Engineering a thermostable chondroitinase for production of specifically distributed low‐molecular‐weight chondroitin sulfate

Abstract Chondroitinase ABC I (csABC I) has attracted intensive attention because of its great potential in heparin refining and the enzymatic preparation of low‐molecular‐weight chondroitin sulfate (LMW‐CS). However, low thermal resistance (<30℃) restricts its applications. Herein, structure‐guided and sequence‐assisted combinatorial engineering approaches were applied to improve the thermal resistance of Proteus vulgaris csABC I. By integrating the deletion of the flexible fragment R166–L170 at the N‐terminal domain and the mutation of E694P at the C‐terminal domain, variant NΔ5/E694P exhibited 247‐fold improvement of its half‐life at 37℃ and a 2.3‐fold increase in the specific activity. Through batch fermentation in a 3‐L fermenter, the expression of variant NΔ5/E694P in an Escherichia coli host reached 1.7 g L −1 with the activity of 1.0 × 10 5 U L −1 . Finally, the enzymatic approach for the preparation of LMW‐CS was established. By modulating enzyme concentration and controlling depolymerization time, specifically distributed LMW‐CS (7000, 3400, and 1900 Da) with low polydispersity was produced, demonstrating the applicability of these processes for the industrial production of LMW‐CS in a more environmentally friendly way.