Action pattern of Sulfolobus O-α-glycoligase for synthesis of highly water soluble resveratrol 3,4'-α-diglucoside.

This study presents the enzymatic synthesis of resveratrol-3,4'-O-α-diglucoside (RDG) using a hyperactive O-α-glycoligase (MalA-D416R/Q450S) and α-glucopyranosyl fluoride as the donor substrate. The transglycosylation rate for resveratrol by MalA-D416R/Q450S was maximized in 100 mM Tris-HCl (pH 9.5) containing 20 % DMSO at 45°C. Because the pKa of the 4'-OH group of resveratrol is lower than that of the 3-OH group, the 4'-OH group is more nucleophilic at the alkaline pH, leading to a preference for glycosylation at the 4'-OH site rather than the 3-OH site. This preference makes resveratrol 3-O-α-glucoside (R3G) as the more efficient acceptor than resveratrol 4'-O-α-glucoside (R4'G), resulting in negligible production of resveratrol 3-O-α-glucoside (R3G) due to its complete consumption in the second transglycosylation reaction when using a 2:1 ratio of donor to acceptor substrates. From a preparative scale reaction, R4'G and RDG were isolated with yields of 41.2 % and 43.3 %, respectively. The water solubility of RDG exceeded 1.67 M, which represents more than a 9,800-fold improvement compared to resveratrol. In a hydrolysis experiment using intestinal α-glycosidase from rat, the α-glucosides of resveratrol (R4'G and RDG) were completely deglycosylated to the aglycone.

Impact of artificial sweeteners and rare sugars on the gut microbiome.

Alternative sugars are often used as sugar substitutes because of their low calories and glycemic index. Recently, consumption of these sweeteners in diet foods and beverages has increased dramatically, raising concerns about their health effects. This review examines the types and characteristics of artificial sweeteners and rare sugars and analyzes their impact on the gut microbiome. In the section on artificial sweeteners, we have described the chemical structures of different sweeteners, their digestion and absorption processes, and their effects on the gut microbiota. We have also discussed the biochemical properties and production methods of rare sugars and their positive and negative effects on gut microbial communities. Finally, we have described how artificial sweeteners and rare sugars alter the gut microbiome and how these changes affect the gut environment. Our observations aim to improve our understanding regarding the potential health implications of the consumption of artificial sweeteners and low-calorie sugars.

Efficient and novel biosynthesis of myricetin α-triglucoside with improved solubility using amylosucrase from Deinococcus deserti.

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 (DdAS) efficiently biosynthesizes a novel MYR α-triglucoside (MYRαG3) using MYR as the acceptor molecule. Comparative homology analysis and computational simulation revealed that DdAS has a different active pocket for the transglycosylation reaction. DdAS 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 DdAS 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 DdAS and highlighted the potential of MYRαG3 as a functional material.

Characterization of a unique pH-dependent amylosucrase from Deinococcus cellulosilyticus.

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.

Efficient biotransformation of naringenin to naringenin α-glucoside, a novel α-glucosidase inhibitor, by amylosucrase from Deinococcus wulumuquiensis.

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 kcat/Km 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.

The branching ratio of enzymatically synthesized α-glucans impacts microbiome and metabolic outcomes of in vitro fecal fermentation.

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.

Characterization and applications of biomacromolecule structurally similar to glycogen as a dispersion aid and skin protection agent.

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.

Enzymatic Synthesis of α-Glucan Microparticles Using Amylosucrases from Bifidobacterium Species and Its Physicochemical Properties.

α-Glucan microparticles (GMPs) have significant potential as high-value biomaterials in various industries. This study proposes a bottom-up approach for producing GMPs using four amylosucrases from Bifidobacterium sp. (BASs). The physicochemical characteristics of these GMPs were analyzed, and the results showed that the properties of the GMPs varied depending on the type of enzymes used in their synthesis. As common properties, all GMPs exhibited typical B-type crystal patterns and poor colloidal dispersion stability. Interestingly, differences in the physicochemical properties of GMPs were generated depending on the synthesis rate of linear α-glucan by the enzymes and the degree of polymerization (DP) distribution. Consequently, we found differences in the properties of GMPs depending on the DP distribution of linear glucans prepared with four BASs. Furthermore, we suggest that precise control of the type and characteristics of the enzymes provides the possibility of producing GMPs with tailored physicochemical properties for various industrial applications.

Correction to: Versatile biotechnological applications of amylosucrase, a novel glucosyltransferase.

[This corrects the article DOI: 10.1007/s10068-019-00686-6.].

Development of Korea Neuroethics Guidelines.

