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Our pursuit of new compounds with enhanced bioavailability and bioactivity prompted us to employ the biotransformation-guided purification (BGP) approach which leverages proficient in vitro biotransformation techniques. Angelica dahurica roots, also called Baizhi in Chinese traditional medicine, are famous for their anti-inflammatory and analgesic properties. Herein, we applied the BGP methodology to Baizhi extracts, employing Deinococcus geothermalis amylosucrase (Dg AS), an enzyme demonstrating catalytic competence across diverse substrates, for biotransformation. Initiating with a 70 % methanol extraction, we obtained the crude extract of commercial Baizhi powder, followed by an additional extraction using ethyl acetate. Notably, reactions performed on this extract yielded limited quantities of novel compounds. Subsequently, the extract underwent partitioning into four fractions based on HPLC profiling, leading to the successful isolation of a compound with significant yield from fraction 2 mixtures upon reaction with Dg AS. Structural elucidation confirmed the compound as byakangelicin-7″- O - α -glucopyranoside (BG-G), a new alpha glycoside derivative of byakangelicin. Furthermore, validation experiments verified the capacity of Dg AS to glycosylate pure byakangelicin, yielding BG-G. Remarkably, the aqueous solubility of BG-G exceeded that of byakangelicin by over 29,000-fold. In conclusion, BGP emerges as a potent strategy combining traditional medicinal insights with robust enzymatic tools for generating new compounds. [ABSTRACT FROM AUTHOR]

Naringenin, a flavanone (flavonoid family) has bioactive effects, such as antioxidant, free radical scavenging, anti-inflammatory, carbohydrate metabolism, and immunoregulatory effects on the human body. However, its low water solubility and bioavailability limit its use in food, pharmaceutical, cosmetic, and drug industries. Amylosucrase (ASase, E. C. 2.4.1.4), uses sucrose as a substrate and is a glycosyltransferase that hydrolyzes glucose and fructose generating a glycosylated product. In this study, the putative ASase gene (accession number: WP_102128133.1) from Deinococcus planocerae KCTC 33809 (DplAS) was expressed in Escherichia coli. The optimum reaction temperature and pH for sucrose hydrolytic capacity of DplAS were determined to be 30 ℃ and 7.0, respectively. DplAS was determined to have a half-life of 256.7 and 42.8 min at 40 ℃ and 45 ℃, respectively. Although it exhibited high similarity with DgAS (80%), DplAS displayed different acceptor specificities to that of DgAS. Uniquely, DplAS generated one major product (naringenin-maltoside) via transglycosylation using sucrose and naringenin as donor and acceptor molecules, respectively. Based on NMR, naringenin-maltoside was identified as naringenin-4'-O-a-D-maltopyranoside, and its conversion rate was optimized using the response surface method (RSM) with central composite design (CCD). This resulted in a donor to acceptor ratio of 25.9:1 at 27.64 ℃, and a 55.69% conversion of sucrose. [ABSTRACT FROM AUTHOR]

Amylosucrase (ASase) from Deinococcus geothermalis (DgAS) is involved in the highest number of glycotransfer reactions with different flavonoids as acceptor molecules among all ASases, overcoming the flavonoid disadvantage; however, its transglycosylation yield is low if the structure of the acceptor molecule does not fit into its active site pocket. Therefore, this study focused on improving the transglycosylation yield and elucidating the underlying mechanism of action by replacing the isoleucine at position 221 of DgAS (I221V) near subsite +1 with a valine residue. Molecular dynamics simulations revealed that the I221V mutation in the protein did not significantly alter its overall conformation but led to decreased flexibility around residues 3 and 7 and an increase in the active site pocket volume. The proportion of hydrolysis and polymerization reactions in I221V increased when reacted with sucrose as the sole substrate, while the proportion of isomerization reactions decreased compared to those of the DgAS wild-type (WT). In addition, I221V showed a higher transglycosylation efficiency for 6- and 4′-hydroxyflavones than for WT, which was also twofold higher for biosynthesized naringenin α-glucoside. Kinetic parameters showed that the higher transglycosylation efficiency of I221V compared to that of WT was due to its increased catalytic rate for sucrose as a donor molecule, rather than its affinity for naringenin as an acceptor molecule. Overall, our findings provide further insight into how DgAS mutation can affect the transglycosylation reaction, which could aid in the development of more efficient industrial applications. [ABSTRACT FROM AUTHOR]

Amylosucrase (ASase) efficiently biosynthesizes α-glucoside using flavonoids as acceptor molecules and sucrose as a donor molecule. Here, ASase from Deinococcus wulumuqiensis (DwAS) biosynthesized more naringenin α-glucoside (NαG) with sucrose and naringenin as donor and acceptor molecules, respectively, than other ASases from Deinococcus sp. The biotransformation rate of DwAS to NαG was 21.3% compared to 7.1–16.2% for other ASases. Docking simulations showed that the active site of DwAS was more accessible to naringenin than those of others. The 217th valine in DwAS corresponded to the 221st isoleucine in Deinococcus geothermalis AS (DgAS), and the isoleucine possibly prevented naringenin from accessing the active site. The DwAS-V217I mutant had a significantly lower biosynthetic rate of NαG than DwAS. The k cat /K m value of DwAS with naringenin as the donor was significantly higher than that of DgAS and DwAS-V217I. In addition, NαG inhibited human intestinal α-glucosidase more efficiently than naringenin. [Display omitted] • Naringenin α-glucoside is efficiently synthesized by D. wulumuquiensis amylosucrase. • Biosynthesized naringenin α-glucoside can be used for treating chronic diseases. • Naringenin α-glucoside might be a better functional food ingredient than naringenin. [ABSTRACT FROM AUTHOR]

The aim of this study was to establish an efficient bioprocess for the synthesis of trehalulose as a novel sweetener. This disaccharide has 70% of the sweetness of sucrose and bioactive properties such as anti-cariogenicity and anti-oxidizing activity. In this study, amylosucrase from the Deinococcus deserti (Dd AS) gene was expressed and purified. When Dd AS was reacted with 2 M sucrose at 35 °C for 120 h, the yield ratio of trehalulose to turanose was approximately 2:1. The trehalulose yield increased when extrinsic fructose was added. Under optimum conditions for trehalulose synthesis, the yield reached 36% (246 g/L, sucrose basis) starting with 2 M sucrose + 0.75 M fructose and showed the highest trehalulose productivity (1.94 g/L/h). As a result, a novel amylosucrase that synthesized trehalulose as the major product was developed, in contrast to other studied amylosucrase-type enzymes. Dd AS could be utilized industrially in a bioprocess for producing trehalulose as a functional sucrose alternative. • A novel amylosucrase was found to synthesize trehalulose as a main product. • The enzyme from Deinococcus sp. was expressed in E. coli. • Yield ratio of trehalulose to turanose was 2:1 with 2 M sucrose at 35 °C and pH7.5. • Maximum trehalulose yield reached 36% with 2 M sucrose + 0.75 M fructose. • Industrial application of this enzyme may lead to development of a valuable sweetener. [ABSTRACT FROM AUTHOR]

