Comparison of the secretory murine DNase1 family members expressed in Pichia pastoris.

Soluble nucleases of the deoxyribonuclease 1 (DNase1) family facilitate DNA and chromatin disposal (chromatinolysis) during certain forms of cell differentiation and death and participate in the suppression of anti-nuclear autoimmunity as well as thrombotic microangiopathies caused by aggregated neutrophil extracellular traps. Since a systematic and direct comparison of the specific activities and properties of the secretory DNase1 family members is still missing, we expressed and purified recombinant murine DNase1 (rmDNase1), DNase1-like 2 (rmDNase1L2) and DNase1-like 3 (rmDNase1L3) using Pichia pastoris. Employing different strategies for optimizing culture and purification conditions, we achieved yields of pure protein between ~3 mg/l (rmDNase1L2 and rmDNase1L3) and ~9 mg/l (rmDNase1) expression medium. Furthermore, we established a procedure for post-expressional maturation of pre-mature DNase still bound to an unprocessed tri-N-glycosylated pro-peptide of the yeast α-mating factor. We analyzed glycosylation profiles and determined specific DNase activities by the hyperchromicity assay. Additionally, we evaluated substrate specificities under various conditions at equimolar DNase isoform concentrations by lambda DNA and chromatin digestion assays in the presence and absence of heparin and monomeric skeletal muscle α-actin. Our results suggest that due to its biochemical properties mDNase1L2 can be regarded as an evolutionary intermediate isoform of mDNase1 and mDNase1L3. Consequently, our data show that the secretory DNase1 family members complement each other to achieve optimal DNA degradation and chromatinolysis under a broad spectrum of biological conditions.

Identification of individual components of a commercial wheat germ acid phosphatase preparation.

Wheat germ acid phosphatase (WGAP) is a commercial preparation of partially purified protein commonly used in laboratory settings for non-specific enzymatic dephosphorylation. It is known that these preparations contain multiple phosphatase isozymes and are still relatively crude. This study therefore aimed to identify the protein components of a commercial preparation of wheat germ acid phosphatase using mass spectroscopy and comparative genomics. After one post-purchase purification step, the most prevalent fifteen proteins in the mixture included heat shock proteins, beta-amylases, glucoseribitol dehydrogenases, enolases, and an aminopeptidase. While not among the most abundant components, eight unique dephosphorylation enzymes were also present including three purple acid phosphatases. Furthermore, it is shown that some of these correspond to previously isolated isozymes; one of which has been also previously shown by transcriptome data to be overexpressed in wheat seeds. In summary, this study identified the major components of WGAP including phosphatases and hypothesizes the most active components towards a better understanding of this commonly used laboratory tool.

Expression in Escherichia Coli, Purification, and Functional Reconstitution of Human Steroid 5α-Reductases.

The potent androgen 5α-dihydrotestosterone irreversibly derives from testosterone via the activity of steroid 5α-reductases (5αRs). The major 5αR isoforms in most species, 5αR1 and 5αR2, have not been purified to homogeneity. We report here the heterologous expression of polyhistidine-tagged, codon-optimized human 5αR1 and 5αR2 cDNAs in Escherichia coli. A combination of the nonionic detergents Triton X-100 and Nonidet P-40 enabled solubilization of these extremely hydrophobic integral membrane proteins and facilitated purification with affinity and cation-exchange chromatography methods. For functional reconstitution, we incorporated the purified isoenzymes into Triton X-100-saturated dioleoylphosphatidylcholine liposomes and removed excess detergent with polystyrene beads. Kinetic studies indicated that the 2 isozymes differ in biochemical properties, with 5αR2 having a lower apparent Km for testosterone, androstenedione, progesterone, and 17-hydroxyprogesterone than 5αR1; however, 5αR1 had a greater capacity for steroid conversion, as reflected by a higher Vmax than 5αR2. Both enzymes preferred progesterone as substrate over other steroids, and the catalytic efficiency of purified reconstituted 5αR2 exhibited a sharp pH optimum at pH 5. Intriguingly, we found that the prostate-cancer drug-metabolite 3-keto-∆ 4-abiraterone is metabolized by 5αR1 but not 5αR2, which may serve as a structural basis for isoform selectivity and inhibitor design. The functional characterization results with the purified reconstituted isoenzymes paralleled trends obtained with HEK-293 cell lines stably expressing native 5αR1 and 5αR2. Access to purified human 5αR1 and 5αR2 will advance studies of these important enzymes and might help to clarify their contributions to steroid anabolism and catabolism.

