Searching for new plastic-degrading enzymes from the plastisphere of alpine soils using a metagenomic mining approach.

Plastic materials, including microplastics, accumulate in all types of ecosystems, even in remote and cold environments such as the European Alps. This pollution poses a risk for the environment and humans and needs to be addressed. Using shotgun DNA metagenomics of soils collected in the eastern Swiss Alps at about 3,000 m a.s.l., we identified genes and their proteins that potentially can degrade plastics. We screened the metagenomes of the plastisphere and the bulk soil with a differential abundance analysis, conducted similarity-based screening with specific databases dedicated to putative plastic-degrading genes, and selected those genes with a high probability of signal peptides for extracellular export and a high confidence for functional domains. This procedure resulted in a final list of nine candidate genes. The lengths of the predicted proteins were between 425 and 845 amino acids, and the predicted genera producing these proteins belonged mainly to Caballeronia and Bradyrhizobium. We applied functional validation, using heterologous expression followed by enzymatic assays of the supernatant. Five of the nine proteins tested showed significantly increased activities when we used an esterase assay, and one of these five proteins from candidate genes, a hydrolase-type esterase, clearly had the highest activity, by more than double. We performed the fluorescence assays for plastic degradation of the plastic types BI-OPL and ecovio® only with proteins from the five candidate genes that were positively active in the esterase assay, but like the negative controls, these did not show any significantly increased activity. In contrast, the activity of the positive control, which contained a PLA-degrading gene insert known from the literature, was more than 20 times higher than that of the negative controls. These findings suggest that in silico screening followed by functional validation is suitable for finding new plastic-degrading enzymes. Although we only found one new esterase enzyme, our approach has the potential to be applied to any type of soil and to plastics in various ecosystems to search rapidly and efficiently for new plastic-degrading enzymes.

Rifampicin synergizes the toxicity of insecticides against the green peach aphid, Myzus persicae.

Myzus persicae is an important pest that has developed resistance to nearly all currently used insecticidal products. The employment of insecticide synergists is one of the effective strategies that need to be developed for the management of this resistance. Our study showed that treatment with a combination of the antibiotic, rifampicin, with imidacloprid, cyantraniliprole, or clothianidin significantly increased their toxicities against M. persicae, by 2.72, 3.59, and 2.41 folds, respectively. Rifampicin treatment led to a noteworthy reduction in the activities of multifunctional oxidases (by 32.64%) and esterases (by 23.80%), along with a decrease in the expression of the CYP6CY3 gene (by 58.57%) in M. persicae. It also negatively impacted the fitness of the aphids, including weight, life span, number of offspring, and elongation of developmental duration. In addition, bioassays showed that the combination of rifampicin and a detoxification enzyme inhibitor, piperonyl butoxide, or dsRNA of CYP6CY3 further significantly improved the toxicity of imidacloprid against M. persicae, by 6.19- and 7.55-fold, respectively. The present study suggests that development of active ingredients such as rifampicin as candidate synergists, show promise to overcome metabolic resistance to insecticides in aphids.

Quantitative proteomic analysis reveals the mechanism and key esterase of ß-cypermethrin degradation in a bacterial strain from fermented food.

Beta-cypermethrin (ß-CY) residues in food are an important threat to human health. Microorganisms can degrade ß-CY residues during fermentation of fruits and vegetables, while the mechanism is not clear. In this study, a comprehensively investigate of the degradation mechanism of ß-CY in a food microorganism was conducted based on proteomics analysis. The ß-CY degradation bacteria Gordonia alkanivorans GH-1 was derived from fermented Pixian Doubanjiang. Its crude enzyme extract could degrade 77.11% of ß-CY at a concentration of 45 mg/L within 24 h. Proteomics analysis revealed that the ester bond of ß-CY is broken under the action of esterase to produce 3-phenoxy benzoic acid, which was further degraded by oxidoreductase and aromatic degrading enzyme. The up-regulation expression of oxidoreductase and esterase was confirmed by transcriptome and quantitative reverse transcription PCR. Meanwhile, the expression of esterase Est280 in Escherichia coli BL21 (DE3) resulted in a 48.43% enhancement in the degradation efficiency of ß-CY, which confirmed that this enzyme was the key enzyme in the process of ß-CY degradation. This study reveals the degradation mechanism of ß-CY by microorganisms during food fermentation, providing a theoretical basis for the application of food microorganisms in ß-CY residues.