4- α -glucanotransferase is used to produce thermoreversible starch gels to alleviate limitations to use of starch gels in repetitively heat-processed foods. However, the gel strength was weakened after this enzyme modification. In the present study, treatment by amylosucrase (NpAS) of corn starch and sucrose was applied to retain the gel thermoreversibility and eliminate the shortcoming caused by 4- α -glucanotransferase (Tu α GT). Changes in molecular structure, rheological and retrogradation properties of modified starch were investigated after NpAS and Tu α GT sequential and one-pot treatment, respectively. The apparent amylose content was reduced and increased by sequential and one-pot treatments, respectively, compared to single Tu α GT modification. Chain length profiles showed higher proportion of degree of polymerization (DP) ≥ 13 by sequential treatment, whereas DP 6–12 was higher after one-pot treatment. All modified starches had reduced molecular weight. G' and G" increased by dual enzyme compared to single Tu α GT treatment having little effect on retrogradation. Interestingly, starch subjected to 3 h one-pot treatment caused G' and G" temperature curves to cross-over, improving thermoreversible properties. The results indicate that NpAS treatment compensated for loss of starch gel strength caused by Tu α GT and offered possibility to provide a wider range of thermoreversible starches. [ABSTRACT FROM AUTHOR]

Enzymatically modified isoquercitrin (EMIQ) is a glyco-chemically modified flavonoid exhibiting notably high biological activity, such as antioxidant, anti-inflammatory and anti-allergic properties. However, the utilization of expensive substrates such as isoquercitrin and cyclodextrin in the conventional approach has hindered the industrial-scale production of EMIQ due to high cost and low yields. Hence, the development of a cost-effective and efficient method is crucial for the biological synthesis of EMIQ. In this study, a natural cascade catalytic reaction system was constructed with α -L-rhamnosidase and amylosucrase using the inexpensive substrates rutin and sucrose. Additionally, a novel approach integrating gradient temperature regulation into biological cascade reactions was implemented. Under the optimal conditions, the rutin conversion reached a remarkable 95.39% at 24 h. Meanwhile, the productivity of quercetin-3- O -tetraglucoside with the best bioavailability reached an impressive 41.69%. This study presents promising prospects for future mass production of EMIQ directly prepared from rutin and sucrose. [Display omitted] • A novel cascade strategy was constructed to produce EMIQ from inexpensive substrates. • Amylosucrase with sucrose as a substrate was used to further reduce the production cost. • Gradient temperature control was innovatively integrated into the biological cascade reaction. • The conversion rate of IQ-G3 with the best bioavailability reached an impressive 41.69%. • The industrial production of EMIQ with rutin and sucrose as materials has good potential. [ABSTRACT FROM AUTHOR]

Although myricetin (3,3′,4′,5,5′,7-hexahydroxyflavone, MYR) has a high antioxidant capacity and health functions, its use as a functional food material is limited owing to its low stability and water solubility. Amylosucrase (ASase) is capable of biosynthesizing flavonol α-glycoside using flavonols as acceptor molecules and sucrose as a donor molecule. Here, ASase from Deinococcus deserti (Dd AS) efficiently biosynthesizes a novel MYR α-triglucoside (MYRαG3) using MYR as the acceptor molecule. Comparative homology analysis and computational simulation revealed that Dd AS has a different active pocket for the transglycosylation reaction. Dd AS produced MYRαG3 with a conversion efficiency of 67.4 % using 10 mM MYR and 50 mM sucrose as acceptor and donor molecules, respectively. The structure of MYRαG3 was identified as MYR 4′- O -4″,6″-tri- O -α-D-glucopyranoside using NMR and LC-MS. In silico analysis confirmed that Dd AS has a distinct active pocket compared to other ASases. In addition, molecular docking simulations predicted the synthetic sequence of MYRαG3. Furthermore, MYRαG3 showed a similar DPPH radical scavenging activity of 49 %, comparable to MYR, but with significantly higher water solubility, which increased from 0.03 μg/mL to 511.5 mg/mL. In conclusion, this study demonstrated the efficient biosynthesis of a novel MYRαG3 using Dd AS and highlighted the potential of MYRαG3 as a functional material. • Distinct active site pocket in Deinococcus deserti amylosucrase (Dd AS) compared with another ASase • Efficient biosynthesis of novel myricetin (MYR) 4′-O-4″,6″-tri-O-α-D-glucopyranoside (MYRαG3) by Dd AS • MYRαG3 showed similar antioxidant capacity and much higher water solubility than MYR. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the impacts of enzymatically synthesized α-glucans possessing α-1,4- and α-1,6-glucose linkages, and varying in branching ratio, on colonic microbiota composition and metabolic function. Four different α-glucans varying in branching ratio were synthesized by amylosucrase from Neisseria polysaccharea and glycogen branching enzyme from Rhodothermus obamensis. The branching ratios were found to range from 0 % to 2.8 % using GC/MS. In vitro fecal fermentation analyses (n = 8) revealed that the branching ratio dictates the short-chain fatty acid (SCFA) generation by fecal microbiota. Specifically, slightly branched (0.49 %) α-glucan resulted in generation of significantly (P < 0.05) higher amounts of propionate, compared to more-branched counterparts. In addition, the amount of butyrate generated from this α-glucan was statistically (P > 0.05) indistinguishable than those observed in resistant starches. 16S rRNA sequencing revealed that enzymatically synthesized α-glucans stimulated Lachnospiraceae and Ruminococcus related OTUs. Overall, the results demonstrated metabolic function of colonic microbiota can be manipulated by altering the branching ratio of enzymatically synthesized α-glucans, providing insights into specific structure-function relationships between dietary fibers and the colonic microbiome. Furthermore, the slightly branched α-glucans could be used as functional carbohydrates to stimulate the beneficial microbiota and SCFAs in the colon. [Display omitted] [ABSTRACT FROM AUTHOR]

The amylosucrase (ASase, EC 2.4.1.4) utilizes sucrose as the sole substrate to catalyze multifunctional reactions. It can naturally synthesize α-1,4-linked glucans such as amylose as well as sucrose isomers with more favorable properties than sucrose with a lower intestinal digestibility and non-cariogenic properties. The amino acid sequence of the asase gene from Deinococcus cellulosilyticus (DceAS) exhibits low homology with those of other ASases from other Deinococcus species. In this study, we cloned and expressed DceAS and demonstrated its high activity at pH 6 and pH 8 and maintained stability. It showed higher polymerization activity at pH 6 than at pH 8, but similar isomerization activity and produced more turanose and trehalulose at pH 6 than at pH 8 and produced more isomaltulose at pH 8. Furthermore, the molecular weight of DceAS was 226.6 kDa at pH 6 and 145.5 kDa at pH 8, indicating that it existed as a trimer and dimer, respectively under those conditions. Additionally, circular dichroism spectra showed that the DceAS secondary structure was different at pH 6 and pH 8. These differences in reaction products at different pHs can be harnessed to naturally produce sucrose alternatives that are more beneficial to human health. • Evaluation of pH-dependent properties of amylosucrase from D. cellulosilyticus. • Understanding differences in product formation by DceAS at pH 6 and 8 • High isomerization activity of DceAS in 50 mM sodium citrate buffer at pH 6 • Increased trehalulose/insoluble glucan ratio in 50 mM sodium citrate buffer at pH 6 [ABSTRACT FROM AUTHOR]