Aggregation and Molecular Properties of ß-Glucosidase Isoform II in Chayote (Sechium edule).

The presence of isoforms of ß-glucosidase has been reported in some grasses such as sorghum, rice and maize. This work aims to extract and characterize isoform II in ß-glucosidase from S. edule. A crude extract was prepared without buffer solution and adjusted to pH 4.6. Contaminating proteins were precipitated at 4 °C for 24 h. The supernatant was purified by chromatography on carboxymethyl cellulose (CMC) column, molecular exclusion on Sephacryl S-200HR, and exchange anionic on QFF column. Electrophoretic analyzes revealed a purified enzyme with aggregating molecular complex on SDS-PAGE, Native-PAGE, and AU-PAGE. Twelve peptides fragments were identified by nano liquid chromatography-tandem mass spectrometry (nano LC-ESI-MS/MS), which presented as 61% identical to Cucurbita moschata ß-glucosidase and 55.74% identical to ß-glucosidase from Cucumis sativus, another Cucurbitaceous member. The relative masses which contained 39% hydrophobic amino acids ranged from 982.49 to 2,781.26. The enzyme showed a specificity to ß-d-glucose with a Km of 4.59 mM, a Vmax value of 104.3 µM∙min-1 and a kcat of 10,087 µM∙min-1 using p-nitrophenyl-ß-D-glucopyranoside. The presence of molecular aggregates can be attributed to non-polar amino acids. This property is not mediated by a ß-glucosidase aggregating factor (BGAF) as in grasses (maize and sorghum). The role of these aggregates is discussed.

Abundance of Lactobacillus plantarum Strains with Beneficial Attributes in Blackberries (Rubus sp.), Fresh Figs (Ficus carica), and Prickly Pears (Opuntia ficus-indica) Grown and Harvested in Algeria.

This first study performed on traditional fruits consumed in North Africa reveals their richness in microorganisms with beneficial attributes like cholesterol lowering capabilities. Blackberries (Rubus sp.), fresh figs (Ficus carica), and prickly pears (Opuntia ficus-indica) are fruits largely and traditionally consumed in Kabylia, a beautiful northern Algerian region. Here, 85 lactic acid bacteria (LAB)-isolates were isolated and identified by MALDI-TOF mass spectrometry. The identified species belong to Lactobacillus and Leuconostoc genera. These 85 LAB-isolates were then assessed for their capabilities to grow under conditions mimicking the gastrointestinal tract, and the resulting data were statistically treated with principal component analysis (PCA). After which, only 26 LAB-isolates were selected and characterized for their genetic relatedness using random amplified polymorphic DNA (RAPD) method. Following the genetic relatedness assessment, only 10 LAB-strains, among which nine Lactobacillus plantarum and one Lactobacillus paracasei were studied for their pathoproperties and some probiotic features. Interestingly, all of these 10 LAB-strains were devoid of adverse effects, but capable to adhere to human epithelial colorectal adenocarcinoma Caco-2 cells. Of note, these 10 LAB-strains exhibited an important in vitro hypocholesteromia effect, in strain-dependent manner. Moreover, the Lactobacillus strains exhibited a high bile salt hydrolase (BSH) activity which was correlated with expression of bsh2, bsh3 and bsh4 genes.

Purification and molecular characterization of phospholipase, antigen 5 and hyaluronidases from the venom of the Asian hornet (Vespa velutina).

