Trehalose Analogues: Latest Insights in Properties and Biocatalytic Production

Abstract

Trehalose (α-D-glucopyranosyl α-D-glucopyranoside) is a non-reducing sugar with unique stabilizing properties due to its symmetrical, low energy structure consisting of two 1,1-anomerically bound glucose moieties. Many applications of this beneficial sugar have been reported in the novel food (nutricals), medical, pharmaceutical and cosmetic industries. Trehalose analogues, like lactotrehalose (α-D-glucopyranosyl α-D-galactopyranoside) or galactotrehalose (α-D-galactopyranosyl α-D-galactopyranoside), offer similar benefits as trehalose, but show additional features such as prebiotic or low-calorie sweetener due to their resistance against hydrolysis during digestion. Unfortunately, large-scale chemical production processes for trehalose analogues are not readily available at the moment due to the lack of efficient synthesis methods. Most of the procedures reported in literature suffer from low yields, elevated costs and are far from environmentally friendly. "Greener" alternatives found in the biocatalysis field, including galactosidases, trehalose phosphorylases and TreT-type trehalose synthases are suggested as primary candidates for trehalose analogue production instead. Significant progress has been made in the last decade to turn these into highly efficient biocatalysts and to broaden the variety of useful donor and acceptor sugars. In this review, we aim to provide an overview of the latest insights and future perspectives in trehalose analogue chemistry, applications and production pathways with emphasis on biocatalysis.

Keywords: enzyme engineering; galactosidase; glycoside phosphorylase; glycosyltransferase; prebiotic.

Figure 1

Structure of trehalose (a) and its analogues lactotrehalose (b); galactotrehalose (c); and 6-azidotrehalose (d). The azide-functional group is indicated in red.

Figure 2

Summary of applications for trehalose and its analogues in food, pharmaceutical, cosmetics and agricultural industries.

Figure 3

Schematic representation of biocatalytic pathways for trehalose synthesis. The traditional trehalose phosphate synthase/phosphorylase (TPS/TPP) route (a); the malto-oligosyltrehalose synthase/hydrolase (MTS/MTH)-coupled route (b); the TreS-type trehalose synthase route (c); the TreT-like trehalose synthase route (d); and the inverting (e) or retaining (f) trehalose phosphorylase (TP) routes coupled with other glycoside phosphorylases, i.e., maltose phosphorylase (MP) and sucrose phosphorylase (SP), respectively. The latter three pathways (d–f) are also amenable to trehalose analogue synthesis. NDP = nucleoside diphosphate.

Figure 4

Pathway for synthesis of trehalose trisaccharide analogues via E. coli β-galactosidase activity starting from the disaccharides lactose and trehalose.

Figure 5

TreT-type trehalose synthase. (a) Natural reaction in trehalose synthesis direction starting from UDP-glucose and d-glucose; (b) active site residues (indicated in dark purple) surrounding the d-glucose moiety (light blue) in the acceptor binding pocket of P. horikoshii TreT [49]. Replacing the histidine at position 92 into an alanine via site-specific mutagenesis resulted in a 10% improvement of the activity on mannose as alternative acceptor monosaccharide.

Figure 6

Enhancing acceptor and donor specificity of T. brockii trehalose phosphorylases (TP) and C. subterraneus TP via random mutagenesis and rational design, respectively. (a) Reversible conversions of β-Glc-1P with d-galactose and β-Gal-1P with d-glucose into lactotrehalose, catalyzed by the T. brockii TP-single mutant (R448S) with enhanced acceptor specificity, and the C. subterraneus TP-triple mutant (L694G/A693Q/W371Y) with enhanced donor specificity, respectively; (b) Active sites of T. brockii TP (left) and C. subterraneus TP (right) with the positions subjected to mutagenesis indicated. For T. brockii TP, the mutated R448-residue is shown in orange/blue amongst other mutated residues from the same study, and the trehalose substrate with the catalytic residue (E498) in green. In the C. subterraneus TP active site, the three mutagenized positions (L694G/A693Q/W371Y) causing a switch in donor specificity are indicated in green with the corresponding wild-type residues shown in yellow. The lactotrehalose substrate is indicated in orange [66,67].