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.