The aim of this study was to purify potential allergenic components of Vespa velutina venom, the yellow legged Asian Hornet, and perform a preliminary characterization of the purified proteins. Starting from the whole venom of V.velutina, several chromatographic steps allowed to purify the phospholipase (named Vesp v 1), as well as the antigen 5 (Vesp v 5, the only allergenic component described as such so far). The two hyaluronidase isoforms found (Vesp v 2A and Vesp v 2B) cannot be separated from each other, but they are partially purified and characterized. Purity of the isolated proteins in shown by SDSPAGE, as well as by the results of the N-terminal sequencing. This characterization and nLC-MS/MS data provide most of the sequence for Vesp v 1 and Vesp v 5 (72 and 84% coverage, respectively), confirming that the whole sequences of the isolated natural components match with the data available in public transcriptomic databases. It is of particular interest that Vesp v 1 is a glycosylated phospholipase, a fact that had only described so far for the corresponding allergen components of Dolichovespula maculata and Solenopsis invicta. The availability of the complete sequences of Vespa velutina components permits comparison with homologous sequences from other Hymenoptera. These data demonstrate the higher similarity among the species of the genera Vespa and Vespula, in comparison to Polistes species, as it is especially observed with the hyaluronidases isoforms: the isoform Vesp v 2A only exists in the former genera, and not in Polistes; in addition, the most abundant isoform (Vesp v 2B) exhibits 93% sequence identity with the Ves v 2 isoform of Vespula vulgaris. Finally, the isolated components might be useful for improving the diagnosis of patients that could be allergic to stings of this invasive Asian hornet, as it has been the case of an improved diagnosis and treatment of other Hymenoptera-sensitized patients.

Fast flow rate processes for purification of alkaline xylanase isoforms from Bacillus pumilus AJK and their biochemical characterization for industrial application purposes.

This study shows the presence of five isozymic forms of alkaline xylanase from Bacillus pumilus using fast flow rate microfiltration, ultrafiltration, Q-sepharose, and phenyl sepharose chromatographic techniques. Polyacrylamide gel electrophoresis, high-performance liquid chromatography, and zymographic studies also revealed the purity of five isoforms of alkaline xylanases. Isoforms-X-I, X-III, and X-V exhibited optimum activity at pH 8.5, whereas X-II, X-IV showed maximum activity at pH 9. All isoforms were optimally active at temperature 55°C. Isoforms were found to be stable at pH 7-11, showed 92-100% residual activity after 3 hr, treatment time for most industrial applications. The isoforms retained nearly 80-86% residual activity after incubating at 45°C for 3 hr. Molecular weights of xylanase I-V, were 13.1, 15.3, 18.4, 20.1, and 21.0 kDa, respectively. Mg2+ ions were found to be potent activator for all isozymic forms. The Km and Vmax values of X-I, X-II, X-III, X-IV, and X-V were 6.71, 6.66, 7.14, 5.88, 6.25 mg/ml and 2,000, 1,695, 1,666.66, 1,428.57, and 1,408.45 IU/mg protein, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed the monomeric nature of all isoforms. The low-molecular masses, significantly enhanced activity in the presence of industrially suitable-low cost activator, better stability of all isoforms at pH 7-11 and at higher temperature, also presence of multiple forms of alkaline xylanase, makes this enzyme suitable for textile-paper industries. This is also the first report mentioning the purification of five isozymic forms of alkaline xylanase using fast flow rate techniques.

Purification and Characterization of Prolyl Hydroxylase 3/Pyruvate Kinase Isoform 2 Protein Complex.

The prolyl hydroxylase 3 (PHD3) protein is less abundant in normal oxygen conditions (normoxia) but increases under deficient oxygen condition (hypoxia). Since cancerous cells often thrive in hypoxic conditions and predominantly express the Pyruvate kinase isoforms 2 (PKM2), the PHD3/PKM2 interaction might be particularly important in cancer development. In the present study, the PHD3/PKM2 complex was co-expressed and purified by size-exclusion chromatography. The interaction of PHD3 with PKM2 was confirmed in Native gel as well as western blot analysis. The PHD3/PKM2 complex formed discreet crystals under suitable conditions, and diffraction data revealed that crystal belonged to the P1 space group with 3.0 Å resolution. This is the first crystal report of PHD3/PKM2 complex as well as this study demonstrates a direct physical binding through protein-protein interaction. The structural analysis of complex will provide the information regarding the amino acid residues critical for the catalytic mechanism. Based on the structural information thus obtained, pharmacological interference with the PHD3/PKM2 interaction could be used as a novel strategy to reduce the cancer progression.

Sorting by interfacial tension (SIFT): Label-free enzyme sorting using droplet microfluidics.