BACKGROUND: Advances in neuroscience and neurotechnology provide great benefits to humans though unknown challenges may arise. We should address these challenges using new standards as well as existing ones. Novel standards should include ethical, legal, and social aspects which would be appropriate for advancing neuroscience and technology. Therefore, the Korea Neuroethics Guidelines were developed by stakeholders related to neuroscience and neurotechnology, including experts, policy makers, and the public in the Republic of Korea. METHOD: The guidelines were drafted by neuroethics experts, were disclosed at a public hearing, and were subsequently revised by opinions of various stakeholders. RESULTS: The guidelines are composed of twelve issues; humanity or human dignity, individual personality and identity, social justice, safety, sociocultural prejudice and public communication, misuse of technology, responsibility for the use of neuroscience and technology, specificity according to the purpose of using neurotechnology, autonomy, privacy and personal information, research, and enhancement. CONCLUSION: Although the guidelines may require a more detailed discussion after future advances in neuroscience and technology or changes in socio-cultural milieu, the development of the Korea Neuroethics Guidelines is a milestone for the scientific community and society in general for the ongoing development in neuroscience and neurotechnology.

Macromolecular α-glucans with α-1,3/α-1,4 branching structures produced using dual glycosyltransferases: Elucidation of physicochemical and slowly digestible properties.

Amylosucrase from Neisseria polysaccharea (NpAS) 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 NpAS 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.

Gas Chromatography with Flame-Ionization Detection-Based Analysis of Sugar Contents in Korean Agricultural Products for Patients with Galactosemia.

Patients with galactosemia accumulate galactose in their bodies, requiring a lifelong galactose-restricted diet. Therefore, accurate information on the galactose content in commercial agro-food resources is essential. The HPLC method generally used for sugar analysis has low separation and detection sensitivity. Here, we sought to establish an accurate analytical method for determining the galactose content in commercial agro-food resources. To that aim, we employed gas chromatography with flame-ionization detection to detect trimethylsilyl-oxime (TMSO) sugar derivatives (concentration: ≤0.1 mg/100 g). The galactose content in 107 Korean agro-food resources reflecting intake patterns was then analyzed. The galactose content in steamed barley rice was 5.6 mg/100 g, which was higher than that in steamed non-glutinous and glutinous rice. Moist-type and dry-type sweet potatoes, blanched zucchini, and steamed Kabocha squash had high galactose content (36.0, 12.8, 23.1, and 61.6 mg/100 g, respectively). Therefore, these foods are detrimental to patients with galactosemia. Among fruits, avocado, blueberry, kiwi, golden kiwifruit, and sweet persimmon had galactose contents of ≥10 mg/100 g. Dried persimmon had 132.1 mg/100 g and should therefore be avoided. Mushrooms, meat, and aquatic products showed low galactose content (≤10 mg/100 g), making them safe. These findings will help patients to manage their dietary galactose intake.

Production of highly branched α-limit dextrins with enhanced slow digestibility by various glycogen-branching enzymes.

α-Limit dextrins (α-LDx) are slowly digestible carbohydrates that attenuate postprandial glycemic response and trigger the secretion of satiety-related hormones. In this study, more highly branched α-LDx were enzymatically synthesized to enhance the slowly digestible property by various origins of glycogen branching enzyme (GBE), which catalyzes the transglycosylation to form α-1,6 branching points after cleaving α-1,4 linkages. Results showed that the proportion of branched α-LDx in starch molecules increased around 2.2-8.1 % compared to α-LDx from starch without GBE treatment as the ratio of α-1,6 linkages increased after different types of GBE treatments. Furthermore, the enzymatic increment of branching points enhanced the slowly digestible properties of α-LDx at the mammalian α-glucosidase level by 17.3-28.5 %, although the rates of glucose generation were different depending on the source of GBE treatment. Thus, the highly branched α-LDx with a higher amount of α-1,6 linkages and a higher molecular weight can be applied as a functional ingredient to deliver glucose throughout the entire small intestine without a glycemic spike which has the potential to control metabolic diseases such as obesity and type 2 diabetes.

Production of branched glucan polymer by a novel thermostable branching enzyme of Bifidobacterium thermophilum via one-pot biosynthesis containing a dual enzyme system.

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 (BtBE) and Neisseria polysaccharea amylosucrase (NpAS). BtBE 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.

Different physicochemical properties of entirely α-glucan-coated starch from various botanical sources.

Amylosucrase from Neisseria polysaccharea (NpAS) synthesizes α-1,4 glucan polymer from sucrose. In this study, we coated various botanical sources of raw starch with an α-glucan layer generated by NpAS to improve physicochemical properties. Field-emission scanning electron microscopy demonstrated that all surfaces of the starch granules were successfully coated via the NpAS reaction. X-ray diffraction analysis revealed that the crystallinity decreased and the crystal pattern changed to C-type as an amylose layer formed around the surface of the starch granules. Based on rapid viscosity and differential scanning calorimetry analyses, the gelatinization resistance of the α-glucan-coated starch increased owing to decreased viscosity and increased melting temperature. Therefore, the α-glucans coated the starches by enzymatic reactions of various botanical sources; these have applicability in the food and starch industries owing to various physicochemical properties such as enhanced thermostability. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01113-z.