Glycogen-like particles (GLPs) were built up from sucrose by applying de novo one-pot enzymatic process of amylosucrase (ASase; 6 U·mL−1) and glycogen branching enzymes (GBEs; 0.001 and 0.005 U·mL−1). Due to different chain-length transferring patterns of GBEs, structurally differentiated GLPs were synthesized. Yields of GLPs synthesized at pH 7.0 and 30 °C were improved by increasing the GBE/ASase ratio. Branching degrees of GLPs obviously was increased along with the ratio of GBEs, of which result was directly supported by shortened branch-chain length with greater GBE activity. Long branch chains seemed to play as efficient acceptor molecules to bind newly transferred branch chains especially at lower ratio of GBE/ASase, resulting in greater molecular weight and size of GLP with higher proportion of them. Molecular weight, size, and density of GLPs were ranged from 7.37 × 105 to 1.94 × 108 g·mol−1, from 23.70 to 52.65 nm, and from 7.99 to 374.32 g·mol−1·nm−3, respectively. By increasing GBE/ASase ratio, more compact GLP architecture was fabricated due to increased weight and reduced size with exception of a unique GBE. GLPs were efficiently synthesized by two different glycosyltransferases, and their chemical structures were controllable by source and ratio of GBEs due to their different branch-chain transferring specificity. • Glycogen-like particle (GLP) was synthesized by one-pot dual enzyme system. • Nano-size GLP structure was highly dependent on type of branching enzymes. • Size and density of GLP were controllable by changing enzyme activity ratio. • GLP yield reached max. 44.5% by branching enzyme activity of Synechocystis sp. [ABSTRACT FROM AUTHOR]

The transglycosylation activity of Deinococcus geothermalis amylosucrase (DgAS) expressed in Corynebacterium glutamicum (cDgAS) was optimized for luteolin glycoside synthesis using purified enzymes {PEs), whole cells (WCs) suspended in buffer (WCB) and WCs in ethanol (WCE). Unlike PE, both WCB and WCE had high transglycosylation yields exceeding 95% over a wide temperature range, with cells in ethanol exhibiting the greatest enzymatic activity for up to 48 h due to membrane degradation and the slow release of cDgAS, which led to lysis and death. Meanwhile, in buffer, protection of the cytoplasmic membrane and ideal pH conditions maintained activity for over 200 h. To improve their performance, WCs were immobilized on agarose and the resulting immobilized cells (!Cs) had high stability in organic solvents, although WCB possessed greater thermal stability at 50 °c. The reaction parameters for PE, WCE, WCB, and IC transglycosylation were optimized using response surface methodology, and !Cs required lower concentrations of substrates while synthesizing higher concentrations of reaction products in addition to producing the novel compound luteolin maltotrioside after several cycles of reuse. This study expanded on the range of possibilities for the industrial application of C. glutamicum in increasing the production and bioavailability of novel and improved products. [ABSTRACT FROM AUTHOR]

Glycogen is a naturally occurring or metabolically synthesized biological macromolecule found in a wide range of living organisms, including animals, microorganisms, and even plants. However, naturally sourced glycogen poses challenges for industrial use. This study focused on a biological macromolecule referred to as glycogen-like particles (GLPs), detailing the production methods and biological properties of these particles. In vitro enzymatic production of GLPs was successfully achieved. GLPs synthesized through a simultaneous enzymatic reaction using sucrose had significant changes in their structure and functionality based on the branching enzyme (BE) to amylosucrase (ASase) ratio. As this ratio increased, the GLPs developed higher molecular weights and greater density, solubility, and branching degree while reducing size and turbidity. Structural changes in these enzymes were not observed beyond a critical BE/ASase ratio. Uniformly dispersed curcumin powder was generated in 50 % (w / v) aqueous GLP solution, and the GLPs were non-toxic to human skin keratinocytes at a concentration of 2.5 mg/mL. GLPs with lower branching inhibited tyrosinase activity and melanin synthesis, while those with more long chains displayed effective UV-blocking. By manipulating the BE/ASase ratio, GLPs were shown to display diverse chemical structures and physical characteristics, suggesting their potential application in the food and cosmetics industries. • GLPs are synthetic particles that are made from sucrose and enzymes. • The properties of GLPs can be tuned by varying the BE/ASase ratio. • GLPs help a uniform dispersion of curcumin. • GLPs with higher long chains give effective UV protection. • GLPs have potential applications in the food and cosmetic industries. [ABSTRACT FROM AUTHOR]

Amylosucrase (ASase) is α-glucan-producing enzyme. Four putative ASase genes (bdas , blas , bpas , and btas) were cloned from Bifidobacterium sp. and expressed in Escherichia coli. All ASases from Bifidobacterium sp. (BAS) displayed typical ASase properties with slightly different characteristics. Among the BASs studied, BdAS and BpAS showed maximal enzyme activities at 35 and 30 °C, respectively, whereas BlAS and BtAS were maximally active at higher temperatures, i.e., 45 and 50 °C, respectively. BpAS exhibited optimum pH under slightly basic conditions (pH 8.0), while BdAS, BlAS, and BtAS preferred weakly acidic conditions (pH 5.0–6.0). All BASs showed higher isomerization activities. Particularly, BlAS produced more trehalulose than turanose. Although polymerization was the highest for BtAS, BtAS synthesized α-1, 4-glucans with a lower degree of polymerization than that of the other BASs. The versatile properties of the BASs described could contribute to the efficient production of highly valuable biomaterials for the agriculture, food, and pharmaceutical industries. • Four amylosucrases from Bifidobacterium (BdAS, BlAS, BpAS, and BtAS) were studied. • BlAS and BpAS have lower substrate affinities for sucrose than BdAS and BtAS. • The lower polymerization of BdAS may link to a six critical residues (HDDIGW). • Trehalulose production of BlAS was superior among four amylosucrases. • The study on BASs contributes to the efficient application of valuable biomaterials. [ABSTRACT FROM AUTHOR]

Modification of chestnut starch with amylosucrase from Deinococcus geothermalis (DGAS) increases the proportion of resistant starch. DGAS-modified chestnut starch (DMCS) attenuates obesity in diet-induced obese mice via a receptor of short chain fatty acids (SCFAs), G-protein-coupled receptor 43. SCFAs are gut microbial metabolites produced by fermenting resistant starch and have key roles in the obesity-ameliorating effects of DMCS. Here, we evaluated the mechanical links among DMCS-induced changes in the gut microbiota, consequent production of microbial metabolites, and host genetic responses. Supplementation with DMCS altered the proportions of cecal microbiota, such as Ruminococcaceae and Bacteroides ; microbial metabolites, such as acetic acid; and some carbohydrate metabolites. DMCS also induced changes in the expression of some genes in cecal epithelial cells, including genes involved in energy production, the cell cycle, and cellular junctions. Changes in the gut microbiota, microbial metabolites, and host gene expression were found to be significantly correlated. Our findings demonstrated the integrated and incorporated association among the gut microbiota, their beneficial metabolites, and the host transcriptome, which contributed to clarifying the anti-obesity effects of DMCS as a prebiotic. Therefore, fortifying resistant starch by modification of chestnut starch using DGAS may be a good strategy in the functional food industry. • Acetic acid as a mediator of the health-benefit was produced by gut microbiota by fermenting resistant starch in DMCS. • Identification of the prebiotic characteristics of DMCS through multi-omics analyses. • Increased expression of genes related to cell cycle and cell components in DMCS-supplemented mice. • The activation of energy production mechanism by gut microbial metabolites acetic acid in DMCS-supplemented mice. [ABSTRACT FROM AUTHOR]

