Biosynthesis of Raffinose and Stachyose from Sucrose via an In Vitro Multienzyme System

Abstract

Herein, we present a biocatalytic method to produce raffinose and stachyose using sucrose as the substrate. An in vitro multienzyme system was developed using five enzymes, namely, sucrose synthase (SUS), UDP-glucose 4-epimerase (GalE), galactinol synthase (GS), raffinose synthase (RS), and stachyose synthase (STS), and two intermedia, namely, UDP and inositol, which can be recycled. This reaction system produced 11.1 mM raffinose using purified enzymes under optimal reaction conditions and substrate concentrations. Thereafter, a stepwise cascade reaction strategy was employed to circumvent the instability of RS and STS in this system, and a 4.2-fold increase in raffinose production was observed. The enzymatic cascade reactions were then conducted using cell extracts to avoid the need for enzyme purification and supplementation with UDP. Such modification further increased raffinose production to 86.6 mM and enabled the synthesis of 61.1 mM stachyose. The UDP turnover number reached 337. Finally, inositol in the reaction system was recycled five times, and 255.8 mM raffinose (128.9 g/liter) was obtained. IMPORTANCE Soybean oligosaccharides (SBOS) have elicited considerable attention because of their potential applications in the pharmaceutical, cosmetics, and food industries. This study demonstrates an alternative method to produce raffinose and stachyose, which are the major bioactive components of SBOS, from sucrose via an in vitro enzyme system. High concentrations of galactinol, raffinose, and stachyose were synthesized with the aid of a stepwise cascade reaction process, which can successfully address the issue of mismatched enzyme characteristics of an in vitro metabolic engineering platform. The biocatalytic approach presented in this work may enable the synthesis of other valuable galactosyl oligosaccharides, such as verbascose and higher homologs, which are difficult to obtain through plant extraction.

Leloir Glycosyltransferases in Applied Biocatalysis: A Multidisciplinary Approach

“…Under thermodynamic control, Leloir glycosyltransferases produce oligosaccharides if the overall glycosylation reaction is exergonic (Figure 7). A one-pot procedure using five enzymes allowed for the production of raffinose and stachyose from sucrose [183], using unpurified cell-free extract formulations and supplementation of UDP with a total-turnover number (TTN) of 337. Thermodynamic constraints were observed in the endergonic nucleotide sugar donor production, while coupling of the galactinol, raffinose, and stachyose were exergonic, thereby driving the overall reaction toward oligosaccharide synthesis.…”

“…( a ). The standard Gibbs free energy changes of the individual reactions (ΔG°s, red) and the total reaction (ΔG°, grey) shown in ( b ) [183]. The Δ r G′° represents the change of Gibbs free energy and was calculated using the eQuilibrator web interface () [155] using the following conditions: ionic strength 0.1 M, pH 7.0, 1 mM of component, 298 K. Abbreviations: UDP- d -glc, UDP- d -glucose; UDP- d -gal, UDP- d -galactose, SuSy, Sucrose synthase; GalE, UDP- d -glucose-4-epimerase; GS, galactinol synthase; RS, raffinose synthase; STS, stachyose synthase.…”

The interaction of intestinal bacteria and feed: a case study of intensive-duck husbandry in Central Java

“…The raffinose molecules are composed of glucose, galactose, and fructose, which bond to each other at C1 and C6 atoms (Gangl and Tenhaken, 2016). Simultaneously, the tetrasaccharide group stachyose comprises two α-D-galactose molecules and one α-D-glucose β-D-fructose molecule each (Gangl and Tenhaken, 2016;Tian et al, 2019). Both types of oligosaccharides cannot be digested by monogastric animals such as ducks but can be fermented into short-chain fatty acids by intestinal bacteria using the α-galactosidase enzyme (Dong et al, 2020).…”

Identification and characterization of a moonlighting protein-enolase for surface display in Streptococcus thermophilus

“…Purified enzymes are often non-reusable and suffer from the environment pressure in the industrial production and storage [18,[43][44][45]. In this study, the EnoM could drive the β-galactosidase on the surface of S. thermophilus cells in vitro, achieving the reutilization of this enzyme.…”