Expression of a nematode chondroitin hydrolase in Pichia pastoris for low-molecular-weight chondroitin preparation

Chondroitin sulfate (CS) is widely used in dietary supplements and pharmaceuticals due to its biological activities. However, the large molecular size of high-molecular-weight CS limits its bioavailability and therapeutic efficacy, necessitating the production of low-molecular-weight CS or oligosaccharides to enhance absorption and bioactivity. Here, we report the heterologous expression of recombinant chondroitin hydrolase (rCeChnase) from Caenorhabditis elegans in the methylotrophic yeast Pichia pastoris . This expression system offers an environmentally friendly and cost-effective alternative to animal-derived hyaluronidases, with rCeChnase demonstrating significantly higher catalytic efficiency. The enzyme exhibited optimal activity at 37 °C and pH 5.0, with the highest stability at 25 °C and pH 5.0. A five-fold increase in enzymatic activity production, reaching over 40,000 U/mL, was achieved by lowering the culture temperature to 25 °C. rCeChnase showed a strong preference for non-sulfated chondroitin, with minimal activity towards CS and hyaluronic acid. The enzyme was used to efficiently degrade high-molecular-weight chondroitin into low-molecular-weight chondroitin and oligosaccharides, which were verified by agarose gel electrophoresis and LC-MS. rCeChnase's endo -β1,4-hydrolysis activity was confirmed, and the major product was chondroitin tetrasaccharides. This study highlights the industrial potential of rCeChnase as a promising tool for the production of native low-molecular-weight chondroitin and oligosaccharides.