Cellulose-Based Hectocycle Nanopolymers: Synthesis, Molecular Docking and Adsorption of Difenoconazole from Aqueous Medium.

The goal of this work was to develop polymer-based heterocycle for water purification from toxic pesticides such as difenoconazole. The polymer chosen for this purpose was cellulose nanocrystalline (CNC); two cellulose based heterocycles were prepared by crosslinking with 2,6-pyridine dicarbonyl dichloride (Cell-X), and derivatizing with 2-furan carbonyl chloride (Cell-D). The synthesized cellulose-based heterocycles were characterized by SEM, proton NMR, TGA and FT-IR spectroscopy. To optimize adsorption conditions, the effect of various variable such as time, adsorbent dose, pH, temperature, and difenoconazole initial concentration were evaluated. Results showed that, the maximum difenoconazole removal percentage was about 94.7%, and 96.6% for Cell-X and Cell-D, respectively. Kinetic and thermodynamic studies on the adsorption process showed that the adsorption of difenoconazole by the two polymers is a pseudo-second order and follows the Langmuir isotherm model. The obtained values of ∆G ° and ∆H suggest that the adsorption process is spontaneous at room temperature. The results showed that Cell-X could be a promising adsorbent on a commercial scale for difenoconazole. The several adsorption sites present in Cell-X in addition to the semi crown ether structure explains the high efficiency it has for difenoconazole, and could be used for other toxic pesticides. Monte Carlo (MC) and Molecular Dynamic (MD) simulation were performed on a model of Cell-X and difenoconazole, and the results showed strong interaction.

New Insight into Justicidin B Pathway and Production in Linum austriacum.

Lignans are the main secondary metabolites synthetized by Linum species as plant defense compounds but they are also valuable for human health, in particular, for novel therapeutics. In this work, Linum austriacum in vitro cultures, cells (Cc), adventitious roots (ARc) and hairy roots (HRc) were developed for the production of justicidin B through elicitation with methyl jasmonate (MeJA) and coronatine (COR). The performances of the cultures were evaluated for their stability, total phenols content and antioxidant ability. NMR was used to identify justicidin B and isojusticidin B and HPLC to quantify the production, highlighting ARc and HRc as the highest productive tissues. MeJA and COR treatments induced the synthesis of justicidin B more than three times and the synthesis of other compounds. RNA-sequencing and a de novo assembly of L. austriacum ARc transcriptome was generated to identify the genes activated by MeJA. Furthermore, for the first time, the intracellular localization of justicidin B in ARc was investigated through microscopic analysis. Then, HRc was chosen for small-scale production in a bioreactor. Altogether, our results improve knowledge on justicidin B pathway and cellular localization in L. austriacum for future scale-up processes.

Immune Modulation by Inhibitors of the HO System.

The heme oxygenase (HO) system involves three isoforms of this enzyme, HO-1, HO-2, and HO-3. The three of them display the same catalytic activity, oxidating the heme group to produce biliverdin, ferrous iron, and carbon monoxide (CO). HO-1 is the isoform most widely studied in proinflammatory diseases because treatments that overexpress this enzyme promote the generation of anti-inflammatory products. However, neonatal jaundice (hyperbilirubinemia) derived from HO overexpression led to the development of inhibitors, such as those based on metaloproto- and meso-porphyrins inhibitors with competitive activity. Further, non-competitive inhibitors have also been identified, such as synthetic and natural imidazole-dioxolane-based, small synthetic molecules, inhibitors of the enzyme regulation pathway, and genetic engineering using iRNA or CRISPR cas9. Despite most of the applications of the HO inhibitors being related to metabolic diseases, the beneficial effects of these molecules in immune-mediated diseases have also emerged. Different medical implications, including cancer, Alzheimer´s disease, and infections, are discussed in this article and as to how the selective inhibition of HO isoforms may contribute to the treatment of these ailments.

Enantioselective Behavior of Chiral Difenoconazole in Apple and Field Soil.

