A Novel Neutral and Mesophilic β-Glucosidase from Coral Microorganisms for Efficient Preparation of Gentiooligosaccharides

Abstract

β-glucosidases can produce gentiooligosaccharides that are lucrative and promising for the prebiotic and alternative food industries. However, the commercial production of gentiooligosaccharides using β-glucosidase is challenging, as this process is limited by the need for high thermal energy and increasing demand for the enzyme. Here, a putative β-glucosidase gene, selected from the coral microbial metagenome, was expressed in Escherichia coli. Reverse hydrolysis of glucose by Blg163 at pH 7.0 and 40 °C achieved a gentiooligosaccharide yield of 43.02 ± 3.20 g·L^-1 at a conversion rate of 5.38 ± 0.40%. Transglycosylation of mixed substrates, glucose and cellobiose, by Blg163 consumed 21.6 U/0.5 g glucose/g cellobiose, achieving a gentiooligosaccharide yield of 70.34 ± 2.20 g·L^-1 at a conversion rate of 15.63%, which is close to the highest yield reported in previous findings. Blg163-mediated synthesis of gentiooligosaccharides is the mildest reaction and the lowest β-glucosidase consumption reported to date.

Keywords: Transglycosylation; coral microorganism; gentiooligosaccharides; reverse hydrolysis; β-glucosidase.

Figure 1

Alignment of amino acid sequence of Blg163 with other β-glucosidases.

Figure 2

SDS-PAGE analysis of the purified Blg163 (A). M protein marker; 1 recombinant Escherichia coli BL21(DE3) harboring pEASY-E1(+) induced with IPTG; 2 recombinant Escherichia coli BL21(DE3) harboring pEASY-E1(+)—Blg163 induced with IPTG; 3 purified Blg163. Effects of temperature on gentiooligosaccharide production (B). The reactions were launched in 50 mM citrate buffer (pH 7.0) at 25–45 °C with 30% glucose and 15% cellobiose.

Figure 3

Effects of pH on the yield of gentiooligosaccharides (A). The reactions were performed at 40 °C in 0.2 M McIlvaine buffer for pH 3.0–8.0 or 0.05 M glycine-NaOH buffer for pH 8.0–11.0 with 30% glucose and 15% cellobiose. Outputs of gentiooligosaccharides at different concentration of Blg163 (B). The reactions were performed in optimum conditions.

Figure 4

Time course of gentiobiose yield by the recombinant Blg163. The reactions were performed in optimum conditions. The HPLC analysis of the products formed by transglycosylation. (●) Glucose; (■) cellobiose; (▲) gentiooligosaccharides.