Monomeric Esterase: Insights into Cooperative Behavior, Hysteresis/Allokairy.

Herein, we present a novel esterase enzyme, Ade1, isolated from a metagenomic library of Amazonian dark earths soils, demonstrating its broad substrate promiscuity by hydrolyzing ester bonds linked to aliphatic groups. The three-dimensional structure of the enzyme was solved in the presence and absence of substrate (tributyrin), revealing its classification within the α/ß-hydrolase superfamily. Despite being a monomeric enzyme, enzymatic assays reveal a cooperative behavior with a sigmoidal profile (initial velocities vs substrate concentrations). Our investigation brings to light the allokairy/hysteresis behavior of Ade1, as evidenced by a transient burst profile during the hydrolysis of substrates such as p-nitrophenyl butyrate and p-nitrophenyl octanoate. Crystal structures of Ade1, coupled with molecular dynamics simulations, unveil the existence of multiple conformational structures within a single molecular state (E̅1). Notably, substrate binding induces a loop closure that traps the substrate in the catalytic site. Upon product release, the cap domain opens simultaneously with structural changes, transitioning the enzyme to a new molecular state (E̅2). This study advances our understanding of hysteresis/allokairy mechanisms, a temporal regulation that appears more pervasive than previously acknowledged and extends its presence to metabolic enzymes. These findings also hold potential implications for addressing human diseases associated with metabolic dysregulation.

Bioorthogonal masked acylating agents for proximity-dependent RNA labelling.

RNA localization is highly regulated, with subcellular organization driving context-dependent cell physiology. Although proximity-based labelling technologies that use highly reactive radicals or carbenes provide a powerful method for unbiased mapping of protein organization within a cell, methods for unbiased RNA mapping are scarce and comparably less robust. Here we develop α-alkoxy thioenol and chloroenol esters that function as potent acylating agents upon controlled ester unmasking. We pair these probes with subcellular-localized expression of a bioorthogonal esterase to establish a platform for spatial analysis of RNA: bioorthogonal acylating agents for proximity labelling and sequencing (BAP-seq). We demonstrate that, by selectively unmasking the enol probe in a locale of interest, we can map RNA distribution in membrane-bound and membrane-less organelles. The controlled-release acylating agent chemistry and corresponding BAP-seq method expand the scope of proximity labelling technologies and provide a powerful approach to interrogate the cellular organization of RNAs.

Methionine inducing carbohydrate esterase secretion of Trichoderma harzianum enhances the accessibility of substrate glycosidic bonds.

BACKGROUND: The conversion of plant biomass into biochemicals is a promising way to alleviate energy shortage, which depends on efficient microbial saccharification and cellular metabolism. Trichoderma spp. have plentiful CAZymes systems that can utilize all-components of lignocellulose. Acetylation of polysaccharides causes nanostructure densification and hydrophobicity enhancement, which is an obstacle for glycoside hydrolases to hydrolyze glycosidic bonds. The improvement of deacetylation ability can effectively release the potential for polysaccharide degradation. RESULTS: Ammonium sulfate addition facilitated the deacetylation of xylan by inducing the up-regulation of multiple carbohydrate esterases (CE3/CE4/CE15/CE16) of Trichoderma harzianum. Mainly, the pathway of ammonium-sulfate's cellular assimilates inducing up-regulation of the deacetylase gene (Thce3) was revealed. The intracellular metabolite changes were revealed through metabonomic analysis. Whole genome bisulfite sequencing identified a novel differentially methylated region (DMR) that existed in the ThgsfR2 promoter, and the DMR was closely related to lignocellulolytic response. ThGsfR2 was identified as a negative regulatory factor of Thce3, and methylation in ThgsfR2 promoter released the expression of Thce3. The up-regulation of CEs facilitated the substrate deacetylation. CONCLUSION: Ammonium sulfate increased the polysaccharide deacetylation capacity by inducing the up-regulation of multiple carbohydrate esterases of T. harzianum, which removed the spatial barrier of the glycosidic bond and improved hydrophilicity, and ultimately increased the accessibility of glycosidic bond to glycoside hydrolases.