Difenoconazole is a universal chiral fungicide which is widely used in apples. Recently, it is still employed as racemic mixtures without distinction of the enantiomers, which may lead to an incomplete risk assessment. Here, we analyzed the stereoselective degradation of difenoconazole in apple fruits and open-field soil using an HPLC-UV system. Different trends were established in various apple varieties under identical environmental conditions. No significant differences were found in its enantioselectivity of the degradation processes applied in the field soil of an apple orchard. However, preferential dissipation of (2R,4R)-difenoconazole and (2R,4S)-difenoconazole was observed in Hanfu and Fuji apples, resulting in the enrichment of stereoisomers of (2S,4S)-difenoconazole and (2S,4R)-difenoconazole. Meanwhile, no significant enantioselectivity was detected in Huahong apples. The present study will provide additional information that contributes to the comprehensive evaluation of the risks posed by the application of chiral difenoconazole in agricultural production practices.

Human Platelets Utilize Cycloxygenase-1 to Generate Dioxolane A3, a Neutrophil-activating Eicosanoid.

Eicosanoids are important mediators of fever, pain, and inflammation that modulate cell signaling during acute and chronic disease. We show by using lipidomics that thrombin-activated human platelets generate a new type of eicosanoid that both stimulates and primes human neutrophil integrin (Mac-1) expression, in response to formylmethionylleucylphenylalanine. Detailed characterization proposes a dioxolane structure, 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (dioxolane A3, DXA3). The lipid is generated in nanogram amounts by platelets from endogenous arachidonate during physiological activation, with inhibition by aspirin in vitro or in vivo, implicating cyclooxygenase-1 (COX). Pharmacological and genetic studies on human/murine platelets revealed that DXA3 formation requires protease-activated receptors 1 and 4, cytosolic phospholipase A2 (cPLA2), Src tyrosine kinases, p38 MAPK, phospholipase C, and intracellular calcium. From data generated by purified COX isoforms and chemical oxidation, we propose that DXA3 is generated by release of an intermediate from the active site followed by oxygenation at C8. In summary, a new neutrophil-activating platelet-derived lipid generated by COX-1 is presented that can activate or prime human neutrophils, suggesting a role in innate immunity and acute inflammation.

Sex-specific effects of difenoconazole on the growth hormone endocrine axis in adult zebrafish (Danio rerio).

Difenoconazole, as one of the most widely used triazole fungicides, is applied to protect crops, fruits, and vegetables. It has been reported that difenoconazole can enter the environment and impair aquatic organisms, but whether difenoconazole can disrupt the growth hormone (GH) balance in adult zebrafish (Danio rerio) is still unclear. In this study, adult female and male zebrafish were exposed to difenoconazole (0, 5, 50, and 500µg/L) for seven days. The results revealed that the bioaccumulation of difenoconazole and its primary metabolite difenoconazole alcohol in females were both larger than that in males. In females, the growth of the liver and ovary were inhibited, which may be due to the decreased transcription of the key genes igf1a, igf2a, and igf2b in both organs. Male fish growth was promoted in response to the increased expression of genes relevant to the GH/insulin-like growth factor axis (GH/IGF) axis in the brain, liver, and testis as well as increased GH levels. It was found that difenoconazole interfered with the growth endocrine system and sex-specifically altered the expression of GH/IGF axis related genes in adult zebrafish after a short-term exposure.

Bioaccumulation and the expression of hepatic cytochrome P450 genes in marine medaka (Oryzias melastigma) exposed to difenoconazole.

This study was conducted to assess the effects of difenoconazole (DFZ), a triazole fungicide, on the hepatic biotransformation system and its bioaccumulation in marine medaka (Oryzias melastigma). Fish were exposed to DFZ (1, 10, 100, 1000ng/L) for 180days. The results showed that: (1) The mRNA levels of hepatic CYP1A1, CYP1B, CYP1C1, CYP27B and CYP3A40 were up-regulated, but those of CYP3A38 and CYP27A1 were down-regulated. (2) The activity of ethoxyresorufin-O-deethylase (EROD) and the content of reduced glutathione (GSH) in the liver were increased in the DFZ-treated groups, and glutathione S-transferase (GST) activity was increased in the 100 and 1000ng/L groups. (3) DFZ was accumulated in the muscle and the biological concentration factors in the 10, 100, and 1000ng/L groups were respectively 149, 81 and 25. These results suggested that long-term exposure to DFZ at low concentrations would result in a bioaccumulation of this compound and disturb the biotransformation system.

Characterization of the product of a nonribosomal peptide synthetase-like (NRPS-like) gene using the doxycycline dependent Tet-on system in Aspergillus terreus.