Droplet microfluidics has the ability to greatly increase the throughput of screening and sorting of enzymes by carrying reagents in picoliter droplets flowing in inert oils. It was found with the use of a specific surfactant, the interfacial tension of droplets can be very sensitive to droplet pH. This enables the sorting of droplets of different pH when confined droplets encounter a microfabricated trench. The device can be extended to sort enzymes, as a large number of enzymatic reactions lead to the production of an acidic or basic product and a concurrent change in solution pH. The progress of an enzymatic reaction is tracked from the position of a flowing train of droplets. We demonstrate the sorting of esterase isoenzymes based on their enzymatic activity. This label-free technology, that we dub droplet sorting by interfacial tension (SIFT), requires no active components and would have applications for enzyme sorting in high-throughput applications that include enzyme screening and directed evolution of enzymes.

Rapid Identification of New Delhi Metallo-ß-Lactamase (NDM) Using Tryptic Peptides and LC-MS/MS.

There is significant interest in the development of mass spectrometry (MS) methods for antimicrobial resistance protein detection, given the ability of these methods to confirm protein expression. In this work, we studied the performance of a liquid chromatography, tandem MS multiple-reaction monitoring (LC-MS/MS MRM) method for the direct detection of the New Delhi metallo-ß-lactamase (NDM) carbapenemase in clinical isolates. Using a genoproteomic approach, we selected three unique peptides (SLGNLGDADTEHYAASAR, AFGAAFPK, and ASMIVMSHSAPDSR) specific to NDM that were efficiently ionized and spectrally well-defined. These three peptides were used to build an assay with turnaround time of 90 min. In a blind set, the assay detected 21/24 blaNDM-containing isolates and 76/76 isolates with negative results, corresponding to a sensitivity value of 87.5% (95% confidence interval [CI], 67.6% to 97.3%) and a specificity value of 100% (95% CI, 95.3% to 100%). One of the missed identifications was determined by protein fractionation to be due to low (∼0.1 fm/µg) NDM protein expression (below the assay limit of detection). Parallel disk diffusion susceptibility testing demonstrated this isolate to be meropenem susceptible, consistent with low NDM expression. Total proteomic analysis of the other two missed identifications did not detect NDM peptides but detected other proteins expressed from the blaNDM-containing plasmids, confirming that the plasmids were not lost. The measurement of relative NDM concentrations over the entire isolate test set demonstrated variability spanning 4 orders of magnitude, further confirming the remarkable range that may be seen in levels of NDM expression. This report highlights the sensitivity of LC-MS/MS to variations in NDM protein expression, with implications for how this technology may be used.

Sulfonate and sulfamate derivatives possessing benzofuran or benzothiophene nucleus as potent carbonic anhydrase II/IX/XII inhibitors.

In the current work, we report the discovery of new sulfonate and sulfamate derivatives of benzofuran- and benzothiophene as potent inhibitors of human carbonic anhydrases (hCAs) II, IX and XII. A set of derivatives, 1a-t, having different substituents on the fused benzofuran and benzothiophene rings (R = alkyl, cyclohexyl, aryl, NH2, NHMe, or NMe2) was designed and synthesized. Most of the derivatives exhibited higher potency than acetazolamide as inhibitors of the purified hCAII, IX and XII isoforms. The most potent inhibitors for hCAII, hCAIX and hCAXII were 1g, 1b and 1d with an IC50 ±â€¯SEM values of 0.14 ±â€¯0.03, 0.13 ±â€¯0.03 and 0.17 ±â€¯0.06 µM, respectively. In addition, compounds 1d and 1n exerted preferential inhibitory effect against hCAXII isozyme with good potencies. Some selected compounds were docked within the active pocket of these isozymes and binding of the molecules revealed that sulfonate and sulfamate rings were located towards the active cavity and compounds coordinated to zinc ions.

Purification and partial biochemical characterization of recombinant lactate dehydrogenase 1 (LDH-1) of the white shrimp Litopenaeus vannamei.