Variations of the NodB Architecture Are Attuned to Functional Specificities into and beyond the Carbohydrate Esterase Family 4.

Enzymes of the carbohydrate esterase family 4 (CE4) deacetylate a broad range of substrates, including linear, branched and mesh-like polysaccharides. Although they are enzymes of variable amino acid sequence length, they all comprise the conserved catalytic domain NodB. NodB carries the metal binding and active site residues and is characterized by a set of conserved sequence motifs, which are linked to the deacetylation activity. Besides a non-structured, flexible peptide of variable length that precedes NodB, several members of the CE4 family contain additional domains whose function or contribution to substrate specificity are not efficiently characterized. Evidence suggests that CE4 family members comprising solely the NodB domain have developed features linked to a variety of substrate specificities. To understand the NodB-based substrate diversity within the CE4 family, we perform a comparative analysis of all NodB domains structurally characterized so far. We show that amino acid sequence variations, topology diversities and excursions away from the framework structure give rise to different NodB domain classes associated with different substrate specificities and particular functions within and beyond the CE4 family. Our work reveals a link between specific NodB domain characteristics and substrate recognition. Thus, the details of the fold are clarified, and the structural basis of its variations is deciphered and associated with function. The conclusions of this work are also used to make predictions and propose specific functions for biochemically/enzymatically uncharacterized NodB-containing proteins, which have generally been considered as putative CE4 deacetylases. We show that some of them probably belong to different enzymatic families.

Synthesis of eco-friendly layered double hydroxide and nanoemulsion for jasmine and peppermint oils and their larvicidal activities against Culex pipiens Linnaeus.

Mosquito-borne diseases represent a growing health challenge over time. Numerous potential phytochemicals are target-specific, biodegradable, and eco-friendly. The larvicidal activity of essential oils, a jasmine blend consisting of Jasmine oil and Azores jasmine (AJ) (Jasminum sambac and Jasminum azoricum) and peppermint (PP) Mentha arvensis and their nanoformulations against 2nd and 4th instar larvae of Culex pipiens, was evaluated after subjecting to different concentrations (62.5, 125, 250, 500, 1000, and 2000 ppm). Two forms of phase-different nanodelivery systems of layered double hydroxide LDH and oil/water nanoemulsions were formulated. The synthesized nanoemulsions showed particle sizes of 199 and 333 nm for AJ-NE and PP-NE, with a polydispersity index of 0.249 and 0.198, respectively. Chemical and physiochemical analysis of TEM, SEM, XRD, zeta potential, drug loading capacity, and drug release measurements were done to confirm the synthesis and loading efficiencies of essential oils' active ingredients. At high concentrations of AJ and PP nanoemulsions (2000 ppm), O/W nanoemulsions showed higher larval mortality than both LDH conjugates and crude oils. The mortality rate reached 100% for 2nd and 4th instar larvae. The relative toxicities revealed that PP nanoemulsion (MA-NE) was the most effective larvicide, followed by AJ nanoemulsion (AJ-NE). There was a significant increase in defensive enzymes, phenoloxidase, and α and ß-esterase enzymes in the treated groups. After treatment of L4 with AJ, AJ-NE, PP, and PP-NE, the levels of phenoloxidase were 545.67, 731.00, 700.00, and 799.67 u/mg, respectively, compared with control 669.67 u/mg. The activity levels of α-esterase were 9.71, 10.32, 8.91, and 10.55 mg α-naphthol/min/mg protein, respectively. It could be concluded that the AJ-NE and PP-NE nanoformulations have promising larvicidal activity and could act as safe and effective alternatives to chemical insecticides.