Genome sequencing of the fungus Aspergillus terreus uncovered a number of silent core structural biosynthetic genes encoding enzymes presumed to be involved in the production of cryptic secondary metabolites. There are five nonribosomal peptide synthetase (NRPS)-like genes with the predicted A-T-TE domain architecture within the A. terreus genome. Among the five genes, only the product of pgnA remains unknown. The Tet-on system is an inducible, tunable and metabolism-independent expression system originally developed for Aspergillus niger. Here we report the adoption of the Tet-on system as an effective gene activation tool in A. terreus. Application of this system in A. terreus allowed us to uncover the product of the cryptic NRPS-like gene, pgnA. Furthermore expression of pgnA in the heterologous Aspergillus nidulans host suggested that the pgnA gene alone is necessary for phenguignardic acid (1) biosynthesis.

Tracing the dissipation of difenoconazole, its metabolites and co-formulants in tomato: A comprehensive analysis by chromatography coupled to high resolution mass spectrometry in laboratory and greenhouse trials.

The study evaluated Ceremonia 25 EC®, a plant protection product (PPP) containing difenoconazole, in tomato crops, to identify potential risks associated with PPPs, and in addition to this compound, known metabolites from difenoconazole degradation and co-formulants present in the PPP were monitored. An ultra high performance liquid chromatography coupled to quadrupole-Orbitrap mass analyser (UHPLC-Q-Orbitrap-MS) method was validated with a working range of 2 µg/kg (limit of quantification, LOQ) to 200 µg/kg. Difenoconazole degradation followed a biphasic double first-order in parallel (DFOP) kinetic model in laboratory and greenhouse trials, with high accuracy (R2 > 0.9965). CGA-205374, difenoconazole-alcohol, and hydroxy-difenoconazole metabolites were tentatively identified and semi-quantified in laboratory trials by UHPLC-Q-Orbitrap-MS from day 2 to day 30. No metabolites were found in greenhouse trials. Additionally, 13 volatile co-formulants were tentatively identified by gas chromatography (GC) coupled to Q-Orbitrap-MS, detectable up to the 7th day after PPP application. This study provides a comprehensive understanding of difenoconazole dissipation in tomatoes, identification of metabolites, and detection of co-formulants associated with the applied PPP.

Enantioselective bioaccumulation, biotransformation and spatial distribution of chiral fungicide difenoconazole in earthworms (Eisenia fetida).

The enantioselective environmental behavior of difenoconazole, a widely utilized triazole fungicide commonly detected in agricultural soils, has yet to be comprehensively explored within the earthworm-soil system. To address this research gap, we investigated the bioaccumulation and elimination kinetics, degradation pathways, biotransformation mechanisms, spatial distribution, and toxicity of chiral difenoconazole. The four stereoisomers of difenoconazole were baseline separated and analyzed using SFC-MS/MS. Pronounced enantioselectivity was observed during the uptake phase, with earthworms exhibiting a preference for (2R,4R)-difenoconazole and (2R,4S)-difenoconazole. A total of five transformation products (TPs) were detected and identified using UHPLC-QTOF/MS in the earthworm-soil system. Four of the TPs were detected in both earthworm and soil, and one TP was produced only in eaerthwroms. Hydrolysis and hydroxylation were the primary transformation pathways of difenoconazole in both earthworms and soil. Furthermore, a chiral TP, 3-chloro, 4-hydroxy difenoconazole, was generated with significant enantioselectivity, and molecular docking results indicate the greater catalytic bioactivity of (2R,4R)- and (2R,4S)-difenoconazole, leading to the preferential formation of their corresponding hydroxylated TPs. Furthermore, Mass Spectrometry Imaging (MSI) was applied for the first time to explore the spatial distribution of difenoconazole and the TPs in earthworms, and the "secretory zone" was found to be the dominant region to uptake and biodegrade difenoconazole. ECOSAR predictions highlighted the potentially hazardous impact of most difenoconazole TPs on aquatic ecosystems. These findings are important for understanding the environmental fate of difenoconazole, evaluating environmental risks, and offering valuable insights for guiding scientific bioremediation efforts.

RNA Adduction Resulting from the Metabolic Activation of Myristicin by P450 Enzymes and Sulfotransferases.