Enzymatically modified starch with controllable digestibility may find unique applications. In the present study, side chains of amylopectin were elongated by amylosucrase, followed by debranching with pullulanase to produce linear chains that self-assembled to form microparticles containing aggregated nanoparticles. Amylosucrase treatment for a longer time resulted in longer branches that were not changed after the subsequent modification by pullulanase. The A-type crystalline structure of native amylopectin was changed to the B-type and the percentage of crystallinity progressively increased after amylosucrase and subsequent pullulanase treatments. Elongation of branch chains favored the formation of thermal stable double helices, which decreased the contents of slowly digestible and rapidly digestible starches and increased the content of resistant starch from 9.1% of native amylopectin to be as much as 67.2% of microparticles. This study may provide an efficient and environmentally benign technology to produce modified starch with controllable physical and digestion properties enabling novel applications. Image 1 • Side chains of amylopectin were progressively elongated by amylosucrase. • Branching points of modified amylopectin were hydrolyzed by pullulanase. • Microparticles produced from linear chains containing aggregated nanoparticles. • Digestibility of amylopectin after sequential enzymatic treatments were evaluated. [ABSTRACT FROM AUTHOR]

Amylosucrase (AS) is a glycosyltransferase that produces linear α-1,4 glucans using sucrose as the sole substrate. In this study, for various applications, α-glucan-coated starch (α-GCS) was produced by AS (20 U/L reactant) from Neisseria polysaccharea to improve the physicochemical properties of raw normal corn starch (NCS) by applying different reaction conditions (i.e., varying the substrate concentration, pH, and temperature). Field-emission scanning electron microscopy (FE-SEM) and X-ray diffraction (XRD) showed that raw NCS was successfully coated by α-glucan. Differential scanning calorimetry (DSC) and rapid viscosity analyses (RVA) of the α-GCS confirmed that the α-glucan coating decreased the degree of retrogradation. Notably, compared to raw NCS as a control, starch retrogradation was significantly (p < 0.05) decreased by 13.7% after five weeks. Therefore, the novel α-GCS can be applied as a functional material for controlled retrogradation in the starch-based food industry for shelf-life extension. • α-Glucan-coated starch (α-GCS) was produced from raw starch by amylosucrase. • α-GCS retained the inherent physicochemical properties of raw starch. • The retrogradation level of α-GCS decreased compared to that of raw starch. • α-GCS can be applied as a functional food ingredient with extended shelf-life. [ABSTRACT FROM AUTHOR]

In this study, we improved the polymerization activity of Deinococcus geothermalis amylosucrase (DGAS) via semi-rational design to support increased potential industrial applications. Structural analysis selected three amino acid residues (Pro219, Phe225, and Ala226) in loop 3 of the B domain that were considered to be related to polymerization activity. Various DGAS variants were constructed through site-saturated mutagenesis, from which two mutants (DGAS-P219Y and DGAS-A226N) with increased polymerization activity were selected by fluorescence screening. DGAS-A226N synthesized longer amylose-like polymers from sucrose than those from wild-type (WT) DGAS. A kinetic study of sucrose as a substrate showed that the ratio of polymerization to hydrolysis activity of DGAS-A226N was significantly higher than that of WT DGAS. DGAS-A226N exhibited its highest enzymatic activity at 50 °C; its half-life was 6.8 min at 60 °C, and its melting temperature was 61.16 ± 0.09 °C. Computational simulation analysis of DGAS-A226N indicated that its conformation was more compact than that of WT DGAS, possibly owing to decreased flexibility of loops 2, 3, 4, 7, and 8. Thus, conformational changes in DGAS-A226N allowed the production of longer oligosaccharides, which may support a broader range of industrial application by affording novel or improved product characteristics. • An improved DGAS polymerization activity variant was made via semi-rational design. • Two mutants (P219Y and A226N) were selected by fluorescence-based screening. • DGAS-A226N synthesized longer amylose-like polymers from sucrose than WT DGAS. • DGAS-A226N loop 2, 3, 4, 7, and 8 flexibilities were decreased compared to WT DGAS. • DGAS-A226N conformational changes increased polymerization activity from WT enzyme. [ABSTRACT FROM AUTHOR]

Abstract Enzymatically-modified resistant starch (RS) was prepared by amylosucrase (ASase) reaction and was applied to develop structurally strengthened rice starch gels. Effects of ASase-modified and commercial RSs were examined for textural and physicochemical properties of rice starch gels. Rice gel hardness increased up to 60% with the presence of ASase-modified RS; the increase was proportional to the substitution level. Gel cohesiveness seemed to decline significantly, while springiness kept intact when RSs were added. A slight decrease in elasticity was observed for the sample with 20% (w/w) of ASase-modified normal corn starch, while 2–5 times higher RS content in this sample was obtained compared to untreated gel. Rice starch gels reinforced with ASase-modified RS decreased up to 40% of water exudation from gel structure, and intact morphological structure was observed after 7-day storage. These results suggested that ASase-modified RS would be applicable for producing rice starch gel foods as a texture enhancer and a functional ingredient. Graphical abstract Image 1 Highlights • Firmness of rice starch gel was improved by enzymatically-made resistant starch (RS). • This RS supplementation greatly increased dietary fiber content in rice starch gels. • Rice gel structure by supplementing RS was greatly stabilized with less syneresis. • Rice gel elasticity was not negatively affected by replacing up to 20% of the RS. [ABSTRACT FROM AUTHOR]

Highlights • A new thermostable amylosucrase was isolated from Bifidobacterium thermophilum (Bt AS). • Sucrose biomass was converted biocatalytically to low-calorie functional turanose at 50 °C. • Molecular size of linear α-glucan produced by Bt AS was not affected by [sucrose]. Abstract A unique thermostable amylosucrase from Bifidobacterium thermophilum was produced as a recombinant protein with the half-life of 577 h at 50 °C. By adding 1.0 M fructose, turanose yield was improved from 22.7% to 43.3% with 1.0 M sucrose, and from 23.7% to 39.4% with 1.5 M sucrose. Sucrose consumption rate was greatest at 55 °C, but the lowest amount of turanose was produced. Thus, turanose yield from sucrose biomass was inversely proportional to reaction temperature and was highly dependent on [fructose]. Meanwhile, insoluble α-glucan yield was clearly reduced as [fructose] increased. With 1.0 M fructose + 1.0 M sucrose, glucan byproduct yield significantly decreased from 29.4% to 1.1%. Molecular weights of linear glucans were almost identical among various [sucrose]s and were homogenous with very low polydispersity. This unique dual reaction patterns of amylosucrase enzyme would be very useful for massive productions of two different biomaterials simply by changing sucrose biomass concentration. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Amylosucrase and CGTases were compared to generate α -glycosyl isoquercitrin. • Amylosucrase has several pros over CGTase to generate α -glycosyl isoquercitrin. • Amylosucrase showed bioconversion yield up to 97%. • Amylosucrase produced quercetin-3- O -tetra-glucoside over 5 folds higher than CGTase. Abstract Isoquercitrin (IQ, quercetin-3- O - β - d -glucopyranoside) has diverse biological functions, such as anti-oxidant and anti-cancer activity, but its use is limited by poor solubility and bioavailability. Enzymatically modified IQ (EMIQ) is a mixture of transglycosylated IQs that have better solubility and bioavailability than do quercetin and IQ. Two different enzymes, cyclodextrin glucanotransferase (CGTase) and amylosucrase (ASase), have the transglycosylation activity to produce EMIQ. Both enzymes produce a variety of EMIQs including IQ, IQ-glucoside (IQ-G1), IQ-diglucoside (IQ-G2), and IQ-triglucoside (IQ-G3). ASase had a higher bioconversion yield from IQ to EMIQ (97.6%) than did CGTase (76.8%). In addition, the yield of IQ-G3, which was the most bioavailable form, was higher with ASase (46%) than with CGTases (8%). Taken together, these results suggest that ASase can be used to synthesize EMIQ in a simple and specific process. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A novel α-amylase from Pseudomonas mendocina was partially characterized. • A dual enzyme strategy was developed to produce MOS from sucrose. • In the one-pot enzymatic reaction, a maximum MOS yield of 22.8% was achieved. • In the two-step enzymatic reaction, the highest MOS production yield was 34.1%. Abstract Maltooligosaccharides (MOS), oligosaccharides composed of 2 to 10 glucopyranose subunits linked by α-1,4 bonds, are promising ingredients possessing low sweetness, low osmolality, high water-holding capacity, and crystallization-prevention ability. For improved the cost effectiveness, a novel process for MOS preparation was developed by utilizing sucrose as a raw material instead of starch. In this work, a novel α-amylase from Pseudomonas mendocina (PM-Amy) was characterized and applied to produce the MOS from α-1,4-glucans obtained by the amylosucrase treatment of sucrose. PM-Amy showed a maximum activity at a pH of 6.0 and 55 °C. In the one-pot enzymatic reaction, a maximum MOS yield of 22.8% (w/w) was achieved, while the highest MOS yield (34.1%, w/w) was achieved from the two-step dual enzyme reaction. [ABSTRACT FROM AUTHOR]