Computer-aided rational design strategy based on protein surface charge to improve the thermal stability of a novel esterase from Geobacillus jurassicus.

OBJECTIVES: Although Geobacillus are significant thermophilic bacteria source, there are no reports of thermostable esterase gene in Geobacillus jurassicus or rational design strategies to increase the thermal stability of esterases. RESULTS: Gene gju768 showed a highest similarity of 15.20% to esterases from Geobacillus sp. with detail enzymatic properties. Using a combination of Gibbs Unfolding Free Energy (∆∆G) calculator and the distance from the mutation site to the catalytic site (DsCα-Cα) to screen suitable mutation sites with elimination of negative surface charge, the mutants (D24N, E221Q, and E253Q) displayed stable mutants with higher thermal stability than the wild-type (WT). Mutant E253Q exhibited the best thermal stability, with a half-life (T1/2) at 65 °C of 32.4 min, which was 1.8-fold of the WT (17.9 min). CONCLUSION: Cloning of gene gju768 and rational design based on surface charge engineering contributed to the identification of thermostable esterase from Geobacillus sp. and the exploration of evolutionary strategies for thermal stability.

Acaricide resistance status of deltamethrin and coumaphos in Hyalomma anatolicum ticks collected from different districts of Haryana.

To study the acaricide resistance status and possible mechanisms of action in conferring resistance to commonly used acaricides (deltamethrin and coumaphos), Hyalomma anatolicum ticks were collected from 6 dairy farms of Hisar and Charkhi Dadri districts of Haryana. By using standard larval packet test, H. anatolicum tick larvae of Charkhi Dadri isolates were found to be susceptible (100% mortality) to both the acaricides. Level-I resistance against coumaphos was recorded from four isolates, whereas, level-II was observed in only one isolate, collected from Hisar. One isolates (Kaimri) from Hisar also showed level-I resistance against deltamethrin. Biochemically, the ticks having higher values of resistance factor (RF) against coumaphos were found to possess increased enzymatic activity of α-esterase, ß-esterase, glutathione-S-transferase (GST) and mono-oxygenase enzymes, whereas, the monoamine oxidase did not show any constant trend. However, the RF showed a statistical significant correlation with GST only. Native PAGE analysis of H. anatolicum ticks revealed the presence of nine types of esterases (EST-1 h to EST-9 h) by using napthyl acetate as substrate. In the inhibitory assay, esterases were found to be inhibited by PMSF, indicating the presence of serine residue at catalytic triad. The partial cds of carboxylesterase and domain II of sodium channel genes were sequenced to determine any proposed mutations in resistant isolates of H. anatolicum ticks, however, no mutations were observed in either gene, indicating that increased expression of detoxification enzymes as a possible mechanism for resistance development, in the current study.

Neutral Sphingomyelinase 2 Inhibition Limits Hepatic Steatosis and Inflammation.