Lactate dehydrogenase (LDH) is a key enzyme to produce energy during hypoxia by anaerobic glycolysis. In the white shrimp Litopenaeus vannamei, two protein subunits (LDH-1 and LDH-2) were previously identified, deduced from two different transcripts that come from the same LDH gene by processing via mutually exclusive alternative splicing. LDH-1 contains exon five and LDH-2 contains exon six and the two proteins differ only in 15 amino acid residues. Both subunits were independently cloned and overexpressed in E. coli as a fusion protein containing a chitin binding domain. Previously, recombinant LDH-2 was successfully purified and characterized, but LDH-1 was insoluble and aggregated forming inclusion bodies. We report the production of soluble LDH-1 by testing different pHs in the buffers used to lyse the bacterial cells before the purification step and the characterization of the purified protein to show that the cDNA indeed codes for a functional and active protein. The recombinant native protein is a homotetramer of approximately 140 kDa composed by 36 kDa subunits and has higher affinity for pyruvate than for lactate. LDH-1 has an optimum pH of 7.5 and is stable between pH 8.0 and 9.0; pH data analysis showed two pKa values of 6.1 ±â€¯0.15 and 8.8 ±â€¯0.15 suggesting a histidine and asparagine, respectively, involved in the active site. The enzyme optimal temperature was 44 °C and it was stable between 20 and 60 °C. LDH-1 was slightly activated by NaCl, KCl and MgCl2 and fully inhibited by ZnCl2.

Development and validation of an absolute protein assay for the simultaneous quantification of fourteen CYP450s in human microsomes by HPLC-MS/MS-based targeted proteomics.

The cytochrome P450 (CYP450) superfamily constitutes the major enzymatic system involved in drug metabolism. CYP450s are highly expressed in the liver and other tissues and limited data on absolute characterization of CYP450s in extra hepatic organs, such as the small intestine, are available. Our objective was to develop and validate an absolute quantification assay by HPLC-MS/MS-based targeted proteomics allowing the simultaneous quantification of fourteen major human CYP450 isoforms (CYP1A1, 1A2, 1B1, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, 3A7 and 4F2) in human liver and intestine microsomes. Absolute protein quantification was performed using two proteotypic peptides for each of the fourteen CYP450s. Peptides were obtained after a tryptic digestion of microsomes and samples were analyzed by high performance liquid chromatography with heated electrospray ionization tandem mass spectrometry (HPLC-HESI-MS/MS). Chromatographic separation was performed on a Biobasic-8 analytical column (5 µm 100 x 1 mm) with a gradient elution using acetonitrile and water both fortified with 0.1% formic acid (flow rate: 75 µL/min). Calibration curves were linear over a wide range of concentrations (0.1-50 nM) and the assay met all requirements of sensitivity, linearity, precision, accuracy and matrix effect. Strong correlations were observed between the two proteotypic peptides for each isoenzyme, corroborating the strength of this method. Twelve CYP450s were detected in commercially available human liver microsomes while seven CYP450s were detected in human intestine microsomes. To our knowledge, this is the most sensitive (0.1 nM) and the first most extensively validated assay that can be applied to the absolute quantification of CYP450s in human tissues.

Characterization of plant glutamine synthetase S-nitrosation.

The identification of S-nitrosated substrates and their target cysteine residues is a crucial step to understand the signaling functions of nitric oxide (NO) inside the cells. Here, we show that the key nitrogen metabolic enzyme glutamine synthetase (GS) is a S-nitrosation target in Medicago truncatula and characterize the molecular determinants and the effects of this NO-induced modification on different GS isoenzymes. We found that all the four M. truncatula GS isoforms are S-nitrosated, but despite the high percentage of amino acid identity between the four proteins, S-nitrosation only affects the activity of the plastid-located enzymes, leading to inactivation. A biotin-switch/mass spectrometry approach revealed that cytosolic and plastid-located GSs share an S-nitrosation site at a conserved cysteine residue, but the plastidic enzymes contain additional S-nitrosation sites at non-conserved cysteines, which are accountable for enzyme inactivation. By site-directed mutagenesis, we identified Cys369 as the regulatory S-nitrosation site relevant for the catalytic function of the plastid-located GS and an analysis of the structural environment of the SNO-targeted cysteines in cytosolic and plastid-located isoenzymes explains their differential regulation by S-nitrosation and elucidates the mechanistic by which S-nitrosation of Cys369 leads to enzyme inactivation. We also provide evidence that both the cytosolic and plastid-located GSs are endogenously S-nitrosated in leaves and root nodules of M. truncatula, supporting a physiological meaning for S-nitrosation. Taken together, these results provide new insights into the molecular details of the differential regulation of individual GS isoenzymes by NO-derived molecules and open new paths to explore the biological significance of the NO-mediated regulation of this essential metabolic enzyme.

Comparison of the Sulfonamide Inhibition Profiles of the α-Carbonic Anhydrase Isoforms (SpiCA1, SpiCA2 and SpiCA3) Encoded by the Genome of the Scleractinian Coral Stylophora pistillata.

