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.
Levansucrases, which belong to the glycoside hydrolase family 68 (GH68), synthesize β (2-6)-linked fructan levan with sucrose as substrate. We described the use of a levansucrase (Bl_SacB) from Bacillus licheniformis 8-37-0-1 for catalysis of fructosyl transfer to obtain high levan yield previously. In the present study, six variants (Y246A, N251A, K372A, R369A, R369S, and R369K) were constructed through sequence alignment and structural analysis to explore the synthesis mechanism of Bl_SacB. The selected residues were predicted to localize to the substrate-entering channel of the active cavity and close to or remote from the catalytic triad. The products of these variants ranged from homopolymers levan to fructo-oligosaccharides (FOSs). The primary FOSs were identified through MS and NMR analyses as neolevan-type neokestose [β-D-Fru-(2-6)-α-D-Glc-(1-2)-β-D-Fru], levan-type 6-kestose [β-D-Fru-(2-6)-β-D-Fru-(2-1)-α-D-Glc], and inulin-type 1-kestose [β-D-Fru-(2-1)-β-D-Fru-(2-1)-α-D-Glc]. The mutation at Tyr located remote from the catalytic triad led to the production of short-chain oligosaccharides with degree of polymerization (DP) of up to 25. The replaced Arg located close to the catalytic triad resulted in either elimination of polysaccharide synthesis or complete change in the dominant linkage of the products. The Michaelis constants (K) of Y246A, N251A, K372A, and R369K were found to be similar to that of the wild type (WT). However, the turnover number (k) and the value of transfructosylation versus hydrolysis activity of the six variants decreased compared with those of the WT. Hence, the residues located on the surface of the substrate-entering channel of Bl_SacB can be critical in product linkage type and/or elongation mechanism.
This work established an integrated utilization of dairy whey in β-galactosidase production from Lactobacillus bulgaricus and prebiotics synthesis by the probiotic enzyme. A cost-effective whey-based medium was newly developed for culturing Lactobacillus bulgaricus to produce β-galactosidase. The medium was optimized through response surface methodology (RSM) involving a series of statistical designs, such as the Plackett–Burman design, steepest ascent experiment, and central composite design. Under the optimized medium, the β-galactosidase activity of L. bulgaricus reached 2034 U/L, which was twice that produced from the traditional MRS medium. The cells of L. bulgaricus harvested from the whey-based medium were subsequently treated with lysozyme. The resulting crude enzyme was used as an efficient catalyst, which catalyzed the synthesis of the prebiotic galacto-oligosaccharides (GOS) in a high yield of 44.7% by using whey (200 g/L) as the substrate. The sugar mixture was further purified by activated charcoal adsorption, thereby yielding a high-purity level of 77.6% GOS.
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