Non-alcoholic fatty liver disease (NAFLD) is manifested by hepatic steatosis, insulin resistance, hepatocyte death, and systemic inflammation. Obesity induces steatosis and chronic inflammation in the liver. However, the precise mechanism underlying hepatic steatosis in the setting of obesity remains unclear. Here, we report studies that address this question. After 14 weeks on a high-fat diet (HFD) with high sucrose, C57BL/6 mice revealed a phenotype of liver steatosis. Transcriptional profiling analysis of the liver tissues was performed using RNA sequencing (RNA-seq). Our RNA-seq data revealed 692 differentially expressed genes involved in processes of lipid metabolism, oxidative stress, immune responses, and cell proliferation. Notably, the gene encoding neutral sphingomyelinase, SMPD3, was predominantly upregulated in the liver tissues of the mice displaying a phenotype of steatosis. Moreover, nSMase2 activity was elevated in these tissues of the liver. Pharmacological and genetic inhibition of nSMase2 prevented intracellular lipid accumulation and TNFα-induced inflammation in in-vitro HepG2-steatosis cellular model. Furthermore, nSMase2 inhibition ameliorates oxidative damage by rescuing PPARα and preventing cell death associated with high glucose/oleic acid-induced fat accumulation in HepG2 cells. Collectively, our findings highlight the prominent role of nSMase2 in hepatic steatosis, which could serve as a potential therapeutic target for NAFLD and other hepatic steatosis-linked disorders.

Reconstructing curcumin biosynthesis in yeast reveals the implication of caffeoyl-shikimate esterase in phenylpropanoid metabolic flux.

Curcumin is a polyphenolic natural product from the roots of turmeric (Curcuma longa). It has been a popular coloring and flavoring agent in food industries with known health benefits. The conventional phenylpropanoid pathway is known to proceed from phenylalanine via p-coumaroyl-CoA intermediate. Although hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase (HCT) plays a key catalysis in the biosynthesis of phenylpropanoid products at the downstream of p-coumaric acid, a recent discovery of caffeoyl-shikimate esterase (CSE) showed that an alternative pathway exists. Here, the biosynthetic efficiency of the conventional and the alternative pathway in producing feruloyl-CoA was examined using curcumin production in yeast. A novel modular multiplex genome-edit (MMG)-CRISPR platform was developed to facilitate rapid integrations of up to eight genes into the yeast genome in two steps. Using this MMG-CRISPR platform and metabolic engineering strategies, the alternative CSE phenylpropanoid pathway consistently showed higher titers (2-19 folds) of curcumin production than the conventional pathway in engineered yeast strains. In shake flask cultures using a synthetic minimal medium without phenylalanine, the curcumin production titer reached up to 1.5 mg/L, which is three orders of magnitude (∼4800-fold) improvement over non-engineered base strain. This is the first demonstration of de novo curcumin biosynthesis in yeast. Our work shows the critical role of CSE in improving the metabolic flux in yeast towards the phenylpropanoid biosynthetic pathway. In addition, we showcased the convenience and reliability of modular multiplex CRISPR/Cas9 genome editing in constructing complex synthetic pathways in yeast.

Antigenic commonality and divergence of hemagglutinin-esterase-fusion protein among influenza D virus lineages revealed using epitope mapping.

Influenza D virus (IDV) is one of the causative agents of bovine respiratory disease complex, which is the most common and economically burdensome disease affecting the cattle industry, and the need for an IDV vaccine has been proposed to enhance disease control. IDVs are classified into five genetic lineages based on the coding sequences of the hemagglutinin-esterase-fusion (HEF) protein, an envelope glycoprotein, which is the main target of protective antibodies against IDV infection. Herein, we prepared a panel of monoclonal antibodies (mAbs) against the HEF protein of viruses of various lineages to investigate the antigenic characteristics of IDVs and found that the mAbs could be largely separated into three groups. The first, second, and third groups demonstrated lineage-specific reactivity, cross-reactivity to viruses of multiple but not all lineages, and cross-reactivity to viruses of all lineages, respectively. Analyzing the escape mutant viruses from virus-neutralizing mAbs revealed that the receptor-binding region of the HEF molecule harbors virus-neutralizing epitopes that are conserved across multiple lineage viruses. In contrast, the apex region of the molecule possessed epitopes unique to each lineage virus. Furthermore, reverse genetics-generated recombinant viruses with point mutations revealed that amino acids within positions 210-214 of the HEF protein determined the antigenic specificity of each lineage virus. Taken together, this study reveals considerable antigenic variation among IDV lineages, although they are presumed to form a single serotype in terms of HEF antigenicity. Characterization of the antigenic epitope structure of HEF may contribute to selecting and creating effective vaccine viruses against IDV.IMPORTANCEInfluenza D viruses (IDVs) are suggested to create cross-reactive single serotypes in hemagglutinin-esterase-fusion (HEF) antigenicity, as indicated by serological analyses among distinct HEF lineage viruses. This is supported by the high identities of HEF gene sequences among strains, unlike the hemagglutinin (HA) genes of the influenza A virus that exhibit HA subtypes. Herein, we analyzed HEF antigenicity using a monoclonal antibody panel prepared from several virus lineages and found the existence of lineage-conserved and lineage-specific epitopes in HEF molecules. These findings confirm the HEF commonality and divergence among IDVs and provide useful information for constructing a vaccine containing a recombinant IDV virus with an engineered HEF gene, thereby leading to broad immunogenicity.

