Enzymatic Preparation of Gentiooligosaccharides by a Thermophilic and Thermostable β-Glucosidase at a High Substrate Concentration

Abstract

Gentiooligosaccharides (GnOS) are a kind of oligosaccharide formed by glucose with β-1-6 glycosidic bonds, which has become a new type of functional oligosaccharide for its unique refreshing bitter taste and valuable probiotic effects. However, the research on the enzymatic preparation of GnOS is not thorough enough. In this study, a GH1 thermophilic β-glucosidase from Thermotoga sp. KOL6 was used as a biocatalyst for the synthesis of GnOS. TsBgl1 exhibited excellent thermophilic and thermostable properties by possessing a melting temperature of 101.5 °C and reacting at 80-90 °C efficiently. Its half-life at 90 °C was approximately 5 h, suggesting its high heat resistance as well. TsBgl1 also showed excellent glucose tolerance with an inhibition constant (K_i) of 1720 mM and was stimulated in the presence of 0-900 mM glucose. TsBgl1 showed the highest hydrolytic activity on laminaribiose (Glc-β-1,3-Glc), but mainly synthetized gentiobiose (Glc-β-1,6-Glc) during transglycosylation. By optimizing the reaction conditions and substrate concentration, the highest yield of GnOS synthesized by TsBgl1 reached 144.3 g·L^-1 when 1000 g·L^-1 glucose was used as a substrate, which was higher than the highest yield ever reported. The thermophilic and thermostable properties of TsBgl1 were considered to be significant advantages in the industrial production of GnOS, where long periods of high-temperature reactions are required. This study was expected to provide an excellent candidate enzyme for industrial production of GnOS and also provide a reference for studying the transglycosylation of GH1 β-glucosidases.

Keywords: gentiobiose; gentiooligosaccharides; thermophilic; transglycosylation; β-glucosidase.

Figure 1

SDS-PAGE (A) and DSC (B) analysis of purified TsBgl1. M, standard protein marker. Lane 1, purified TsBgl1. Aw, water activity. Tm, melting temperature. ΔH, enthalpy change.

Figure 2

Enzymatic properties of recombinant TsBgl1. (A) effect of temperature on enzyme activity. (B) effect of pH on enzyme activity. (C) thermostability. (D) pH stability. The data of pH stability was shown as the residual enzyme activities measured under optimal conditions of enzymes incubated in buffers of different pH values at 4 °C for seven days. The activity assays were performed using 4 mM pNP-β-Glc as substrate.

Figure 3

Glucose inhibition curve of recombinant TsBgl1. The activity assays were performed under the optimum condition of 90 °C and pH 6.0 using 4 mM pNP-β-Glc as substrate in the presence of different concentrations of glucose.

Figure 4

Effects of reaction parameters and substrate concentration on GnOS yield. (A) effect of reaction temperature on GnOS yield. (B) effect of reaction pH on GnOS yield. (C) effect of enzyme amount on GnOS yield. (D) effect of substrate concentration on GnOS yield.

Figure 5

Transglycosylation product analysis of recombinant TsBgl1. (A) HPLC chromatograms of transglycosylation products and formulas of the disaccharides synthetized by TsBgl1. Lam, laminaribiose (Glc-β-1,3-Glc). Cel, cellobiose (Glc-β-1,4-Glc). Sop, sophorose (Glc-β-1,2-Glc). Gen, gentiobiose (Glc-β-1,6-Glc). The formulas of the disaccharides were shown in the boat conformation, and the position of the linked hydroxyl group from the non-reducing end to the reducing end were annotated by arrow. (B) Time course of TsBgl1 catalyzed transglycosylation. The reaction was performed by adding 500 U·g glucose⁻¹ TsBgl1 and using 1000 g·L⁻¹ glucose as substrates at 80 °C and pH 6.0.