Myristicin (MYR) mainly occurs in nutmeg and belongs to alkoxy-substituted allylbenzenes, a class of potentially toxic natural chemicals. RNA interaction with MYR metabolites in vitro and in vivo has been investigated in order to gain a better understanding of MYR toxicities. We detected two guanosine adducts (GA1 and GA2), two adenosine adducts (AA1 and AA2), and two cytosine adducts (CA1 and CA2) by LC-MS/MS analysis of total RNA extracts from cultured primary mouse hepatocytes and liver tissues of mice after exposure to MYR. An order of nucleoside adductions was found to be GAs > AAs > CAs, and the result of density functional theory calculations was in agreement with that detected by the LC-MS/MS-based approach. In vitro and in vivo studies have shown that MYR was oxidized by cytochrome P450 enzymes to 1'-hydroxyl and 3'-hydroxyl metabolites, which were then sulfated by sulfotransferases (SULTs) to form sulfate esters. The resulting sulfates would react with the nucleosides by SN1 and/or SN2 reactions, resulting in RNA adduction. The modification may alter the biochemical properties of RNA and disrupt RNA functions, perhaps partially contributing to the toxicities of MYR.

Effect of growth stage on essential-oil yield and composition of Daucus sahariensis.

The essential oils obtained by hydrodistillation from Daucus sahariensis Murb. harvested at three different growth stages were characterized by GC/MS analysis. In total, 88 compounds were identified, with myristicin (29.8-51.7%), myrcene (6.7-31.1%), α-pinene (11.6-14.8%), and limonene (5.3-11.5%) as main constituents. Monoterpene hydrocarbons were the most represented compounds in the oils of the plant samples collected during the flower-budding and full-flowering periods. On the contrary, during the fruiting stage, the oils were dominated by phenylpropanoids. The essential oils were subject of considerable variation in their composition during the various developmental stages, particularly concerning the content of myrcene that decreased significantly passing from the vegetative to the fruiting stage. Conversely, for myristicin, the opposite trend was observed. Furthermore, the essential-oil yields were quite low during the flower-budding phase (0.27%), but rapidly increased during plant development (0.63 and 0.68% for the flowering and fruiting phases, resp.).

Stereospecific metabolism of itraconazole by CYP3A4: dioxolane ring scission of azole antifungals.

Itraconazole (ITZ) is a mixture of four cis-stereoisomers that inhibit CYP3A4 potently and coordinate CYP3A4 heme via the triazole nitrogen. However, (2R,4S,2'R)-ITZ and (2R,4S,2'S)-ITZ also undergo stereoselective sequential metabolism by CYP3A4 at a site distant from the triazole ring to 3'-OH-ITZ, keto-ITZ, and N-desalkyl-ITZ. This stereoselective metabolism demonstrates specific interactions of ITZ within the CYP3A4 active site. To further investigate this process, the binding and metabolism of the four trans-ITZ stereoisomers by CYP3A4 were characterized. All four trans-ITZ stereoisomers were tight binding inhibitors of CYP3A4-mediated midazolam hydroxylation (IC(50) 16-26 nM), and each gave a type II spectrum upon binding to CYP3A4. However, instead of formation of 3'-OH-ITZ, they were oxidized at the dioxolane ring, leading to ring scission and formation of two new metabolites of ITZ. These two metabolites were also formed from the four cis-ITZ stereoisomers, although not as efficiently. The catalytic rates of dioxolane ring scission were similar to the dissociation rates of ITZ stereoisomers from CYP3A4, suggesting that the heme iron is reduced while the triazole moiety coordinates to it and no dissociation of ITZ is necessary before catalysis. The triazole containing metabolite [1-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethanone] also inhibited CYP3A4 (IC(50) >15 µM) and showed type II binding with CYP3A4. The dioxolane ring scission appears to be clinically relevant because this metabolite was detected in urine samples from subjects that had been administered the mixture of cis-ITZ isomers. These data suggest that the dioxolane ring scission is a metabolic pathway for drugs that contain this moiety.

Specific 5-hydroxylation of piperlongumine by Beauveria bassiana ATCC 7159.

A new hydroxylated derivative was efficiently prepared by transforming the natural anti-cancer product, piperlongumine, with Beauveria bassiana ATCC 7159. Its structure was determined to be 5-hydroxylpiperlongumine on the basis of the spectroscopic data. The absolute configuration at C-5 was established as R by Mosher's method.