Characterization of a novel amidohydrolase with promiscuous esterase activity from a soil metagenomic library and its application in degradation of amide herbicides.

Amide herbicides have been extensively used worldwide and have received substantial attention due to their adverse environmental effects. Here, a novel amidohydrolase gene was identified from a soil metagenomic library using diethyl terephthalate (DET) as a screening substrate. The recombinant enzyme, AmiH52, was heterologously expressed in Escherichia coli and later purified and characterized, with the highest activity occurring at 40 ℃ and pH 8.0. AmiH52 was demonstrated to have both esterase and amidohydrolase activities, which exhibited highly specific activity for p-nitrophenyl butyrate (2669 U/mg) and degrading activity against several amide herbicides. In particular, it displayed the strongest activity against propanil, with a high degradation rate of 84% at 8 h. A GC-MS analysis revealed that propanil was transformed into 3,4-dichloroaniline (3,4-DCA) during this degradation. The molecular interactions and binding stability were then analyzed by molecular docking and molecular dynamics simulation, which revealed that several key amino acid residues, including Tyr164, Trp66, Ala59, Val283, Arg58, His33, His191, and His226, are involved in the specific interactions with propanil. This study provides a function-driven screening method for amide herbicide hydrolase from the metagenomic libraries and a promising propanil-degrading enzyme (AmiH52) for potential applications in environmental remediation.

Flow cytometric assessments of metabolic activity in bacterial assemblages provide insight into ecosystem condition along the Buffalo National River, Arkansas.

The Buffalo National River (BNR), on karst terrain in Arkansas, is considered an extraordinary water resource. Water collected in Spring 2017 along BNR was metagenomically analyzed using 16S rDNA, and for 17 months (5/2017-11/2018), bacterial responses were measured in relation to nutrients sampled along a stretch of BNR near a concentrated animal feed operation (CAFO) on Big Creek. Because cell count and esterase activity can increase proportionally with organic enrichment, they were hypothesized to be elevated near the CAFO. Counts (colony forming units; CFUs) were different among sites for 73 % of the months; Big Creek generated highest CFUs 27 % of the time, with the closest downstream site at 13.3 %. Esterase activity was different among sites 94 % of the time, with Big Creek exhibiting lowest activity 71 % of the time. Over the months, activity was similar across sites at ~70 % active, except at Big Creek (56 %). The α-diversity of BNR microbial consortia near a wastewater treatment plant (WWTP) and the CAFO was related to distance from the WWTP and CAFO. The inverse relationship between high CFUs and low esterase activity at Big Creek (r = -0.71) actuated in vitro exposures of bacteria to organic wastewater contaminants (OWC) previously identified in the watershed. Exponential-phase Escherichia coli (stock strain), Streptococcus suis (avirulent, from swine), and S. dysgalactiae (virulent, from silver carp, Hypophthalmichthys molitrix) were incubated with atrazine, pharmaceuticals (17 α-ethynylestradiol and trenbolone), and antimicrobials (tylosin and butylparaben). Bacteria were differentially responsive. Activity varied with exposure time and OWC type, but not concentration; atrazine decreased it most. Taken together - the metagenomic taxonomic similarities along BNR, slightly higher bacterial growth and lower bacterial esterase at the CAFO, and the lab exposures of bacterial strains showing that OWC altered metabolism - the results indicated that bioactive OWC entering the watershed can strongly influence microbial processes in the aquatic ecosystem.

