Differentiated digestion resistance and physicochemical properties of linear and α-1,2/α-1,3 branched isomaltodextrins prepared by 4,6-α-glucanotransferase and branching sucrases.

Isomaltodextrins (IMDs) are starch-based dietary fibers (DF) prepared enzymatically, which show great potential as a functional food ingredient. In this study, a series of novel IMDs with diverse structures were generated by 4,6-α-glucanotransferase GtfBΔN from Limosilactobacillus fermentum NCC 3057, combined with two α-1,2 and α-1,3 branching sucrases. Results indicated that α-1,2 and α-1,3 branching significantly improved the DF contents of α-1,6 linear products up to 60.9-62.8%. When altering the ratios of [sucrose]/[maltodextrin], IMDs containing 25.8-89.0% α-1,6 bonds, 0-59.6% α-1,2 bonds and 0-35.1% α-1,3 bonds and Mw ranged from 1967 to 4876 Da were obtained. Physicochemical property analysis showed that grafting with α-1,2 or α-1,3 single glycosyl branches can improve the solubility of the α-1,6 linear product, in which α-1,3 branched products were better. Moreover, α-1,2 or α-1,3 branching did no effect on the viscosity of the products but Mw did, the larger Mw the greater viscosity. In addition, α-1,6 linear and α-1,2 or α-1,3 branched IMDs all exhibited strong acid-heating stabilities, freeze-thaw stabilities, and good resistance to browning caused by the Maillard reaction. Branched IMDs showed excellent storage stabilities at room temperature for one year at a concentration of 60%, whereas 45% α-1,6 linear IMD precipitated quickly within 12 h. Most importantly, α-1,2 or α-1,3 branching remarkably increased the contents of resistant starch in the α-1,6 linear IMDs to 74.5-76.8%. These clear qualitative assessments demonstrated the outstanding processing and application properties of the branched IMDs and were expected to provide valuable perspectives toward the technological innovation of functional carbohydrates.

Structural characterization of slow digestion dextrin synthesized by a combination of α-glucosidase and cyclodextrin glucosyltransferase and its prebiotic potential on the gut microbiota in vitro.

Starch-based dietary fibers are at the forefront of functional ingredient research. In this study, a novel water-soluble slow digestion dextrin (SDD) was synthesized by synergy of α-glucosidase and cyclodextrin glucosyltransferase and characterized. Results showed that SDD exhibited high solubility, low viscosity, and resistance to digestive enzymes, and also showed an increased dietary fiber content of 45.7% compared with that of α-glucosidase catalysis alone. Furthermore, SDD was used as the sole carbon source to ferment selected intestinal strains and human fecal microflora in vitro to investigate its prebiotic effects. It was found that SDD could markedly enriched the abundance of Bifidobacterium, Veillonella, Dialister, and Blautia in human gut microflora and yielded higher total organic acid. The combination of α-glucosidase and cyclodextrin glucosyltransferase in this study showed valuable potential for the preparation of a novel slow digestion dextrin with good physicochemical properties and improved prebiotic effects.

Enhancing 2-O-α-D-glucopyranosyl-L-ascorbic acid synthesis by weakening the acceptor specificity of CGTase toward glucose and maltose.

2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) is a stable derivative of L-ascorbic acid (L-AA), which has been widely used in food and cosmetics industries. Sugar molecules, such as glucose and maltose produced by cyclodextrin glycosyltransferase (CGTase) during AA-2G synthesis may compete with L-AA as the acceptors, resulting in low AA-2G yield. Multiple sequence alignment combined with structural simulation analysis indicated that residues at positions 191 and 255 of CGTase may be responsible for the difference in substrate specificity. To investigate the effect of these two residues on the acceptor preference and the AA-2G yield, five single mutants Bs F191Y, Bs F255Y, Bc Y195F, Pm Y195F and Pm Y260F of three CGTases from Bacillus stearothermophilus NO2 (Bs), Bacillus circulans 251 (Bc) and Paenibacillus macerans (Pm) were designed for AA-2G synthesis. Under optimal conditions, the AA-2G yields of the mutants Bs F191Y and Bs F255Y AA-2G were 34.3% and 7.9% lower than that of Bs CGTase, respectively. The AA-2G yields of mutant Bc Y195F, Pm Y195F and Pm Y260F were 45.8%, 36.9% and 12.6% higher than those of wild-type CGTases, respectively. Kinetic studies revealed that the residues at positions 191 and 255 of the three CGTases were F, which decreased glucose and maltose specificity and increased L-AA specificity. This study not only proposes for the first time that the AA-2G yield can be improved by weakening the acceptor specificity of CGTase toward sugar byproducts, but also provides new insight on the modification of CGTase that catalyze the double-substrate transglycosylation reaction.