Amylosucrase (EC 2.4.1.4, ASase), an outstanding sucrose-utilizing transglucosylase in the glycoside hydrolase family 13, can produce glucans with only α-1,4 linkages. Generally, on account of a double-displacement mechanism, ASase can catalyze polymerization, isomerization, and hydrolysis reactions with sucrose as the sole substrate, and has transglycosylation capacity to attach glucose molecules from sucrose to extra glycosyl acceptors. Based on extensive enzymology research, this review presents the characteristics of various ASases, including their microbial metabolism, preparation, and enzymatic properties, and exhibits structure-based strategies in the improvement of activity, specificity, and thermostability. As a vital transglucosylation tool of producing sugars, carbohydrate-based bioactive compounds, and materials, the bioengineering applications of ASases are also systematically summarized. [ABSTRACT FROM AUTHOR]

In this study, a novel recombinant amylosucrase from Truepera radiovictrix DSM 17093 was characterized and found to produce α -glucans from sucrose. The enzyme showed maximum total and transglucosylation activities at pH 7.5 and maximum hydrolysis activity at pH 5.5. The optimum temperature for total, transglucosylation, and hydrolysis activities were determined to be 45, 45, and 50 °C, respectively. When the conversion of 100 mM sucrose was catalyzed at 35, 45, and 55 °C for 24 h, TR-ASase produced α -(1,4) glucans with average DPs of 59, 45, and 37, respectively. TR-ASase displayed more than 90% of its original activity after incubation at 55 °C for 5 h, which was much higher than that of all other reported ASases. Melting temperature determination and homology modeling were also adopted to analyze the extreme thermostability of this enzyme, TR-ASase. [ABSTRACT FROM AUTHOR]

Few reports about the biosynthesis of α-arbutin using amylosucrase are available. In this work, an amylosucrase from Cellulomonas carboniz T26, CcAS, was identified for highly efficient synthesis of α-arbutin using 20 mM sucrose and 5 mM hydroquinone as a glucosyl donor and acceptor, respectively. The optimum conditions for transglucosylation are a pH of 7.0 and a temperature of 40 °C. Under these conditions, 40%–44.7% of molar production yield of α-arbutin can be obtained at low concentration of hydroquinone (5 mM) without addition of ascorbic acid after 2 h, which saves time and raw material compared with the reported amylosucrase DGAS. In spite of oxidation of hydroquinone at high concentration of 23.6 mM, 16.1% of molar production yield of α-arbutin is acquired using CcAS without addition of ascorbic acid, while only 1.3% is obtained using DGAS. This work presents that higher molar production yield of α-arbutin at low concentrations of hydroquinone can be obtained using amylosucrase, because hydroquinone at high concentrations is easily oxidized, which inhibits the synthesis of α-arbutin. Simultaneously, CcAS likely further glycosylates the synthesized α-arbutin after a 2-h reaction, which is not reported using DGAS. CcAS exhibits a potential in industrialized application. [ABSTRACT FROM AUTHOR]

Amylosucrase is a valuable enzymatic tool for the preparation of resistant starch (RS). This study investigated the morphology, thermal properties and molecular structure of the RS components resulting from the digestion of amylosucrase modified starches (MSs). Morphology of starch samples suggests that the surface of MSs is partially attacked by the digestion enzymes. Besides, the peak melting temperature of RSs shows a considerable increase compared with that of the corresponding MSs. Branch chain length distributions data reveals that the long branch chains of MSs are prone to being hydrolyzed, and the branch chains with degree polymerization of 22–32 are the largest populations among the RS components. Moreover, the RS content in MSs decreases significantly after thermal treatment. Overall results demonstrates that the existence of crystalline structure with thermostable double helices is responsible for the anti-digestion properties of modified starches. [ABSTRACT FROM AUTHOR]

Amylosucrase (ASase) is a glucosyltransferase, which catalyzes the de novo synthesis of amylose-like polymers from sucrose. In the present study, ASase from Neisseria subflava ( Ns AS) was cloned, sequenced, and expressed in Escherichia coli . The production of Ns AS was achieved by inducting gene expression with 0.2 mM isopropyl-β- d -thiogalactopyranoside. The molecular mass of the Ni-NTA column purified Ns AS analyzed by SDS-PAGE was determined to be 72 kDa. Ns AS exhibited maximal activity at 45 °C and pH 8.0, and showed strong thermal stability at 40 °C with a half-life of 385 h. The reaction pattern of Ns AS at [sucrose] range of 0.1–1.0 M showed that at 0.7 M of [sucrose], the production yield of insoluble linear α-(1,4)-glucans reached 24% maximum, and any further increase in [sucrose] resulted in a slight decrease in yield. Meanwhile, the production yield of turanose significantly increased from 16 to 29% by increasing [sucrose] from 0.1 to 1.0 M. The synthesized glucan had degrees of polymerization (DP); for 0.1, 0.4, 0.7, and 1.0 M sucrose, the DP values were 77, 49, 39, and 31 respectively. These results suggested that Ns AS would be a promising candidate for food industrial production of linear α-(1,4)-glucans and turanose as a next generation sweetener. [ABSTRACT FROM AUTHOR]