The ubiquitous metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1) are responsible for the reversible hydration of CO2 to bicarbonate (HCO3-) and protons (H⁺). Bicarbonate may subsequently generate carbonate used in many functional activities by marine organisms. CAs play a crucial role in several physiological processes, e.g., respiration, inorganic carbon transport, intra and extra-cellular pH regulation, and bio-mineralization. Multiple transcript variants and protein isoforms exist in the organisms. Recently, 16 α-CA isoforms have been identified in the coral Stylophora pistillata. Here, we focalized the interest on three coral isoforms: SpiCA1 and SpiCA2, localized in the coral-calcifying cells; and SpiCA3, expressed in the cytoplasm of the coral cell layers. The three recombinant enzymes were heterologously expressed and investigated for their inhibition profiles with sulfonamides and sulfamates. The three coral CA isoforms differ significantly in their susceptibility to inhibition with sulfonamides. This study provides new insights into the coral physiology and the comprehension of molecular mechanisms involved in the bio-mineralization processes, since CAs interact with bicarbonate transporters, accelerating the trans-membrane bicarbonate movement and modulating the pH at both sides of the plasma membranes.

Molecular cloning, expression, and functional analysis of the chitin synthase 1 gene and its two alternative splicing variants in the white-backed planthopper, Sogatella furcifera (Hemiptera: Delphacidae).

Chitin synthase is responsible for chitin synthesis in the cuticles and cuticular linings of other tissues in insects. We cloned two alternative splicing variants of the chitin synthase 1 gene (SfCHS1) from the white-backed planthopper, Sogatella furcifera. The full-length cDNA of the two variants (SfCHS1a and SfCHS1b) consists of 6408 bp, contains a 4719-bp open reading frame encoding 1572 amino acids, and has 5' and 3' non-coding regions of 283 and 1406 bp, respectively. The two splicing variants occur at the same position in the cDNA sequence between base pairs 4115 and 4291, and consist of 177 nucleotides that encode 59 amino acids but show 74.6% identity at the amino acid level. Analysis in different developmental stages showed that expression of SfCHS1 and SfCHS1a were highest just after molting, whereas SfCHS1b reached its highest expression level 2 days after molting. Further, SfCHS1 and SfCHS1a were mainly expressed in the integument, whereas SfCHS1b was predominately expressed in the gut and fat body. RNAi-based gene silencing inhibited transcript levels of the corresponding mRNAs in S. furcifera nymphs injected with double-stranded RNA of SfCHS1, SfCHS1a, and SfCHS1b, resulted in malformed phenotypes, and killed most of the treated nymphs. Our results indicate that SfCHS1 may be a potential target gene for RNAi-based S. furcifera control.

Isoenzymes of Aedes albopictus (Diptera: Culicidae) Glutathione S-transferases: Isolation and expression after acute insecticide treatment.

Glutathione S-transferases (GSTs) from susceptible Aedes albopictus larvae were partially isolated using two different purification strategies (GSTrap™ HP and GSH-agarose affinity columns) and the effects of permethrin and DDT on expression of the GSTs were investigated. Distinct double bands on SDS-PAGE with molecular weights between 20 and 25 kDa were successfully purified using GSTrap™ HP while a single band of 24.5 kDa was purified using GSH-agarose. The isolated GSTs belonged to the Delta, Sigma and Theta GST classes. When exposed to permethrin, one isoform of Theta, four isoforms of Sigma and thirteen isoforms of Delta GSTs showed an increased expression between 1.4-fold and 2.5-fold while DDT treatment resulted in between 1.4-fold and 3.2-fold increased expression in one isoform of Theta, four isoforms of Sigma and eleven isoforms of Delta GSTs (p < .05). This study indicated that GSTrap™ HP was more competent in isolating Ae. albopictus GSTs compared to GSH-agarose and also variable expression of GST isoforms occur in response to different insecticides. This information may be useful for improving insecticide resistance management strategies in aspect of molecular resistant and evolutionary tolerant detoxification enzyme.

Expression, purification and virucidal activity of two recombinant isoforms of phospholipase A2 from Crotalus durissus terrificus venom.

