Enzymatic Production of Chondroitin Oligosaccharides and Its Sulfate Derivatives

Abstract

Chondroitin sulfate (CS) has a wide range of physiological functions and clinical applications. However, the biosynthesis of chondroitin oligosaccharides (o-CHs) and sulfate derivatives with specific length is always challenging. Herein, we report enzymatic strategies for producing homogeneous o-CHs and its sulfate derivatives from microbial sourced chondroitin. Chondroitin disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, and decasaccharides with defined structure were produced by controllably depolymerizing microbial sourced chondroitin with an engineered chondroitinase ABC I. The highest conversion rates of the above corresponding o-CHs were 65.5%, 32.1%, 12.7%, 7.2%, and 16.3%, respectively. A new efficient enzymatic sulfation system that directly initiates from adenosine 5'-triphosphate (ATP) and sulfate was developed and improved the sulfation of chondroitin from 8.3% to 85.8% by optimizing the temperature, sulfate and ATP concentration. o-CHs decasaccharide, octasaccharide, hexasaccharide, tetrasaccharide and disaccharide were modified and the corresponding sulfate derivatives with one sulfate group were prepared. The enzymatic approaches constructed here for preparing o-CHs and its sulfate derivatives pave the way for the study of structure-activity relationship and applications.

Keywords: chondroitin; chondroitin sulfate; chondroitinase ABC I; oligosaccharides; sulfation system.

FIGURE 1

Chondroitinase ABC I depolymerizes chondroitin chain by breaking the β-1,4 glycoside bond.

FIGURE 2

Production and characterization of chondroitin oligosaccharides. (A) Time course of chondroitin depolymerization with different concentrations of csABC I. (B) Fraction of chondroitin oligosaccharides were linearly eluted with 0–140 mM NaCl from a Q HP column at a flow rate of 3 ml/min. (C) Purity analysis of the CH2, CH4, CH6, CH8, and CH10 via HPLC. (D) Ultraviolet absorption spectrum of chondroitin oligosaccharides. M _w, molecular weight; CH2, disaccharide; CH4, tetrasaccharide; CH6, hexasaccharide; CH8, octasaccharide; CH10, decasaccharide. The data are expressed as the mean ± SD from three (n = 3) biologically independent replicates.

FIGURE 3

Orthogonalizing csABC I activities and depolymerization time to maximized the yield of o-CHs. (A) CH10, (B) CH8, (C) CH6, (D) CH4, (E) CH2. The csABC I activity and reaction time spanned 200–800 U/L and 2–24 h, respectively. CH2, disaccharide; CH4, tetrasaccharide; CH6, hexasaccharide; CH8, octasaccharide; CH10, decasaccharide. The data are expressed from three (n = 3) biologically independent replicates.

FIGURE 4

Construction and optimization of the sulfation system from ATP and sulfate. (A) Schematic diagram of sulfation-modification system for CSA biosynthesis. (B) The effect of temperature on sulfation in the reaction system with 10 mM ATP, 20 mM MgSO₄, 0.5 g/L ASAK^S5, 1.0 g/L C4ST, and 2.0 g/L chondroitin for 48 h. (C) The effect of ATP concentration and MgSO₄ concentration on the sulfation in the system with ASAK^S5 (0.5 g/L), C4ST (1.0 g/L) and chondroitin (2.0 g/L) at a reaction temperature of 35°C for 48 h. ATP, adenosine 5′-triphosphate; ADP, adenosine 5′-diphosphate; PAPS, 3′-phosphoadenosine-5′-phosphosulfate; PPi, pyrophosphate; ASAK^S5, a bifunctional PAPS synthase that converts ATP to PAPS. The data are expressed as the mean ± SD from three (n = 3) biologically independent replicates. The statistical analysis was performed by two-sided t-test. *p < 0.05, **p < 0.01, ***p < 0.001; NS not significant (p ≥ 0.05).

FIGURE 5

Characterization of chondroitin sulfate A oligosaccharides via UPLC-MS. (A) The ion chromatogram of Di-4S was determined by UPLC-MS. (B) MS spectra of chondroitin disaccharide (Di-0S) (C) MS spectra of chondroitin sulfate A disaccharide (Di-4S). Di-0S, chondroitin disaccharide; Di-4S, chondroitin sulfate A disaccharide; CHS2, chondroitin disaccharide sulfate derivative; CHS4, chondroitin tetrasaccharide sulfate derivative; CHS6, chondroitin hexasaccharide sulfate derivative; CHS8, chondroitin octasaccharide sulfate derivative; CHS10, chondroitin decasaccharide sulfate derivative.

FIGURE 6

Mass spectrometry identification of sulfate derivatives of chondroitin oligosaccharides (A) Mass spectrometric identification of CH10 sulfated to CHS10. (B) Mass spectrometric identification of CH8 sulfated to CHS8. (C) Mass spectrometric identification of CH6 sulfated to CHS6. (D) Mass spectrometric identification of CH4 sulfated to CHS4. CHS2, chondroitin disaccharide sulfate derivative; CHS4, chondroitin tetrasaccharide sulfate derivative; CHS6, chondroitin hexasaccharide sulfate derivative; CHS8, chondroitin octasaccharide sulfate derivative; CHS10, chondroitin decasaccharide sulfate derivative.