The ameliorative effect of enzymatically modified chestnut starch on high-fat diet (HFD)-induced obesity in a mouse model and the underlying mechanism were investigated. Chestnut starch was enzymatically modified by amylosucrase from Deinococcus geothermalis (DGAS). In vitro analyses including Englyst's assay and determination of the starch digestive pattern to investigate starch digestibility revealed that chestnut starch naturally contained higher slowly digestible starch and resistant starch than corn starch. Modification of chestnut native starch by DGAS increased the proportion of resistant starch, rendering it unavailable for catalysis by small-intestinal mucosal α-glucosidase. The amylose ratio and branch-chain of amylopectin in chestnut starch were increased by DGAS. In an in vivo study, HFD (45% kcal from fat)-induced obese C57BL/6 model mice were orally administered DGAS-modified chestnut starch at the dose of 1500 mg/kg b·w for 10 weeks. Supplementation of DGAS-modified chestnut starch to obese mice significantly reduced features of obesity as compared with HFD- or native chestnut starch-fed mice. Food intake and the gut hormones PYY and GLP-1 were not significantly changed. However, we found that feeding DGAS-modified chestnut starch led to a significant increase in empty cecum weight, indicating short-chain fatty acid production in the cecum. Additionally, DGAS-modified chestnut starch induced the short-chain fatty acid receptor GPR43-mediated suppression of insulin signaling. Changes in all these factors were resistant starch content-dependent. In conclusion, DGAS modification of chestnut starch increases non-digestible resistant starch and this ameliorates diet-induced obesity via GPR43-mediated suppression of insulin signaling, thereby presumably reducing fat accumulation in white adipose tissue. [ABSTRACT FROM AUTHOR]

Here, we report a simple and non-emulsion based approach to prepare starch-based magnetic polymer beads with well-defined size, shape and dispersibility in aqueous environment through co-precipitation of enzymatically synthesized short chain amylose (SCA) and dextran-coated iron oxide nanoparticles (Dex@IONPs). The size and morphology of magnetic polymer beads (MPBs) were controlled by employing Dex@IONPs as a seeding agent. The resulting superparamagnetic amylose microbeads (SAMBs) were readily functionalized with antibody through one step reaction using a linker protein, which showed great capture efficiency (>90%) and specificity for target bacteria present in complicated food matrix. The excellent magnetic sensitivity also enabled the SAMBs readily assembled into ordered 1D arrays by external magnetic field whose structure could permanently be fixed by SCA-mediated precipitation process. [ABSTRACT FROM AUTHOR]

Amylosucrase from Neisseria polysaccharea (Np AS) produces the linear amylose-like α-glucans by the elongation property from sucrose, and 4,3-α-glucanotransferase from Lactobacillus fermentum NCC 2970 (4,3-αGT) newly synthesizes the α-1,3 linkages after cleaving the α-1,4 linkages by the glycosyltransferring property. This study focused on the synthesis of high molecular α-1,3/α-1,4-linked glucans by combining Np AS and 4,3-αGT and analyzed their structural and digestive properties. The enzymatically synthesized α-glucans have a molecular weight of >1.6 × 107 g mol−1, and the α-4,3 branching ratios on the structures increased as the amount of 4,3-αGT increased. The synthesized α-glucans were hydrolyzed to linear maltooligosaccharides and α-4,3 branched α-limit dextrins (α-LDx) by human pancreatic α-amylase, and the amounts of produced α-LDx were increased depending on the ratio of synthesized α-1,3 linkages. In addition, approximately 80 % of the synthesized products were partially hydrolyzed by mammalian α-glucosidases, and the glucose generation rates decelerated as the amounts of α-1,3 linkages increased. In conclusion, new types of α-glucans with α-1,4 and α-1,3 linkages were successfully synthesized by a dual enzyme reaction. These can be utilized as slowly digestible and prebiotic ingredients in the gastrointestinal tract due to their novel linkage patterns and high molecular weights. [ABSTRACT FROM AUTHOR]

Glycogen-like particles (GLPs) are applied in food, pharmaceutical, and cosmetics. The large-scale production of GLPs is limited by their complicated multi-step enzymic processes. In this study, GLPs were produced in a one-pot dual-enzyme system using Bifidobacterium thermophilum branching enzyme (Bt BE) and Neisseria polysaccharea amylosucrase (Np AS). Bt BE showed excellent thermal stability (half-life of 1732.9 h at 50 °C). Substrate concentration was the most influential factor during GLPs production in this system: GLPs yield and [sucrose] ini decreased from 42.4 % to 17.4 % and 0.3 to 1.0 M, respectively. Molecular weight and apparent density of GLPs decreased significantly with increasing [sucrose] ini. Regardless of the [sucrose] ini , the DP 6 of branch chain length was predominantly occupied. GLP digestibility increased with increasing [sucrose] ini , indicating that the degree of GLP hydrolysis may be negatively related to its apparent density. This one-pot biosynthesis of GLPs using a dual-enzyme system could be useful for the development of industrial processes. [Display omitted] [ABSTRACT FROM AUTHOR]

Amylosucrase from Neisseria polysaccharea naturally catalyzes the synthesis of α-1,4 glucans from sucrose. The product profile is quite polydisperse, ranging from soluble chains called maltooligosaccharides to high-molecular weight insoluble amylose. This enzyme was recently subjected to engineering of its active site to enable recognition of non-natural acceptor substrates. Libraries of variants were constructed and screened on sucrose, allowing the identification of a mutant that showed a 6-fold enhanced activity toward sucrose compared to the wild-type enzyme. Furthermore, its product profile was unprecedented, as only soluble maltooligosaccharides of controlled size chains (2 < DP < 21) with a narrow polydispersity were observed. This variant, containing 9 mutations in the active site, was characterized at both biochemical and structural levels. Its x-ray structure was determined and further investigated by molecular dynamics to understand the molecular origins of its higher activity on sucrose and higher production of small maltooligosaccharides, with a totally abolished insoluble glucan synthesis. [ABSTRACT FROM AUTHOR]

Classification of starch into rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) has been in controversy. Therefore, this study aimed to ascertain the individual existence of RDS and SDS using amylosucrase-modified starch. Enzymatic hydrolysis curves of modified waxy corn starches were obtained and fitted to a logarithm of slope (LOS) plot. LOS plots for amylosucrase-modified starches revealed a discontinuity, reflecting the change of rate constant k during digestion, such that distinguishable k s for each of the phases were defined ( k RDS , k SDS ). The LOS plot approach could be utilized as a persuasive basis for the classification of the starch fractions. The structure of amylosucrase-modified starches was characterized before and after removal of RDS and/or SDS. Branch chains with certain lengths contributed to the organization of each fraction and determined the primary crystallite configuration of amylosucrase-modified starches causing the diversity of digestion properties. [ABSTRACT FROM AUTHOR]

Recombinant Amylosucrase (AS) from Neisseria polysaccharea was used to modify waxy corn starch, and the reaction process was monitored by the turbidity and transglycosylation degree (TD). Results showed that the modification by AS significantly elongated the chain length of the starches, resulting in the formation of precipitate. Based on regression equation, AS-modified starches with defined TD values were prepared, and chain length distributions of AS-modified starches indicated that the non-reducing ends of the branch chains were mostly elongated before the occurrence of precipitation. Differ from the native starch, all of the AS-modified starches showed a B-type crystal structure, and thermal analysis suggested that the AS-modified starches containing longer chains possessed a higher thermostability. High resistant starch content (33.8%–73.6%) of AS-modified starches was attributed to the formation of long-ranged double helices. Moreover, the very long chains, produced after the precipitation, might not generate the double helical structure during the retrogradation. [ABSTRACT FROM AUTHOR]

