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Hyaluronidases have been a subject of great interest in medical and cosmeceutical applications. Previously, our group demonstrated that the venom glands of Vespa tropica contain hyaluronidase enzymes (VesT2s), and heterologous expression of the corresponding gene (VesT2a) in E. coli systems results in inclusion bodies, necessitating functional folding using urea. Here, we report the successful heterologous expression of VesT2a in the Pichia pastoris expression system, with gene construction achieved using GoldenPiCS. After confirming gene integration in the yeast genome, methanol-induced cultures yielded an exceptional amount of VesT2a, approximately two-fold higher than that obtained with the constitutive expression vector (PGAP). Upon culturing in a bioreactor, yeast cells harboring pAOX1-αMF-VesT2a produced secreted proteins with a total yield of 96.45 mg/L. The secreted VesT2a has a molecular weight of 59.35 kDa, significantly higher than the expected molecular weight (~40.05 kDa), presumably due to endogenous glycosylation by the yeast cells. It exhibits optimal activity at 37 °C and pH 3, showing a specific activity of 4238.37 U/mg, and remains active across a broad range of pH and temperature. Notably, it exhibits higher hyaluronidase activity than the crude venom and E. coli-expressed protein, likely due to improved folding via endogenous post-translational modifications, such as disulfide bonds and N-glycosylation; this underscores the potential of heterologous systems for producing venomous hyaluronidases from other species. In silico docking-based analyses further support its catalytic activity and provide insights into seeking natural inhibitors from phenolic-rich plant extracts to alleviate symptoms in patients suffering from insect bites and stings.

Our group previously demonstrated that Caesalpinia mimosoides Lamk exhibits many profound biological properties, including anticancer, antibacterial, and antioxidant activities. However, its antiviral activity has not yet been investigated. Here, the aqueous extract of C. mimosoides was prepared from the aerial parts (leaves, stalks, and trunks) to see whether it exerts anti-influenza (H1N1) effects and to reduce the organic solvents consumed during extraction, making it a desirable approach for the large-scale production for medical uses. Our plant extract was quantified to contain 7 g of gallic acid (GA) per 100 g of a dry sample, as determined using HPLC analysis. It also exerts potent antioxidant activities comparable to those of authentic GA. According to untargeted metabolomics (UPLC-ESI(-)-QTOF-MS/MS) with the aid of cheminformatics tools (MetFrag (version 2.1), SIRIUS (version 5.8.3), CSI:FingerID (version 4.8), and CANOPUS), the major metabolite was best annotated as “gallic acid”, phenolics (e.g., quinic acid, shikimic acid, and protocatechuic acid), sugar derivatives, and dicarboxylic acids were deduced from this plant species for the first time. The aqueous plant extract efficiently inhibited an influenza A (H1N1) virus infection of MDCK cells with an IC50 of 5.14 µg/mL. Of equal importance, hemolytic activity was absent for this plant extract, signifying its applicability as a safe antiviral agent. Molecular docking suggested that GA interacts with conserved residues (e.g., Arg152 and Asp151) located in the catalytic inner shell of the viral neuraminidase (NA), sharing the same pocket as those of anti-neuraminidase drugs, such as laninamivir and oseltamivir. Additionally, other metabolites were also found to potentially interact with the active site and the hydrophobic 430-cavity of the viral surface protein, suggesting a possibly synergistic effect of various phytochemicals. Therefore, the C. mimosoides aqueous extract may be a good candidate for coping with increasing influenza virus resistance to existing antivirals.

The increasing antimicrobial-resistant prevalence has become a severe health problem. It has led to the invention of a new antimicrobial agent such as antimicrobial peptides. Heteroscorpine-1 is an antimicrobial peptide that has the ability to kill many bacterial strains. It consists of 76 amino acid residues with a cecropin-like region in N-terminal and a defensin-like region in the C-terminal. The cecropin-like region from heteroscorpine-1 (CeHS-1) is similar to cecropin B, but it lost its glycine-proline hinge region. The bioinformatics prediction was used to help the designing of mutant peptides. The addition of glycine-proline hinge and positively charged amino acids, the deletion of negatively charged amino acids, and the optimization of the hydrophobicity of the peptide resulted in two mutant peptides, namely, CeHS-1 GP and CeHS-1 GPK. The new mutant peptide showed higher antimicrobial activity than the native peptide without increasing toxicity. The interaction of the peptides with the membrane showed that the peptides were capable of disrupting both the inner and outer bacterial cell membrane. Furthermore, the SEM analysis showed that the peptides created the pore in the bacterial cell membrane resulted in cell membrane disruption. In conclusion, the mutants of CeHS-1 had the potential to develop as novel antimicrobial peptides.

Abstract Background: Crude venom of the banded tiger waspVespa affinis contains a variety of enzymes including hyaluronidases, commonly known as spreading factors. Methods: The cDNA cloning, sequence analysis and structural modelling of V. affinis venom hyaluronidase (VesA2) were herein described. Moreover, heterologous expression and mutagenesis of rVesA2 were performed. Results: V. affinis venom hyaluronidase full sequence is composed of 331 amino acids, with four predicted N-glycosylation sites. It was classified into the glycoside hydrolase family 56. The homology modelling exhibited a central core (α/β)7 composed of Asp107 and Glu109, acting as the catalytic residues. The recombinant protein was successfully expressed in E. coli with hyaluronidase activity. A recombinant mutant type with the double point mutation, Asp107Asn and Glu109Gln, completely lost this activity. The hyaluronidase from crude venom exhibited activity from pH 2 to 7. The recombinant wild type showed its maximal activity at pH 2 but decreased rapidly to nearly zero at pH 3 and was completely lost at pH 4. Conclusion: The recombinant wild-type protein showed its maximal activity at pH 2, more acidic pH than that found in the crude venom. The glycosylation was predicted to be responsible for the pH optimum and thermal stability of the enzymes activity.