Production of Vespa tropica Hyaluronidase by Pichia pastoris

Hyaluronidases have been a subject of great interest in medical and cosmeceutical applications. Previously, our group demonstrated that the venom glands of <i>Vespa tropica</i> contain hyaluronidase enzymes (VesT2s), and heterologous expression of the corresponding gene (<i>VesT2a</i>) in <i>E. coli</i> systems results in inclusion bodies, necessitating functional folding using urea. Here, we report the successful heterologous expression of VesT2a in the <i>Pichia pastoris</i> expression system, with gene construction achieved using Golden<i>PiCS</i>. 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 (P_GAP). Upon culturing in a bioreactor, yeast cells harboring pAOX1-αMF-<i>VesT2a</i> 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 <i>E. coli</i>-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.