Penta-, hexa-, and heptasaccharide acceptor products of alternansucrase

In the presence of suitable acceptor molecules, dextransucrase makes a homologous series of oligosaccharides in which the isomers differ by a single glucosyl unit, whereas alternansucrase synthesizes one trisaccharide, two tetrasaccharides, etc. For the example of maltose as the acceptor, if one considers only the linear, unbranched possibilities for alternansucrase, the hypothetical number of potential products increases exponentially as a function of the degree of polymerization (DP). Experimental evidence indicates that far fewer products are actually formed. We show that only certain isomers of DP >4 are formed from maltose in measurable amounts, and that these oligosaccharides belong to the oligoalternan series rather than the oligodextran series. When the oligosaccharide acceptor products from maltose were separated by size-exclusion chromatography and HPLC, only one pentasaccharide was isolated. Its structure was alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. Two hexasaccharides were formed in approximately equal quantities: alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc and alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. Just one heptasaccharide was isolated from the reaction mixture, alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. We conclude that the enzyme is incapable of forming two consecutive alpha-(1-->3) linkages, and does not form products with more than two consecutive alpha-(1-->6) linkages. The distribution of products may be kinetically determined.     

Starch and α-glucan acting enzymes, modulating their properties by directed evolution

Starch is the major food reserve in plants and forms a large part of the daily calorie intake in the human diet. Industrially, starch has become a major raw material in the production of various products including bio-ethanol, coating and anti-staling agents. The complexity and diversity of these starch based industries and the demand for high quality end products through extensive starch processing, can only be met through the use of a broad range of starch and α-glucan modifying enzymes. The economic importance of these enzymes is such that the starch industry has grown to be the largest market for enzymes after the detergent industry. However, as the starch based industries expand and develop the demand for more efficient enzymes leading to lower production cost and higher quality products increases. This in turn stimulates interest in modifying the properties of existing starch and α-glucan acting enzymes through a variety of molecular evolution strategies. Within this review we examine and discuss the directed evolution strategies applied in the modulation of specific properties of starch and α-glucan acting enzymes and highlight the recent developments in the field of directed evolution techniques which are likely to be implemented in the future engineering of these enzymes.     

Biotechnological potential of novel glycoside hydrolase family 70 enzymes synthesizing α-glucans from starch and sucrose

Transglucosidases belonging to the glycoside hydrolase (GH) family 70 are promising enzymatic tools for the synthesis of α-glucans with defined structures from renewable sucrose and starch substrates. Depending on the GH70 enzyme specificity, α-glucans with different structures and physicochemical properties are produced, which have found diverse (potential) commercial applications, e.g. in food, health and as biomaterials. Originally, the GH70 family was established only for glucansucrase enzymes of lactic acid bacteria that catalyze the synthesis of α-glucan polymers from sucrose. In recent years, we have identified 3 novel subfamilies of GH70 enzymes (designated GtfB, GtfC and GtfD), inactive on sucrose but converting starch/maltodextrin substrates into novel α-glucans. These novel starch-acting enzymes considerably enlarge the panel of α-glucans that can be produced. They also represent very interesting evolutionary intermediates between sucrose-acting GH70 glucansucrases and starch-acting GH13 α-amylases. Here we provide an overview of the repertoire of GH70 enzymes currently available with focus on these novel starch-acting GH70 enzymes and their biotechnological potential. Moreover, we discuss key developments in the understanding of structure-function relationships of GH70 enzymes in the light of available three-dimensional structures, and the protein engineering strategies that were recently applied to expand their natural product specificities.     

Acceptor products of alternansucrase with gentiobiose. Production of novel oligosaccharides for food and feed and elimination of bitterness

In the presence of suitable acceptor molecules, dextransucrase makes a homologous series of oligosaccharides in which the isomers differ by a single glucosyl unit, whereas alternansucrase synthesizes one trisaccharide, two tetrasaccharides, etc. Previously, we showed that alternansucrase only forms certain isomers of DP > 4 from maltose in measurable amounts, and that these oligosaccharides belong to the oligoalternan series rather than the oligodextran series. We now demonstrate that the acceptor products from gentiobiose, also formed in good yields (nearly 90% in unoptimized reactions), follow a pattern similar to those formed from maltose. The initial product is a single trisaccharide, α-d-Glcp-(1→6)-β-d-Glcp-(1→6)-d-Glc. Two tetrasaccharides were formed in approximately equal quantities: α-d-Glcp-(1→3)-α-d-Glcp-(1→6)-β-d-Glcp-(1→6)-d-Glc and α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-β-d-Glcp-(1→6)-d-Glc. Just one pentasaccharide was isolated from the reaction mixture, α-d-Glcp-(1→6)-α-d-Glcp-(1→3)-α-d-Glcp-(1→6)-β-d-Glcp-(1→6)-d-Glc. Our hypothesis that the enzyme is incapable of forming two consecutive α-(1→3) linkages, and does not form products with more than two consecutive α-(1→6) linkages, apparently applies to other acceptors as well as to maltose. The glucosylation of gentiobiose reduces or eliminates its bitter taste.     