Bifidobacteria uses insoluble resistant starch (RS) fractions as prebiotics in the colon, consequently improving human intestinal health. In this study, various α-1,6 linkage ratios of insoluble α-glucans were synthesized by amylosucrase from Neisseria polysaccharea and glycogen branching enzyme from Rhodothermus obamensis to investigate the effect of branching patterns on Bifidogenic property. Different ratios (0%–4%) of α-1,6 branched insoluble α-glucans with α-1,4 linked backbones were effectively synthesized. Additionally, the proportions of RS fraction in the synthesized α-glucans were 69.5%–81.9% after the cooking process, which are higher amounts than amylomaize VII (36.8%). As various Bifidobacteria used the insoluble branched α-glucans , the structure with 2.5% of 1,6 linkage showed the highest consumption yield, more than 70%, among all tested strains. Therefore, the insoluble α-glucans with different α-1,6 linkage ratios can be applied as prebiotic ingredients to modulate the Bifidobacterium species to enhance colon health and related outcomes. [Display omitted] • Amylosucrase and glycogen branching enzyme was applied to synthesize α-glucans. •Insoluble α-glucans with different α-1,6 linkage ratios were successfully produced. •Insoluble branched α-glucans contain high amounts of resistant starch fraction. •Bifidobacterium species broadly used the insoluble α-glucans as prebiotics. [ABSTRACT FROM AUTHOR]

Amylose microparticles can be produced by self-assembly of amylose molecules through an amylosucrase-mediated synthesis. Here we investigated the role of short-chain fatty acids in the formation of amylose microparticles and the fate of these fatty acids at the end of the reaction. The rate of self-assembly and production yields of amylose microparticles were significantly enhanced in the presence of fatty acids. The effect was dependent on the length of the fatty acid carbon tail; butanoic acid (C4) was the most effective, followed by hexanoic acid (C6) and octanoic acid (C8). The amylose microparticles were investigated by carrying out SEM, XRD, Raman, NMR, FT-IR and DSC analysis. The size, morphology and crystal structure of the resulting amylose microparticles were comparable with those of amylose microparticles produced without fatty acids. The results indicated the carboxyl group of the fatty acid to be responsible for promoting the self-assembly of amylose chains to form microparticles. The fatty acids were eventually removed from the microstructure through the tight association of amylose double helices to form the amylose microparticles. [ABSTRACT FROM AUTHOR]

Flavonoids possess a wide range of biological activities. Their glycosylation is of considerable interest, as it often positively influences their pharmacokinetic and other molecular properties. We recently showed that two non-Leloir glycosyltransferases that use sucrose as carbohydrate donor, an amylosucrase from Neisseria polysaccharea (Ams-Np) and a glucansucrase from Streptococcus oralis (GtfR-So), were hardly able to glucosylate flavones, but accepted flavanes as substrates. We now examined compounds from two other flavonoid classes, flavanones and isoflavones for glucose transfer by these enzymes. Taxifolin was investigated as a flavanone analogue of both, the accepted pentahydroxyflavane catechin and the non-accepted pentahydroxyflavone quercetin. It was glucosylated by both enzymes, but much better by GtfR-So than by Ams-Np due to apparent strong inhibition of the latter. The acceptance of a collection of isoflavones strongly depended on the substitution pattern of the core. Only two of the 10 compounds examined yielded glucosides in satisfactory amounts. With these substrates, both enzymes catalyzed formation of a range of products, differing in the number of saccharide units. The structures of mono- and diglycosylated compounds obtained in higher amounts were elucidated. While GtfR-So attached glucose to taxifolin in the B ring at O4′, both enzymes glucosylated the isoflavones in the A ring at O7. All products were α-glucosides. Interglycosidic linkages formed by Ams-Np were α1-4. To our knowledge, this is the first report of glucosylation of flavanone and isoflavone aglycones by an amylosucrase. All characterized compounds have not previously been described. [ABSTRACT FROM AUTHOR]

To investigate the action mode of amylosucrase (AS) on amylopectin, waxy corn starch (WCS) was selected as an acceptor. The effects of WCS dissolution method, reaction temperature, sucrose concentration and AS activity on transglycosylation degree (TD) were investigated. Under the selected reaction condition, the enzymatic reaction process was divided into two stages, i.e. before and after 0.25 h, of which the relations between TD value and reaction time were successfully described using a linear and a logarithmic function, respectively. Then, the elongated WCSs with different TDs were produced according to the theoretical reaction time calculated based on the regression equations. The chain length distribution of the elongated WCSs indicated that all of the branch chains of WCS were greatly elongated by AS before occurrence of starch precipitation. Afterwards, however, AS merely elongated the branch chains whose non-reducing ends were exposed on the surface of the precipitate. [ABSTRACT FROM AUTHOR]

Sweet potato Daeyumi starch was dually modified using glycogen branching enzyme (BE) from Streptococcus mutans and amylosucrase (AS) from Neisseria polysaccharea to prepare slowly digestible starch (SDS). Dually modified starches had higher SDS and resistant starch (RS) contents than control starch. The branched chain length distributions of the BE-modified starches indicated an increase in short side-chains [degree of polymerization (DP) ≤ 12] compared with native starch. AS treatment of the BE-modified starches decreased the proportion of short side-chains and increased the proportion of long side-chains (DP ≥ 25) and molecular mass. It also resulted in a B-type X-ray diffraction pattern and an increased relative crystallinity. Regarding thermal properties, the BE-modified starches showed no endothermic peak, whereas the BEAS-modified starches had a broader melting temperature range and lower melting enthalpy compared to native starch. The combined enzymatic treatment resulted in novel glucan polymers with slow digestion properties. [ABSTRACT FROM AUTHOR]

Amylosucrase (ASase; EC 2.4.1.4) synthesizes α-1,4-glucans using sucrose as a sole substrate. The aim of this study was to compare the enzymatic properties of four recombinant ASase genes to determine the underlying mechanisms thereof. Following cloning and expression in Escherichia coli , we determined that the ASase enzyme from Deinococcus geothermalis (DGAS) had the highest thermostability whereas ASase from Neisseria polysaccharea (NPAS) showed the greatest polymerization activity. Chimeric ASases were constructed using dgas and npas genes by overlap extension polymerase chain reaction. Two of the six chimeric ASases generated, NPAS-B′ and DGAS-B, showed ASase activity using sucrose as the sole substrate. However, DGAS-B was not able to produce longer α-1,4-glucans; the highest degree of polymerization was <12. In the kinetic study, not only the substrate binding affinity but also the production rate of DGAS-B was greater than those of DGAS. Molecular dynamic computational simulation suggested that DGAS-B could not synthesize longer glucan chains because of the change in flexibilities of loops 4, 7, and 8 as compared to those of DGAS. [ABSTRACT FROM AUTHOR]

The ileal brake mechanism which induces a potentially beneficial slower gastric emptying rate and increased satiety is triggered by macronutrients including glucose from glycemic carbohydrates. For optimization of this diet-induced health benefit, there is the need for a way to determine the location of glucose deposition in the small intestine. Labeled 2-deoxyglucose (2-DG) can be used to trace the location of glucose absorption due to its accumulative property in the small intestine enterocytes. However, because pure glucose, or 2-DG, is directly absorbed in the proximal small intestine, we designed 2-DG containing maltooligosaccharides (2-DG-MOs) that can be used with a mild α-glucosidase inhibitor to attain an analytical method for determining location-specific delivery of glucose and its physiological effect. [ABSTRACT FROM AUTHOR]