Co(II) Substitution Enhances the Esterase Activity of a de Novo Designed Zn(II) Carbonic Anhydrase.

Carbonic Anhydrases (CAs) have been a target for de novo protein designers due to the simplicity of the active site and rapid rate of the reaction. The first reported mimic contained a Zn(II) bound to three histidine imidazole nitrogens and an exogenous water molecule, hence closely mimicking the native enzymes' first coordination sphere. Co(II) has served as an alternative metal to interrogate CAs due to its d7 electronic configuration for more detailed solution characterization. We present here the Co(II) substituted [Co(II)(H2O/OH-)]N(TRIL2WL23H)3 n+ that behaves similarly to native Co(II) substituted human-CAs. Like the Zn(II) analogue, the cobalt-derivative at slightly basic pH is incapable of hydrolyzing p-nitrophenylacetate (pNPA); however, as the pH is increased a significant activity develops, which at pH values above 10 eventually yields a catalytic efficiency that exceeds that of the [Zn(II)(OH-)]N(TRIL2WL23H)3 + peptide complex. X-ray absorption analysis is consistent with an octahedral species at pH 7.5 that converts to a 5-coordinate species by pH 11. UV-vis spectroscopy can monitor this transition, giving a pKa for the conversion of 10.3. We assign this conversion to the formation of a 5-coordinate Co(II)(Nimid)3(OH)(H2O) species. The pH dependent kinetic analysis indicates the maximal rate (kcat), and thus the catalytic efficiency (kcat/Km), follow the same pH profile as the spectroscopic conversion to the pentacoordinate species. This correlation suggests that the chemically irreversible ester hydrolysis corresponds to the rate determining process.

Production of acetic acid from wheat bran by catalysis of an acetoxylan esterase.

In this study, a gene encoding for acetylxylan esterase was cloned and expressed in E. coli. A single uniform band with molecular weight of 31.2 kDa was observed in SDS-PAGE electrophoresis. Served as the substrate, p-nitrophenol butyrate was employed to detect the recombinant enzyme activity. It exhibited activity at a wide temperature range (30-100 °C) and pH (5.0-9.0) with the optimal temperature of 70 °C and pH 8.0. Acetylxylan esterase showed two substrates' specificities with the highest Vmax of 177.2 U/mg and Km of 20.98 mM against p-nitrophenol butyrate. Meanwhile, the Vmax of p-nitrophenol acetate was 137.0 U/mg and Km 12.16 mM. The acetic acid yield of 0.39 g/g was obtained (70 °C and pH 8.0) from wheat bran pretreated using amylase and papain. This study showed the highest yield up to date and developed a promising strategy for acetic acid production using wheat bran.

Metabolite profiling and in-silico studies show multiple effects of insecticidal actinobacterium on Spodoptera littoralis.