Alternansucrase acceptor reactions with D-tagatose and L-glucose

Alternansucrase (EC 2.4.1.140) is a d-glucansucrase that synthesizes an alternating alpha-(1-->3), (1-->6)-linked d-glucan from sucrose. It also synthesizes oligosaccharides via d-glucopyranosyl transfer to various acceptor sugars. Two of the more efficient monosaccharide acceptors are D-tagatose and L-glucose. In the presence of d-tagatose, alternansucrase produced the disaccharide alpha-d-glucopyranosyl-(1-->1)-beta-D-tagatopyranose via glucosyl transfer. This disaccharide is analogous to trehalulose. We were unable to isolate a disaccharide product from L-glucose, but the trisaccharide alpha-D-glucopyranosyl-(1-->6)-alpha-d-glucopyranosyl-(1-->4)-l-glucose was isolated and identified. This is analogous to panose, one of the structural units of pullulan, in which the reducing-end D-glucose residue has been replaced by its L-enantiomer. The putative L-glucose disaccharide product, produced by glucoamylase hydrolysis of the trisaccharide, was found to be an acceptor for alternansucrase. The disaccharide, alpha-D-glucopyranosyl-(1-->4)-L-glucose, was a better acceptor than maltose, previously the best known acceptor for alternansucrase. A structure comparison of alpha-D-glucopyranosyl-(1-->4)-L-glucose and maltose was performed through computer modeling to identify common features, which may be important in acceptor affinity by alternansucrase.     

Residue Leu940 Has a Crucial Role in the Linkage and Reaction Specificity of the Glucansucrase GTF180 of the Probiotic Bacterium Lactobacillus reuteri 180

Highly conserved glycoside hydrolase family 70 glucansucrases are able to catalyze the synthesis of α-glucans with different structure from sucrose. The structural determinants of glucansucrase specificity have remained unclear. Residue Leu⁹⁴⁰ in domain B of GTF180, the glucansucrase of the probiotic bacterium Lactobacillus reuteri 180, was shown to vary in different glucansucrases and is close to the +1 glucosyl unit in the crystal structure of GTF180-ΔN in complex with maltose. Herein, we show that mutations in Leu⁹⁴⁰ of wild-type GTF180-ΔN all caused an increased percentage of (α1→6) linkages and a decreased percentage of (α1→3) linkages in the products. α-Glucans with potential different physicochemical properties (containing 67–100% of (α1→6) linkages) were produced by GTF180 and its Leu⁹⁴⁰ mutants. Mutant L940W was unable to form (α1→3) linkages and synthesized a smaller and linear glucan polysaccharide with only (α1→6) linkages. Docking studies revealed that the introduction of the large aromatic amino acid residue tryptophan at position 940 partially blocked the binding groove, preventing the isomalto-oligosaccharide acceptor to bind in an favorable orientation for the formation of (α1→3) linkages. Our data showed that the reaction specificity of GTF180 mutant was shifted either to increased polysaccharide synthesis (L940A, L940S, L940E, and L940F) or increased oligosaccharide synthesis (L940W). The L940W mutant is capable of producing a large amount of isomalto-oligosaccharides using released glucose from sucrose as acceptors. Thus, residue Leu⁹⁴⁰ in domain B is crucial for linkage and reaction specificity of GTF180. This study provides clear and novel insights into the structure-function relationships of glucansucrase enzymes.     

Leuconostoc mesenteroides glucansucrase synthesis of flavonoid glucosides by acceptor reactions in aqueous-organic solvents

The enzymatic glucosylation of luteolin was attempted using two glucansucrases: the dextransucrase from Leuconostoc mesenteroides NRRL B-512F and the alternansucrase from L. mesenteroides NRRL B-23192. Reactions were carried out in aqueous-organic solvents to improve luteolin solubility. A molar conversion of 44% was achieved after 24h of reaction catalysed by dextransucrase from L. mesenteroides NRRL B-512F in a mixture of acetate buffer (70%)/bis(2-methoxyethyl) ether (30%). Two products were characterised by nuclear magnetic resonance (NMR) spectroscopy: luteolin-3'-O-alpha-d-glucopyranoside and luteolin-4'-O-alpha-d-glucopyranoside. In the presence of alternansucrase from L. mesenteroides NRRL B-23192, three additional products were obtained with a luteolin conversion of 8%. Both enzymes were also able to glucosylate quercetin and myricetin with conversion of 4% and 49%, respectively.     