The global emergence and re-emergence of arthropod-borne viruses (arboviruses) over the past four decades have become a public health crisis of international concern, especially in tropical and subtropical countries. A limited number of vaccines against arboviruses are available for use in humans; therefore, there is an urgent need to develop antiviral compounds. Snake venoms are rich sources of bioactive compounds with potential for antiviral prospection. The major component of Crotalus durissus terrificus venom is a heterodimeric complex called crotoxin, which is constituted by an inactive peptide (crotapotin) and a phospholipase A2 (PLA2-CB). We showed previously the antiviral effect of PLA2-CB against dengue virus, yellow fever virus and other enveloped viruses. The aims of this study were to express two PLA2-CB isoforms in a prokaryotic system and to evaluate their virucidal effects. The sequences encoding the PLA2-CB isoforms were optimized and cloned into a plasmid vector (pG21a) for recombinant protein expression. The recombinant proteins were expressed in the E. coli BL21(DE3) strain as insoluble inclusion bodies; therefore, the purification was performed under denaturing conditions, using urea for protein solubilization. The solubilized proteins were applied to a nickel affinity chromatography matrix for binding. The immobilized recombinant proteins were subjected to an innovative protein refolding step, which consisted of the application of a decreasing linear gradient of urea and dithiothreitol (DTT) concentrations in combination with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) as a protein stabilizer. The refolded recombinant proteins showed phospholipase activity and virucidal effects against chikungunya virus, dengue virus, yellow fever virus and Zika virus.

Comprehensive Snake Venomics of the Okinawa Habu Pit Viper, Protobothrops flavoviridis, by Complementary Mass Spectrometry-Guided Approaches.

The Asian world is home to a multitude of venomous and dangerous snakes, which are used to induce various medical effects in the preparation of traditional snake tinctures and alcoholics, like the Japanese snake wine, named Habushu. The aim of this work was to perform the first quantitative proteomic analysis of the Protobothrops flavoviridis pit viper venom. Accordingly, the venom was analyzed by complimentary bottom-up and top-down mass spectrometry techniques. The mass spectrometry-based snake venomics approach revealed that more than half of the venom is composed of different phospholipases A2 (PLA2). The combination of this approach and an intact mass profiling led to the identification of the three main Habu PLA2s. Furthermore, nearly one-third of the total venom consists of snake venom metalloproteinases and disintegrins, and several minor represented toxin families were detected: C-type lectin-like proteins (CTL), cysteine-rich secretory proteins (CRISP), snake venom serine proteases (svSP), l-amino acid oxidases (LAAO), phosphodiesterase (PDE) and 5'-nucleotidase. Finally, the venom of P. flavoviridis contains certain bradykinin-potentiating peptides and related peptides, like the svMP inhibitors, pEKW, pEQW, pEEW and pENW. In preliminary MTT cytotoxicity assays, the highest cancerous-cytotoxicity of crude venom was measured against human neuroblastoma SH-SY5Y cells and shows disintegrin-like effects in some fractions.

A novel homodimer laccase from Cerrena unicolor BBP6: Purification, characterization, and potential in dye decolorization and denim bleaching.

The white-rot fungus Cerrena unicolor BBP6 produced up to 243.4 U mL-1 laccase. A novel laccase isoform LacA was purified; LacA is a homodimer with an apparent molecular mass of 55 kDa and an isoelectric point of 4.7. Its optimal pH was 2.5, 4.0, and 5.5 when 2, 2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), guaiacol, and 2, 6-dimethoxyphenol (2, 6-DMP) were used as the substrates, respectively. The optimal temperature was 60°C for ABTS and 80°C for both guaiacol and 2, 6-DMP. LacA retained 82-92% activity when pH was greater than 4 and 42%-92% activity at or below 50°C. LacA was completely inhibited by 0.1 mM L-cysteine, 1 mM Dithiothreitol, and 10 mM metal ions, Ca2+, Mg2+ and Co2+. LacA had good affinity for ABTS, with a Km of 49.1 µM and a kcat of 3078.9 s-1. It decolorized synthetic dyes at 32.3-87.1%. In the presence of 1-hydroxybenzotriazole (HBT), LacA decolorized recalcitrant dyes such as Safranine (97.1%), Methylene Blue (98.9%), Azure Blue (96.6%) and simulated textile effluent (84.6%). With supplemented manganese peroxidase (MnP), Mn2+ and HBT, the purified LacA and BBP6 fermentation broth showed great potential in denim bleaching, with an up to 5-fold increase in reflectance values.