Effects of a naturally occurring and a synthetic synergist on toxicity of three insecticides and a phytochemical to navel orangeworm (Lepidoptera: Pyralidae).

The navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae), is the most destructive lepidopteran pest of almonds [Prunus dulcis (Mill.) D.A.Webb] and pistachios (Pistacia vera L.) in California and is a serious problem in figs (Ficus carica L.) and walnuts (Juglans spp.). In addition to direct damage, larval feeding leaves nuts vulnerable to infection by Aspergillus spp., fungi that produce toxic aflatoxins. A potentially safe and sustainable approach for managing navel orangeworm in orchards may be to use natural essential oil synergists to interfere with this insect's ability to detoxify insecticides and phytochemicals. We tested the effects of a naturally occurring plant-derived chemical, myristicin, and a synthetic inhibitor of cytochrome P450 monooxygenases (P450s), piperonyl butoxide, on the toxicity of three insecticides (alpha-cypermethrin, tau-fluvalinate, and methoxyfenozide [Intrepid]) and a phytochemical (xanthotoxin) to A. transitella. Piperonyl butoxide significantly synergized alpha-cypermethrin and tau-fluvalinate, whereas myristicin synergized only alpha-cypermethrin. Piperonyl butoxide synergized the toxicity of xanthotoxin as early as 72 h after exposure, whereas myristicin synergized xanthotoxin after 120 h. In view of these findings and the limited availability of environmentally safe synthetic insecticides for sustainable management, particularly in organic orchards, myristicin is a potential field treatment in combination with insecticides to reduce both navel orangeworm survival and aflatoxin contamination of nuts. In addition, this study demonstrates that in A. transitella the insect growth regulator methoxyfenozide is not detoxified by P450s.

Piperlongumine inhibits diethylnitrosamine induced hepatocellular carcinoma in rats.

BACKGROUND: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Piperlongumine (PL) has been claimed to have cytotoxic and HCC inhibitory effects in various cancer cell lines and xenograft models, but the chemopreventive potential of PL has not been studied in experimentally induced HCC yet. RESEARCH DESIGN: Twenty-four Wistar male rats were divided into four groups of six each, Group A: untreated control; Group B: Diethylnitrosamine (DEN) control (200 mg/kg), Group C: DEN + PL 10 mg/kg; and Group D: DEN + PL 20 mg/kg. Rats from all groups were assessed for liver cancer progression or inhibition by evaluating biochemical, cytokines, tumor markers, lipid peroxidation, and histological profiles. RESULTS: The liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP) levels, and lipid peroxidation were significantly decreased in Group C and Group D compared to Group B. Upregulation in the level of pro-inflammatory cytokines IL-1B, TNF-α, inflammatory mediator (NF-κB) and tumour marker alpha-fetoprotein (AFP) in Group B were brought down upon treatment with piperlongumine in a dose-dependent manner. Antitumor cytokine (IL-12) was upregulated in PL-treated rats compared to DEN control rats. DEN treated group (Group B) showed histological features of HCC, and in rats treated with PL (Groups C, D) partial to complete reversal to normal liver histoarchitecture was observed. CONCLUSIONS: The potential chemopreventive actions of piperlongumine may be due to its free radical scavenging and antiproliferative effect. Therefore, piperlongumine may serve as a novel therapeutic agent for the treatment of hepatocellular carcinoma.

Piperlongumine inhibits the progression of osteosarcoma by downregulating the SOCS3/JAK2/STAT3 pathway via miR-30d-5p.

AIMS: The present study evaluated the functions of Piperlongumine (PL) in osteosarcoma (OS) cell growth and metastasis both in vitro and in vivo. MAIN METHODS: MTT assay was conducted to test the cytotoxic effects of PL on the human osteoblasts line HFOB1.19 and the human normal chondrocyte line C28/I2T. FITC-Annexin V and propidium iodide (PI) were used to examine cell apoptosis. The migration, invasion and relative epithelial-mesenchymal transition were examined by Transwell assay and Western blotting. Reverse transcription-quantitative PCR (RT-qPCR) was performed to analyze the cytokine signaling 3 (SOCS3) mRNA expression. TargetScan database was used to predict the target of SOCS3. The binding association between miR-30d-5p and SOCS3 in U2OS and MG63 cells was evaluated by the dual-luciferase reporter assay. A xenograft model was constructed to evaluate the effect of PL on OS cell growth in vivo. KEY FINDINGS: The results revealed that PL inhibited the growth, migration, invasion, epithelial-mesenchymal transition, and promoted the apoptosis of OS cells dose-dependently. In addition, PL upregulated the protein levels of suppressor of SOCS3, while it inactivated the JAK2/STAT3 pathway, which was accompanied by a decreased level of microRNA (miR)-30d-5p. Furthermore, SOCS3was confirmed as a novel target of miR-30d-5p. Overexpression of miR-30d-5p not only led to decreased expression of SOCS3, but also dampened the antitumor effect of PL on OS. SIGNIFICANCE: The present data demonstrated that PL inhibited the progression of OS via downregulation of the SOCS3-mediated JAK2/STAT3 pathway by inhibiting miR-30d-5p.

