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. 2022 Apr 27;14(9):1770.
doi: 10.3390/polym14091770.

A Highly Active Chondroitin Sulfate Lyase ABC for Enzymatic Depolymerization of Chondroitin Sulfate

Xiao-Man Fan  1 , Jia-Ying Huang  1 , Xiao-Min Ling  1 , Wei Wei  1 , Wen-Bin Su  1 , Ye-Wang Zhang  1
Affiliations

A Highly Active Chondroitin Sulfate Lyase ABC for Enzymatic Depolymerization of Chondroitin Sulfate

Xiao-Man Fan et al. Polymers (Basel). .

Abstract

Enzymatic preparation of low-molecular-weight chondroitin sulfate (LMWCS) has received increasing attention. In this work, a chondroitin sulfate lyase ABC (Chon-ABC) was successfully cloned, expressed, and characterized. The Km and Vmax of the Chon-ABC were 0.54 mM and 541.3 U mg-1, respectively. The maximal activity was assayed as 500.4 U mg-1 at 37 °C in pH 8.0 phosphate buffer saline. The half-lives of the Chon-ABC were 133 d and 127 min at 4 °C and 37 °C, respectively. Enzymatic preparation of LMWCS was performed at room temperature for 30 min. The changes between the substrate and product were analyzed with mass spectrometry (MS), high-performance liquid chromatography (HPLC), gel permeation chromatography (GPC), and nuclear magnetic resonance (NMR). Overall, the Chon-ABC from Bacteroides thetaiotaomicron is competitive in large-scale enzymatic preparation of LMWCS for its high activity, stability, and substrate specificity.

Keywords: Bacteroides thetaiotaomicron; characterization; depolymerization; high activity; low-molecular-weight chondroitin sulfate.

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Conflict of interest statement

The authors declare that they have no known competing financial interest or personal relationship that could have appeared to influence the work reported in this paper.

Figures

Figure 1
Figure 1
SDS-PAGE of Chon-ABC with 4–12% gradient. M: protein marker; Lane 1: cell lysate before induction; Lane 2: cell lysate after induction with IPTG; Lane 3: the supernatant after sonification; Lane 4: the precipitate after sonification; Lane 5: the purified Chon-ABC by Ni-NTA column.
Figure 2
Figure 2
Biochemical characterization of the recombinant Chon-ABC. The effects of temperature (A), pH (B), PBS concentration (C), and metal ions and surfactant (D) on the enzyme activity at 37 °C.
Figure 2
Figure 2
Biochemical characterization of the recombinant Chon-ABC. The effects of temperature (A), pH (B), PBS concentration (C), and metal ions and surfactant (D) on the enzyme activity at 37 °C.
Figure 3
Figure 3
The stability of the recombinant Chon-ABC at 37 °C (A) and 4 °C (B) in 75 mM pH 8.0 PBS.
Figure 4
Figure 4
The effect of chondroitin sulfate concentration on the activity of the Chon-ABC at 37 °C in 75 mM pH 8.0 PBS.
Figure 5
Figure 5
GPC analysis of the CS (A) and hydrolyzed CS (B) and the weight-average molecular weight (Mw) and the distributions (C). The 0 min and 30 min in C are the reaction time. Mn and nd mean the number-average molecular weight and not determined, respectively.
Figure 6
Figure 6
SAX-HPLC (A) and MS (B) analysis of the CS and hydrolyzed CS after 30 min enzymatic reaction. ΔDi0S = ΔUA-GalNAc; ΔDi6S = ΔUA-GalNAc6S.
Figure 7
Figure 7
1H NMR (A) and 13C NMR (B) spectra of the CS and hydrolyzed CS. GlcA: glucuronic acid; GalNAc: N-acetylgalactosamine. The 1H NMR and 13C NMR spectra were obtained using an Agilent DD2 spectrometer at 25 °C, and the nuclear magnetic frequency was 600 MHz.
Figure 7
Figure 7
1H NMR (A) and 13C NMR (B) spectra of the CS and hydrolyzed CS. GlcA: glucuronic acid; GalNAc: N-acetylgalactosamine. The 1H NMR and 13C NMR spectra were obtained using an Agilent DD2 spectrometer at 25 °C, and the nuclear magnetic frequency was 600 MHz.
Figure 8
Figure 8
Visualization of molecular docking of the Chon-ABC and CS. The Chon-ABC and CS (A), the pv-cABC (B), and the superimposition of the Chon-ABC and pv-cABC (C). Molecular docking was performed with AutoDock4 (Lamarckian genetic algorithm, number of runs 100).
See this image and copyright information in PMC

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