Production of mannose-containing oligosaccharides by glucansucrase E81 and determination of their functional characteristics

Abstract

Oligosaccharides are one of the functional ingredients to be used in food technology. In this study, by using an active glucansucrase GTFA-ΔN E81 in the acceptor reaction of mannose, mannose-containing oligosaccharides were produced and their functionality was tested. The formation of the oligosaccharides were visualised by TLC analysis and mannose-containing oligosaccharides up to DP 7 were determined by LC-MS analysis. The presence of the (1,6)Glc and (1,3)Glc units within the oligosaccharides were determined by NMR analysis. The in vitro immune-modulatory functions of the mannose-containing oligosaccharides were determined but no induction in IL-4, IL-10, IL-12 and TNF-α cytokine levels were detected. Importantly this oligosaccharide mixture showed prebiotic effect by triggering the growth of tested probiotics and not affecting the growth of pathogen strains. Our findings reveals the potential of the role of glucansucrases for the production of functional oligosaccharides.     

Bifidogenic effect and in vitro immunomodulatory roles of melibiose-derived oligosaccharides produced by the acceptor reaction of glucansucrase E81

Glucansucrases are responsible for the production of α-glucans using sucrose as the substrate. Glucansucrases may also produce different oligosaccharides by transferring the glucose moiety from sucrose to a variety carbohydrates acting as acceptor nucleophile. In this study, melibiose-derived oligosaccharides were produced by the glucansucrase from Lactobacillus reuteri E81 expressed without the N-terminal region (GtfA-ΔN). The reaction products were characterized by TLC, LC-MS and NMR analysis and it was found that GtfA-ΔN synthesized melibiose-derived oligosaccharides (DP3 and DP4) by adding glucose units through alpha 1->3 or 1->6 glycosidic bond. The functional characteristics of these melibiose-derived oligosaccharides were determined by testing the immune-modulatory functions in HT-29 cells and testing their growth promoting effects for important probiotic and pathogenic strains. The melibiose-derived oligosaccharides triggered the production of anti-inflammatory cytokine IL-10 and pro-inflammatory cytokine TNF-α depending on their concentrations. Finally, melibiose-derived oligosaccharides showed bifidogenic effect as potential prebiotics.     

Alternansucrase acceptor reactions with methyl hexopyranosides

Alternansucrase (EC 2.4.1.140, sucrose: (1-->6), (1-->3)-alpha-D-glucan 6(3)-alpha-D-glucosyltransferase) is a D-glucansucrase that synthesizes an alternating alpha-(1-->3), (1-->6)-linked D-glucan from sucrose. It also synthesizes oligosaccharides via D-glucopyranosyl transfer to various acceptor sugars. We have studied the acceptor products arising from methyl glycosides as model compounds in order to better understand the specificity of alternansucrase acceptor reactions. The initial product arising from methyl beta-D-glucopyranoside was methyl beta-isomaltoside, which was subsequently glucosylated to yield methyl beta-isomaltotrioside and methyl alpha-D-glucopyranosyl-(1-->3)-alpha-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside. These products are analogous to those previously described from methyl alpha-D-glucopyranoside. The major initial acceptor product from methyl alpha-D-mannopyranoside was methyl alpha-D-glucopyranosyl-(1-->6)-alpha-D-mannopyranoside, but several minor products were also isolated and characterized, including a 3,6-di-O-substituted mannopyranoside. Methyl alpha-D-galactopyranoside yielded two initial products, methyl alpha-D-glucopyranosyl-(1-->3)-alpha-D-galactopyranoside and methyl alpha-D-glucopyranosyl-(1-->4)-alpha-D-galactopyranoside, in a 2.5:1 molar ratio. Methyl D-allopyranosides were glucosylated primarily at position 6, yielding methyl alpha-D-glucopyranosyl-(1-->6)-D-allopyranosides. The latter subsequently gave rise to methyl alpha-D-glucopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1-->6)-D-allopyranosides. In general, the methyl alpha-D-hexopyranosides were better acceptors than the corresponding beta-glycosides.