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°C) 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°C 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.

Editorial: Perineuronal Nets as Therapeutic Targets for the Treatment of Neuropsychiatric Disorders

“…The level of CSPG degradation is limited by Ch-ABC's profile of thermal instability, short half-life and repeated dosing requirements to maintain therapeutically relevant knockdown of PNNs (Muir et al, 2019 ). Numerous groups have addressed this issue, stabilizing the protein either by site directed mutagenesis or by covalent molecules, such as glycerol, sorbitol or polyethylene glycol (PEGylation), which improved the half-life, aggregation and duration of PNN removal from the site of action (Hettiaratchi et al, 2019 ; Takashima et al, 2021 ; Wang et al, 2021 ). Local delivery of stabilized Ch-ABC has shown the greatest success in preclinical models of spinal cord injury (SCI) (Muir et al, 2019 ).…”

Improvement of the stability and catalytic efficiency of heparan sulfate N-sulfotransferase for preparing N-sulfated heparosan

“…Through a combination of protein truncation guided by the B-factor analysis and site-directed mutagenesis, the specific activity and half-life at 37°C of chondroitinase ABC I were significantly improved by 2.3-fold and 247-fold, respectively. (Wang et al., 2021 ). More recently, by integrating the Protein Repair One Stop Shop (PROSS, http://pross.weizmann.ac.il ) (Goldenzweig et al., 2016 ), consensus design and forcefield-based optimization strategies, Hettiaratchi et al., redesigned and constructed chondroitinase ABC variants with much higher efficacy and stability (Hettiaratchi et al., 2020 ).…”

Enzymatic Production of Chondroitin Oligosaccharides and Its Sulfate Derivatives

“…In the past decades, great efforts have been dedicated to the synthesis of CS oligosaccharides with chemical ( Mende et al, 2016 ; Ramadan et al, 2020 ; Wang H. et al, 2020 ; Zhang et al, 2020 ) and chemical enzymatic ( Boltje et al, 2009 ; Gao et al, 2019 ; Zhang et al, 2019 ) methods. As an alternative route, an enzymatic method for synthesizing o-CHs and CS oligosaccharides has been constructed, which depends on the consumption of UDP-GalNAc and UDP-GlcA monomers ( Li J. et al, 2017 ).…”