Dataset Information

Purification and Biochemical Characterization of TsMS 3 and TsMS 4: Neuropeptide-Degrading Metallopeptidases in the Tityus serrulatus Venom.

ABSTRACT: Although omics studies have indicated presence of proteases on the Tityus serrulatus venom (TsV), little is known about the function of these molecules. The TsV contains metalloproteases that cleave a series of human neuropeptides, including the dynorphin A (1-13) and the members of neuropeptide Y family. Aiming to isolate the proteases responsible for this activity, the metalloserrulase 3 and 4 (TsMS 3 and TsMS 4) were purified after two chromatographic steps and identified by mass spectrometry analysis. The biochemical parameters (pH, temperature and cation effects) were determined for both proteases, and the catalytic parameters (Km, kcat, cleavage sites) of TsMS 4 over fluorescent substrate were obtained. The metalloserrulases have a high preference for cleaving neuropeptides but presented different primary specificities. For example, the Leu-enkephalin released from dynorphin A (1-13) hydrolysis was exclusively performed by TsMS 3. Neutralization assays using Butantan Institute antivenoms show that both metalloserrulases were well blocked. Although TsMS 3 and TsMS 4 were previously described through cDNA library studies using the venom gland, this is the first time that both these toxins were purified. Thus, this study represents a step further in understanding the mechanism of scorpion venom metalloproteases, which may act as possible neuropeptidases in the envenomation process.

SUBMITTER: Cajado-Carvalho D

PROVIDER: S-EPMC6520902 | biostudies-other | 2019 Mar

REPOSITORIES: biostudies-other

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Purification and Biochemical Characterization of TsMS 3 and TsMS 4: Neuropeptide-Degrading Metallopeptidases in the <i>Tityus serrulatus</i> Venom.

Cajado-Carvalho Daniela D da Silva Cristiane Castilho Fernandes CCF Kodama Roberto Tadashi RT Mariano Douglas Oscar Ceolin DOC Pimenta Daniel Carvalho DC Duzzi Bruno B Kuniyoshi Alexandre Kazuo AK Portaro Fernanda Vieira FV

Toxins 20190331 4

Although omics studies have indicated presence of proteases on the <i>Tityus serrulatus</i> venom (TsV), little is known about the function of these molecules. The TsV contains metalloproteases that cleave a series of human neuropeptides, including the dynorphin A (1-13) and the members of neuropeptide Y family. Aiming to isolate the proteases responsible for this activity, the metalloserrulase 3 and 4 (TsMS 3 and TsMS 4) were purified after two chromatographic steps and identified by mass spect ...[more]

PMID: 30935107

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Project description:BackgroundA lot of kitchen waste oil is produced every day worldwide, leading to serious environmental pollution. As one of the environmental protection methods, microorganisms are widely used treating of various wastes. Lipase, as one of the cleaning agents can effectively degrade kitchen waste oil. The composting process of pig carcasses produces many lipase producing microorganisms, rendering compost products an excellent source for isolating lipase producing microorganisms. To our knowledge, there are no reports isolating of lipase producing strains from the high temperature phase of pig carcass compost.MethodologyLipase producing strains were isolated using a triglyceride medium and identified by 16S rRNA gene sequencing. The optimal fermentation conditions for maximum lipase yield were gradually optimized by single-factor tests. The extracellular lipase was purified by ammonium sulfate precipitation and Sephadex G-75 gel isolation chromatography. Amino acid sequence analysis, structure prediction, and molecular docking of the purified protein were performed. The pure lipase's enzymatic properties and application potential were evaluated by characterizing its biochemical properties.ResultsIn this study, a lipase producing strain of Bacillus sp. ZF2 was isolated from pig carcass compost products, the optimal fermentation conditions of lipase: sucrose 3 g/L, ammonium sulfate 7 g/L, Mn2+ 1.0 mmol/L, initial pH 6, inoculum 5%, temperature 25 ℃, and fermentation time 48 h. After purification, the specific activity of the purified lipase reached 317.59 U/mg, a 9.78-fold improvement. Lipase had the highest similarity to the GH family 46 chitosanase and molecular docking showed that lipase binds to fat via two hydrogen bonds at Gln146 (A) and Glu203 (A). Under different conditions (temperature, metal ions, organic solvents, and surfactants), lipase can maintain enzymatic activity. Under different types of kitchen oils, lipase has low activity only for 'chicken oil', in treating other substrates, the enzyme activity can exceed 50%.ConclusionsThis study reveals the potential of lipase for waste oil removal, and future research will be devoted to the application of lipase.

Dataset Information

Purification and Biochemical Characterization of TsMS 3 and TsMS 4: Neuropeptide-Degrading Metallopeptidases in the Tityus serrulatus Venom.

Publications

Purification and Biochemical Characterization of TsMS 3 and TsMS 4: Neuropeptide-Degrading Metallopeptidases in the <i>Tityus serrulatus</i> Venom.

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