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

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 K_m and V_max 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.

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

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

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

The stability of the recombinant Chon-ABC at 37 °C (A) and 4 °C (B) in 75 mM pH 8.0 PBS.

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

GPC analysis of the CS (A) and hydrolyzed CS (B) and the weight-average molecular weight (M_w) and the distributions (C). The 0 min and 30 min in C are the reaction time. M_n and nd mean the number-average molecular weight and not determined, respectively.

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

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

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

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).