Enzyme immobilization offers a robust tool to scale up the production of longer, diverse, natural glycosaminoglycan oligosaccharides

Abstract

Although structurally diverse, longer glycosaminoglycan (GAG) oligosaccharides are critical to understand human biology, few are available. The major bottleneck has been the predominant production of oligosaccharides, primarily disaccharides, upon enzymatic depolymerization of GAGs. In this work, we employ enzyme immobilization to prepare hexasaccharide and longer sequences of chondroitin sulfate in good yields with reasonable homogeneity. Immobilized chondroitinase ABC displayed good efficiency, robust operational pH range, broad thermal stability, high recycle ability and excellent distribution of products in comparison to the free enzyme. Diverse sequences could be chromatographically resolved into well-defined peaks and characterized using LC-MS. Enzyme immobilization technology could enable easier access to diverse longer GAG sequences.

Keywords: chondroitinase; glycosaminoglycans; immobilization; oligosaccharides.

Fig. 1

Effect of pH (A) and temperature (B) on the catalytic activity of free and immobilized chABC using chondroitin sulfate from bovine cartilage (CS_bc). Results with CS substrates from other sources are shown in Supplementary Figures S1 and S2. Average A₂₃₂ (n = 3) for CS_bc (5 mg/mL) incubated with either 0.1 IU free or immobilized chABC at 37°C is being reported. (C) Lineweaver–Burk plot and linear fit (solid line) for CS_bc digestion with free and immobilized chABC. (D) Catalytic activity of immobilized chABC as a function of repeat cycles of reaction in batch and flow processes. Average A₂₃₂ values for CS_bc (5 mg/mL) were measured at the end of incubation time (24 h). Percent residual activity was calculated by setting the measured activity in first run as 100% (n = 3). The red dotted line shows 80% activity. (E) Preparative size-exclusion chromatography of CS_bc digested chABC under either immobilized (flow) or free enzyme conditions at pH 6.5 and 37°C. The numbers on peaks correspond to degree of polymerization (dp).

Fig. 2

ESI-MS analysis of dp18 fractions obtained from preparative size-exclusion chromatography of CS_bc digested under flow conditions with immobilized chABC (see Methods for details and Figures S8 and S9 for dp6 and dp8 results). Three relatively homogeneous (>80%) fractions obtained from a single digestion experiment correspond to different octadecasaccharide sequences. Δ refers to unsaturation at the nonreducing end. Numbers following dp (degree of polymerization) refer to length of chain (octasaccharide), number of sulfate groups and number of acetyl groups, respectively, in the sequence. # refers to the mass peak corresponding to the primary oligosaccharide identified in the fraction.