Characterization of Recombinant Hyaluronidase Derived from Great Banded Wasp (Vespa tropica) Venom Produced in Bacterial System

Abstract

The greater banded wasp (Vespa tropica) venom composed of various bioactive molecules, including hyaluronidase (HAase). The enzyme catalyzes the hydrolysis of hyaluronic acid found in the extracellular matrix (ECM) of vertebrates. In this study, HAase was synthesized by using cDNA as template, which prepared from venom glands of V. tropica. Site directed mutagenesis for mutant of HAase (mHAase(N109L)) had been done by substitution an amino acid in active site from asparagine (N) to leucine (L) at position 109. The wild type and mutant type were separately cloned into pET32a expression vector and expressed in E. coli BL21. The heterologous expression was induced with 0.1 mM IPTG at 18^oC for 16 hrs. The recombinant proteins, Re-HAase and Re-mHAase(N109L), were 58 kDa in size, and found in the insoluble fraction as inclusion body. For re-solubilizing, the recombinant proteins were dissolved in 4 M urea and then subjected for dialysis prior to hyaluronidase activity assay by turbidimetric method. The wild type (Re-HAase) and mutant type (Re-mHAase(N109L)) showed the optimal pH around 5.0. The Re-HAase had hyaluronidase activity at the pH ranging from 3 to 6. Whereas, the Re-mHAase(N109L) showed 50% lower catalytic activity than the wild type at the range of 5 to 6. The mutant type showed no activity at pH 3 to 4.  Asparagine located at the active site may play a crucial role for the binding activity of the enzyme. The other amino acid residues might be participated in the catalytic process.