Investigation on the interaction of prulifloxacin with human serum albumin: a spectroscopic analysis.

The interaction between prulifloxacin (PUFX) and human serum albumin (HSA) was investigated under simulated physiologic conditions with fluorescence spectra. The fluorescence quenching process of HSA may be mainly governed by a static quenching mechanism. The apparent binding constant K(b) between PUFX and HSA at different temperatures were 2.08+/-1.04, 2.74+/-0.50, and 4.98+/-1.61x10(8) l/mol. The thermodynamic parameters, with a negative value of DeltaG(0), revealed that the binding is a spontaneous process. A binding distance R of 1.19 nm between donor and acceptor was obtained from the Forster energy transfer theory.

Bioaccessibility of Difenoconazole in Rice Following Industry Standard Processing and Preparation Procedures.

For pesticide registration a post application assessment is made on the safety of any residue remaining in the edible portion of the treated crop. This assessment does not typically consider the bioaccessibility of pesticide residues. The effects of this on potential exposure to incurred difenoconazole residues passing through the human gastrointestinal tract were studied, including the impact of commodity processing. It has previously been demonstrated that solvent extraction methods have the potential to overestimate the bioaccessible fraction, so in vitro simulated gut systems may offer a better approach to determine residue bioaccessibility to refine the risk assessment process. The bioaccessibility of difenoconazole residues associated with processed rice samples was assessed using in vitro intestinal extraction and colonic fermentation methods. The mean bioaccessibility following intestinal digestion was 33.3% with a range from 13% to 70.6%. Quantification of the colonic bioaccessible fraction was not possible due to compound metabolism. Mechanical processing methods generally increased the residue bioaccessibility, while chemical methods resulted in a decrease. Both mechanical and chemical processing methods reduced the total difenoconazole residue level by ca. 50%.

Purification and Characterization of Anabaena flos-aquae Phenylalanine Ammonia-Lyase as a Novel Approach for Myristicin Biotransformation.

Phenylalanine ammonia-lyase (PAL) catalyzes the reversible deamination of phenylalanine to cinnamic acid and ammonia. Algae have been considered as biofactories for PAL production, however, biochemical characterization of PAL and its potency for myristicin biotransformation into MMDA (3-methoxy-4, 5-methylenedioxyamphetamine) has not been studied yet. Thus, PAL from Anabaena flos-aquae and Spirulina platensis has been purified, comparatively characterized and its affinity to transform myristicin was assessed. The specific activity of purified PAL from S. platensis (73.9 µmol/mg/min) and A. flos-aquae (30.5 µmol/mg/min) was increased by about 2.9 and 2.4 folds by gel-filtration comparing to their corresponding crude enzymes. Under denaturing-PAGE, a single proteineous band with a molecular mass of 64 kDa appeared for A. flos-aquae and S. platensis PAL. The biochemical properties of the purified PAL from both algal isolates were determined comparatively. The optimum temperature of S. platensis and A. flos-aquae PAL for forward or reverse activity was reported at 30°C, while the optimum pH for PAL enzyme isolated from A. flos-aquae was 8.9 for forward and reverse activities, and S. platensis PAL had maximum activities at pH 8.9 and 8 for forward and reverse reactions, respectively. Luckily, the purified PALs have the affinity to hydroaminate the myristicin to MMDA successfully in one step. Furthermore, a successful method for synthesis of MMDA from myristicin in two steps was also established. Gas chromatography-mass spectrometry (GC-MS) analysis was conducted to track the product formation.