Trehalose promotes high-level heterologous expression of 4,6-α-glucanotransferase GtfR2 in Escherichia coli and mechanistic analysis.

4,6-α-Glucanotransferases (4,6-α-GTs) hold great potential for applications in the food and medical industries because of their efficient transglycosylation ability. However, it is relatively difficult to achieve high soluble expression because of their high molecular weight and multidomain nature. In this study, 4,6-α-GT of Burkholderia sp. (GtfR2) was successfully expressed in E. coli, and the activity attained 1.55 × 104 U/mL by traditional fermentation optimization. However, a large number of inactive inclusion bodies of GtfR2 were still present due to aggregation and precipitation. The trehalose-mediated strategy was first proposed and applied in the fermentation process of GtfR2. Trehalose addition significantly reduced inclusion bodies, resulting in an increase in GtfR2 activity (6.48 × 104 U/mL), which was 4.20 times higher than that of the control group. Our molecular dynamics simulations revealed that trehalose could spontaneously stabilize the conformational dynamics of GtfR2 by binding to the groove, loop, α-helix and N-terminal unstable regions on the surface. This strategy was also available to enhance the soluble expression of other 4,6/4,3-α-GTs, which were increased by 3.03-77.19 times. This study is the first to observe that trehalose can inhibit the aggregation and precipitation of GtfR2, which provides a new perspective for the recombinant expression of 4,6/4,3-α-GTs.

Diverse prebiotic effects of isomaltodextrins with different glycosidic linkages and molecular weights on human gut bacteria in vitro.

Isomaltodextrin (IMD) is a novel dietary fiber enzymatically produced by reconstructing the molecular chain structure of starch using glycosyltransferases. In this study, the specific prebiotic effects of α-1,6 linear and α-1,2 or α-1,3 branched IMDs with different molecular weights (Mw) on human intestinal bacteria were investigated by pure culture of single strains and mixed fermentation of human fecal microflora in vitro. The results showed that α-1,6 linear IMDs markedly promoted beneficial Bifidobacterium and Lactobacillus in both pure culture and mixed fermentation. α-1,3 branching exhibited similar selectivity with α-1,6 linkage but yielded more butyrate in pure cultures. In contrast, IMDs containing α-1,2 branches were utilized efficiently only during mixed fermentation, which was speculated to result from metabolic cross-feeding. Regarding Mw, IMDs with lower Mw showed better prebiotic effects in pure cultures but no differences in mixed culture. These findings provide a theoretical basis for their application as functional foods.

Enhanced stability of manganese superoxide dismutase by amino acid replacement designed via molecular dynamics simulation.

In order to improve manganese-SOD stability, three mutations were constructed via site-directed mutagenesis, and the root mean square fluctuation (RMSF) and root mean square deviation (RMSD) were used as stability assessment indexes. The amino acids of V140, E155 and E215 from wild-type mouse Mn-SOD was replaced to L140, W155 and W215, and a recombinant plasmid containing DNA segment coding wild-type and mutant Mn-SOD protein was transformed into Escherichia coli BL21 for expression. The highest enzyme activity of the mutations-MnSOD was 2050 U/mg. In addition, the recombinant protein, TM-MnSODV140L, E155W, E215W exhibited higher working temperature and improved stability compared with the wild-type Mn-SOD. Furthermore, CD spectrum analysis of the improved mutants and wild-type enzyme showed that there was no significant change in their secondary structures. This study not only expands the scope of the application of enzymes, but also helps us understand the relationship between protein structure and function.

Chiral N-tert-butanesulfinyl α,ß-unsaturated ketimine: a simple and highly effective olefin/sulfinimide hybrid ligand for asymmetric 1,4-additions.

One novel type of chiral olefin/sulfinimide hybrid ligands has been developed through a simple one-step condensation of α,ß-unsaturated ketones with tert-butanesulfinamide and utilized successfully for rhodium-catalyzed asymmetric conjugated additions to furnish the desired adducts in high yields with excellent ee's.

Simple N-sulfinyl-based chiral sulfur-olefin ligands for rhodium-catalyzed asymmetric 1,4-additions.

A variety of N-sulfinyl-based chiral sulfur-olefin ligands has been successfully developed for the first time for rhodium-catalyzed highly efficient and enantioselective 1,4-additions. The ease of synthesis and needless consideration of the carbon chirality makes this novel type of ligands attractive for asymmetric catalysis.