Glycosylation is one of the most important tailoring reactions for natural products. It typically exerts profound direct or indirect effects on their biological activity. The dihydrochalcone phloretin and its known sugar derivatives, particularly phlori(d)zin, have been shown to influence various cellular processes. We found that a non-Leloir glycosyltransferase, amylosucrase from Neisseria polysaccharea, is an excellent catalyst for the stereospecific glucosylation of phloretin at the 4’ position. Three novel phloretin derivatives were obtained, the first ones in which the sugar-aglycone bond possesses the configuration. A first biological characterization in a cell viability assay showed that each sugar attachment reduced the compound toxicity approximately two-fold. [ABSTRACT FROM AUTHOR]

Physicochemical properties of Neisseria polysaccharea amylosucrase ( Np AS)-treated rice and barley starches were investigated at scale-up production. Pre-gelatinized rice and barley starches were treated with significantly lower Np AS dose (0.1 U/mL) but 100 times larger reaction volume (3500 mL), compared to the analytical scale (35 mL) used in the previous study. Np AS-treated starches in this scale-up production were characterized with respect to reaction efficiency (RE), resistant starch (RS) content, amylopectin (AP) branch-chain length distribution, solubility, swelling power, pasting viscosity, and thermal transition properties. The RE enhanced up to 1.8 times by increasing the reaction volume, which improved the RS content and AP branch-chain lengths of Np AS-treated starches. Compared with the native starch, Np AS-treated starches exhibited lower solubility and swelling power, lower pasting viscosity, and a large increase in the melting peak temperature. Consequently, Np AS treatment of pre-gelatinized starches in this study would be a potential way of replacing commercial RS production. [ABSTRACT FROM AUTHOR]

Baicalein-6- α -glucoside (BG), a glycosylated derivative of baicalein, was synthesized by using sucrose and the amylosucrase of Deinococcus geothermalis and tested for its solubility, chemical stability, and anti-inflammatory activity. BG was 26.3 times more soluble than baicalein and highly stable in buffered solutions and Dulbecco׳s modified Eagle medium containing 10% fetal bovine serum. BG treatment decreased the production of nitric oxide in RAW 264.7 cells treated with lipopolysaccharide (LPS). Luciferase reporter assays, western blots, reverse transcription-polymerase chain reaction, and flow cytometric analyses indicated that BG activated nuclear factor erythroid 2-related factor 2 (Nrf2), an antioxidant transcription factor that confers protection from various inflammatory diseases, induced Nrf2-dependent gene expression, and suppressed the production of reactive oxygen species elicited by LPS more effectively than baicalein. Cellular uptake of BG assessed by confocal microscopy and HPLC analysis of the cell-free extracts of RAW 264.7 cells demonstrated that BG was gradually converted to baicalein inside the cells. These results explain that glycosylation increased the bioavailability of baicalein by helping to protect this vital molecule from chemical or enzymatic oxidation. Therefore, BG, a glycosylated derivative of baicalein, can be an alternative to baicalein as a therapeutic drug. [ABSTRACT FROM AUTHOR]

Highlights: [•] The first functional characterization of AMS and FRK encoding genes from a cyanobacterium was achieved. [•] AMS could be responsible for catalyzing Suc hydrolysis in Synechococcus sp. PCC 7002. [•] Genes involved in Suc synthesis and, AMS and FRK encoding genes are grouped in a transcriptional unit, named Suc cluster. [•] Transcription of the Suc-cluster genes is increased after a salt-treatment. [Copyright &y& Elsevier]

Highlights: [•] Identification of novel starches having a high content of slowly digestible starch fraction. [•] Elongation of branched chains of amylopectin by amylosucrase treatment. [•] Positive relationship between SDS content and degree of elongation of branched chains. [•] Possibility of producing tailored starchy foods with a high SDS content. [Copyright &y& Elsevier]

Highlights: [•] Sucrose phosphorylase was compared with related enzymes (sequence and structure). [•] Several acceptor site residues were substituted by alanine to investigate specificity. [•] Sucrose phosphorylase is highly optimised for recognising its natural acceptors. [•] Hydrolytic side activity is not controlled by residues in the acceptor site. [•] Changing specificity will probably require drastic remodelling of the acceptor site. [ABSTRACT FROM AUTHOR]

Increasing α-1,6 linkages in starch molecules generates a large amount of α-limit dextrins (α-LDx) during α-amylolysis, which decelerate the release of glucose at the intestinal α-glucosidase level. This study synthesized highly branched α-glucans from sucrose using Neisseria polysaccharea amylosucrase and Rhodothermus obamensis glycogen branching enzyme to enhance those of slowly digestible property. The synthesized α-glucans (Mw: 1.7–4.9 × 107 g mol−1) were mainly composed of α-1,4 linkages and large proportions of α-1,6 linkages (7.5%–9.9%). After treating the enzymatically synthesized α-glucans with the human α-amylase, the quantity of branched α-LDx (36.2%–46.7%) observed was higher than that for amylopectin (26.8%) and oyster glycogen (29.1%). When the synthetic α-glucans were hydrolyzed by mammalin α-glucosidases, the glucose generation rate decreased because the amount of embedded branched α-LDx increased. Therefore, the macro-sized branched α-glucans with high α-LDx has the potential to be used as slowly digestible material to attenuate postprandial glycemic response. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract: Amylosucrase (AS), maltooligosyltrehalose synthase (MTS), and maltooligosyltrehalose trehalohydrolase (MTH) were used in combined cross-linked enzyme aggregates (combi-CLEAs) to achieve one-step bioconversion of sucrose to trehalose. Combi-CLEAs of three enzymes were successfully established with acetone and glutaraldehyde (GA) as a precipitant and a cross-linker, respectively. The optimum enzyme ratio was 8:0.5:0.5 (AS, 4mg:MTS, 0.25mg:MTH, 0.25mg). To improve trehalose production, bovine serum albumin was co-aggregated with enzymes as a proteic feeder. The trehalose production yield of combi-CLEAs was about 8% in each cycle on the basis of substrate added up to 400mM. Finally, the combi-CLEAs used in this experiment showed reusability of about five cycles without any activity loss. [Copyright &y& Elsevier]

Abstract: Turanose, a structural isomer of sucrose, is a non-cariogenic and low-calorigenic disaccharide. It has a promising potential to be a high-quality functional sweetener, but there has been no competitive way to produce turanose until now. In this study, a recombinant amylosucrase, originating from Neisseria polysaccharea (NpAS), was utilised to efficiently convert sucrose to turanose. With 2.5M sucrose and 400U/l of NpAS, the production yield of turanose maximally reached to 56.2% at 35°C after a 120h reaction. Main transglycosylating reaction pathway of NpAS clearly shifted from α-(1,4)-glucan synthesis to turanose production by simply increasing sucrose substrate concentration. Purification, by a preparative recycling HPLC, effectively separated turanose from the reaction mixture with a high purity of 94.7% and a high recovery yield of 97.5%. Chemical structure of the purified turanose was confirmed by NMR analysis, and three tautomers, namely 3-O-α-d-glucopyranosyl-α-d-fructofuranose, 3-O-α-d-glucopyranosyl-β-d-fructofuranose, and 3-O-α-d-glucopyranosyl-β-d-fructopyranose, were identified with a ratio of approximately 1:2:2. [Copyright &y& Elsevier]