structures of 3-PBA and intermediate metabolites and fungal enzyme system, the candidate enzymes could be speculated. Then, compared the enzymes activities in the treatments with and without 3-PBA and analyzed the degrading-enzymes. One and half mL of the inoculum of degrading-strain was added to a 250 mL Erlenmeyer flask containing 30 mL of medium with 0.24 mM 3-PBA. The concentrations of 3-PBA and its potential metabolites were measured after incubation at 180 rpm and 30 C for 24, 48, 72 and 96 h, respectively. The sample with the greatest shift in concentrations was selected. The cultures of degrading-strain were filtered through a paper filter, and both the mycelial pellets and filter liquor were collected to determine the activity of the following enzymes. Cytochrome P450 (CYP450) activity was measured using previously described methods [15]. The activity of lignin peroxidase (LiP), laccase, and manganese peroxidase (MnP) was determined according to Guo et al. (2014) [9]. Dioxygenase activity was measured according to Sari et al. (2012) [16]. The candidate enzymes could be speculated by the location of enzymes, the inducible or noninducible nature of enzymes, and the chemical structures of intermediate metabolites.
In order to identify the enzymes involving 3-PBA or its intermediate metabolites from the candidate enzymes, the degradation of these compounds was studied in presence of the inhibitors and inducers of these enzymes. The inhibitors and inducers of CYP450, LiP, laccase, MnP, and dioxygenase were selected according to previous reporters [17][18][19][20]. Some solutions of enzymes inhibitors were prepared. CuSO 4 , NaN 3 , and AgNO 3 were dissolved in water, piperonyl butoxide (PBO) dissolved in EtOH, and EDTA was dissolved in NaOH solution (10 %, w/v). The degradation was proceeded in five

Conclusion
In this study, a new method for investigating the degradation enzymes of 3-PBA and its intermediate metabolites was present. The candidate enzymes were analyzed based on the chemical structure of 3-PBA and its intermediate metabolites and the reported property of fungi. The location and induction of enzymes were used to evaluate the candidate enzymes, and predict the enzymes inhibitors and inducers. Further, the effects of inhibitors and inducers on degradation of 3-PBA and intermediate metabolites were used to analyzed the degrading-enzymes. These procedures will be a helpful guide for researchers to analyze the co-metabolic enzymes of 3-PBA degradation by fungi