Heterologous expression of a papain-like protease inhibitor (SnuCalCpI17) in the E. coli and its mode of inhibition.

The effect of the Escherichia coli (E. coli) Rosetta (DE3) system on the expression of recombinant papain-like cysteine protease inhibitors (SnuCalCpIs) was evaluated, and the inhibition mode of the expressed inhibitor was determined. SnuCalCpI08 and SnuCalCpI17, which previously had not been expressed in the E. coli BL21 (DE3) system due to rare codons of more than 10%, were successfully expressed in E. coli Rosetta (DE3) since the strain provides tRNAs for six rare codons. Initially, both inhibitors were expressed as inclusion bodies; however, the water solubility of SnuCalCpI17 could be improved by lowering the incubation temperature, reducing the IPTG concentration, and increasing the induction time. In contrast, the other inhibitor could not be solubilized in water. To validate whether the inhibitor was expressed with correct protein folding, a papain inhibition assay was performed with SnuCalCpI17. SnuCalCpI17 showed a half-maximal inhibitory concentration (IC50) of 105.671 ± 9.857 µg/mL and a slow-binding inhibition mode against papain at pH 7.0 with a Kiapp of 75.80 µg/mL. The slow-binding inhibitor has a slow dissociation from the inhibitor-target complex, resulting in a long residence time in vivo, and thus can effectively inhibit the target at doses far below the IC50 of the inhibitor. KEY POINTS: • Propeptide inhibitor (SnuCalCpI17) containing rare codons was expressed in E. coli Rosetta (DE3). • The slow-binding inhibition was shown by plotting the apparent first-order rate constant (kobs). • Protein-protein interaction between SnuCalCpIs and papain was verified by docking simulation.

A unique biochemical reaction pathway towards trehalulose synthesis by an amylosucrase isolated from Deinococcus deserti.

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 (DdAS) gene was expressed and purified. When DdAS 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. DdAS could be utilized industrially in a bioprocess for producing trehalulose as a functional sucrose alternative.

Effect of oximation reagents on gas chromatographic separation of eight different kinds of mono- and di-saccharides.

The objective of this study was to investigate the effect of oximation reagents in simultaneous analysis of mono and di-saccharides using gas chromatography. Sugar oximation with O-ethylhydroxylamine separated all the mono- and di-saccharides while hydroxylamine and O-benzylhydroxylamine could make most of the saccharides separable except for xylose and arabinose. Resolution of xylose: arabinose, galactose: glucose, and fructose: galactose oximated by O-ethylhydroxylamine in DB-1ms column were 1.66, 2.15, and 6.19, respectively, which were above 1.5 and were officially acceptable for quantitative analysis according to the AOAC guideline. The applied method was then verified by the method validation parameters; LOD (0.011-0.02 mg/100 g), LOQ (0.032-0.061 mg/100 g), linearity (R2 = 0.9991-1.0000) and precision (repeatability RSD: 1.4-3.3%, reproducibility RSD: 1.7-7.6%). The greatest amounts of xylose (19.03 ± 0.38 mg/100 g), maltose (6,274.48 ± 173.59 mg/100 g) were found in the oyster sauce, and the contents of glucose (10,565.00 ± 125.31 mg/100 g), galactose (170.40 ± 4.62 mg/100 g) were greatest in soybean paste.

Cryoprotective effect of turanose on lyophilized Lactobacillus paracasei subsp. paracasei, L. casei 431.

The lyophilization process is the most convenient and successful method to preserve probiotics, although microorganisms are exposed to conditions of extremely low freezing temperatures as well as dehydration. In this study, we evaluated the cryoprotective effect of turanose on Lactobacillus paracasei subsp. paracasei, L. casei 431 (L. casei 431) as a method to increase survival rate by improving cell viability. The results indicated that the viability of L. casei 431 was 9.6% without the cryoprotective agent, whereas bacterial cell viability was increased to 67.1% with the addition of 12% turanose. When turanose-treated freeze-dried cells were stored at 4 °C for 30 days, the survival rate decreased from 67.1 to 53.4%. Furthermore, cell viability significantly decreased by 50% after 30 days when stored at 25 °C with the same amount of turanose. Overall, turanose may be used as an effective cryoprotectant to preserve probiotics against the freeze-drying process and for extended storage at 4 °C.