The polyphagous pest, Spodoptera littoralis (Boisduval), poses a significant global economic threat by gregariously feeding on over a hundred plant species, causing substantial agricultural losses. Addressing this challenge requires ongoing research to identify environmentally safe control agents. This study aimed to elucidate the insecticidal activity of the metabolite (ES2) from a promising endophytic actinobacterium strain, Streptomyces sp. ES2 EMCC2291. We assessed the activity of ES2 against the eggs and fourth-instar larvae of S. littoralis through spectrophotometric measurements of total soluble protein, α- and ß-esterases, polyphenol oxidase (PPO), and catalase enzyme (CAT). The assessments were compared to commercial Biosad® 22.8% SC. Untargeted metabolomics using LC-QTOF-MS/MS identified 83 metabolic compounds as chemical constituents of ES2. The median lethal concentration (LC50) of ES2 (165 mg/mL) for treated Spodoptera littoralis eggs showed significant differences in polyphenol oxidase and catalase enzymatic activities, while the LC50 of ES2 (695 mg/mL) for treated S. littoralis fourth instar larvae showed lower significance in α- and ß-esterase activities. Molecular docking of ES2 identified seven potent biocidal compounds, showing strong affinity to PPO and catalase CAT proteins in S. littoralis eggs while displaying limited binding to alpha and beta esterase proteins in the larvae. The results contribute to the understanding of ES2 as a promising alternative biopesticide, providing insights for future research and innovative applications in sustainable pest management strategies.

A new and promiscuous α/ß hydrolase from Acinetobacter tandoii DSM 14970 T inactivates the mycotoxin ochratoxin A.

The presence of ochratoxin A (OTA) in food and feed represents a serious concern since it raises severe health implications. Bacterial strains of the Acinetobacter genus hydrolyse the amide bond of OTA yielding non-toxic OTα and L-ß-phenylalanine; in particular, the carboxypeptidase PJ15_1540 from Acinetobacter sp. neg1 has been identified as an OTA-degrading enzyme. Here, we describe the ability to transform OTA of cell-free protein extracts from Acinetobacter tandoii DSM 14970 T, a strain isolated from sludge plants, and also report on the finding of a new and promiscuous α/ß hydrolase (ABH), with close homologs highly distributed within the Acinetobacter genus. ABH from A. tandoii (AtABH) exhibited amidase activity against OTA and OTB mycotoxins, as well as against several carboxypeptidase substrates. The predicted structure of AtABH reveals an α/ß hydrolase core composed of a parallel, six-stranded ß-sheet, with a large cap domain similar to the marine esterase EprEst. Further biochemical analyses of AtABH reveal that it is an efficient esterase with a similar specificity profile as EprEst. Molecular docking studies rendered a consistent OTA-binding mode. We proposed a potential procedure for preparing new OTA-degrading enzymes starting from promiscuous α/ß hydrolases based on our results. KEY POINTS: • AtABH is a promiscuous αß hydrolase with both esterase and amidohydrolase activities • AtABH hydrolyses the amide bond of ochratoxin A rendering nontoxic OTα • Promiscuous αß hydrolases are a possible source of new OTA-degrading enzymes.

The weakened physiological functions of human serum albumin in presence of polystyrene nanoplastics.

Due to the widespread presence of nanoplastics (NPs) in daily essentials and drinking water, the potential adverse effects of NPs on human health have become a global concern. Human serum albumin (HSA), the most abundant and multi-functional protein in plasma, has been chosen to understand the biological effects of NPs after entering the blood. The esterase activity and the transport of bisphenol A in the presence of polystyrene nanoplastics (PSNPs) under physiological conditions (pH 4.0 and 7.4) have been investigated to evaluate the possible biological effects. The interactions between PSNPs and HSA have also been systematically studied by multispectral methods and dynamic light scattering techniques. The esterase activity of HSA presented a decreased trend with increasing PSNPs; conversely, higher permeabilities are accompanied by higher amounts of PSNPs. Compared with the unchanged hydrodynamic diameter and weaker interactions at pH 7.4, stronger binding between HSA and PSNPs at pH 4.0 led to a significant increase in the particle size of the PSNPs-HSA complex. The quenching mechanism belonged to the static quenching type. The electrostatic force is proposed to be the dominant factor for PSNPs binding to HSA. The work provides some information about the toxicity of NPs when exposed to humans.