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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]

Sucrose accumulation has been observed in some methylotrophic bacteria utilizing methane, methanol, or methylated amines as a carbon and energy source. In this work, we have investigated the biochemical pathways for sucrose metabolism in the model halotolerant methanotroph Methylomicrobium alcaliphilum 20Z. The genes encoding sucrose-phosphate synthase (Sps), sucrose-phosphate phosphatase (Spp), fructokinase (FruK), and amylosucrase (Ams) were co-transcribed and displayed similar expression levels. Functional Spp and Ams were purified after heterologous expression in Escherichia coli. Recombinant Spp exhibited high affinity for sucrose-6-phosphate and stayed active at very high levels of sucrose ( K = 1.0 ± 0.6 M). The recombinant amylosucrase obeyed the classical Michaelis-Menten kinetics in the reactions of sucrose hydrolysis and transglycosylation. As a result, the complete metabolic network for sucrose biosynthesis and re-utilization in the non-phototrophic organism was reconstructed for the first time. Comparative genomic studies revealed analogous gene clusters in various Proteobacteria, thus indicating that the ability to produce and metabolize sucrose is widespread among prokaryotes. [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]

Amylosucrase (AS), a glucosyltransferase of the glycoside hydrolase 13 family, does not require a nucleotide-activated sugar as a glucosyl-donor. A gene ( drpas) of Deinococcus radiopugnans encoding a putative AS was identified and cloned for characterization. The amino acid sequence revealed that drpas exhibited 76 and 74% identities with AS genes from D. geothermalis and D. radiodurans, respectively. Recombinant DRpAS exhibited AS activities. The ratios of hydrolysis, polymerization, and isomerization reactions were 5.8:82.7:11.5 at 40°C. The DRpAS was highly thermostable and produced more polymerization products than AS from D. radiodurans although the optimum growth temperature of both strains is 30°C. [ABSTRACT FROM AUTHOR]

ABSTRACT Amylosucrase from Neisseria polysaccharea is a remarkable transglucosylase that synthesizes an insoluble amylose-like polymer from sole substrate sucrose. One particular amino acid, Arg226, was proposed from molecular modeling studies to play an important role in the formation of the active site topology and in the accessibility of ligands to the catalytic site. The systematic mutation of this Arg residue by all 19 other possible amino acids revealed that all single-mutants had a higher activity on sucrose compared to the wild-type enzyme. An extensive kinetic investigation showed that catalytic efficiencies are greatly impacted by the presence of natural acceptors in the reaction media, their chain length and the nature of the amino acid at position 226. Compared to the wild-type enzyme, the R226N mutant showed a 10-fold enhancement in the catalytic efficiency and a nearly twofold higher production of an insoluble amylose-like polymer that can be of interest for biotechnological applications. Biotechnol. Bioeng. 2014;111: 1719-1728. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Main conclusion: Expression of amylosucrase in potato resulted in larger starch granules with rough surfaces and novel physico-chemical properties, including improved freeze-thaw stability, higher end viscosity, and better enzymatic digestibility. Starch is a very important carbohydrate in many food and non-food applications. In planta modification of starch by genetic engineering has significant economic and environmental benefits as it makes the chemical or physical post-harvest modification obsolete. An amylosucrase from Neisseria polysaccharea fused to a starch-binding domain (SBD) was introduced in two potato genetic backgrounds to synthesize starch granules with altered composition, and thereby to broaden starch applications. Expression of SBD-amylosucrase fusion protein in the amylose-containing potato resulted in starch granules with a rough surface, a twofold increase in median granule size, and altered physico-chemical properties including improved freeze-thaw stability, higher end viscosity, and better enzymatic digestibility. These effects are possibly a result of the physical interaction between amylosucrase and starch granules. The modified larger starches not only have great benefit to the potato starch industry by reducing losses during starch isolation, but also have an advantage in many food applications such as frozen food due to its extremely high freeze-thaw stability. [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]

A putative α-amylase gene (accession number, CP000284) of Methylobacillus flagellatus KT ATCC51484 was cloned in Escherichia coli, and its gene product was expressed and characterized. The purified recombinant enzyme (MFAS) displayed a typical amylosucrase (ASase) activity by the demonstration of multiple activities of hydrolysis, isomerization, and polymerization although it was designated as an α-amylase. The optimal reaction temperature and pH for the sucrose hydrolysis activity of MFAS were determined to be 45 °C and pH 8.5, respectively. MFAS has relatively high thermostable characteristics compared with other ASases, as demonstrated by a half-life of 19.3 min at 50 °C. MFAS also showed polymerization activity using sucrose as a sole substrate. Glycerol was transglycosylated by the intermolecular transglycosylation activity of MFAS. Two major products were observed by thin-layer chromatography and isolated by paper chromatography and recycling HPLC. Using H and C NMR, their chemical structures were determined to be (2S)-1- O-α- d-glucosyl-glycerol or (2R)-1- O-α- d-glucosyl-glycerol and 2- O-α- d-glucosyl-glycerol, in which a glucose molecule is linked to glycerol via an α-glycosidic linkage. [ABSTRACT FROM AUTHOR]

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]

Asymmetrical flow field flow fractionation (AF4) has proven to be a very powerful and quantitative method for the determination of the macromolecular structure of high molar mass branched biopolymers, when coupled with multi-angle laser light scattering (MALLS). This work describes a detailed investigation of the macromolecular structure of native glycogens and hyperbranched α-glucans (HBPs), with average molar mass ranging from 2 × 10 to 4.3 × 10 g mol, which are not well fractionated by means of classical size-exclusion chromatography. HBPs were enzymatically produced from sucrose by the tandem action of an amylosucrase and a branching enzyme mimicking in vitro the elongation and branching steps involved in glycogen biosynthesis. Size and molar mass distributions were studied by AF4, coupled with online quasi-elastic light scattering (QELS) and transmission electron microscopy. AF4-MALLS-QELS has shown a remarkable agreement between hydrodynamic radii obtained by online QELS and by AF4 theory in normal mode with constant cross flow. Molar mass, size, and dispersity were shown to significantly increase with initial sucrose concentration, and to decrease when the branching enzyme activity increases. Several populations with different size range were observed: the amount of small size molecules decreasing with increasing sucrose concentration. The spherical and dense global conformation thus highlighted was partly similar to native glycogens. A more detailed study of HBPs synthesized from low and high initial sucrose concentrations was performed using complementary enzymatic hydrolysis of external chains and chromatography. It emphasized a more homogeneous branching pattern than native glycogens with a denser core and shorter external chains. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Understanding in vitro digestibility and release experiments with starch-entrapped calcium alginate microspheres provide the possibility of controlling starch digestibility in the human gastrointestinal tract. Native and amylosucrase (AS)-treated waxy corn starches (5000 U, 20 000 U, and control) were encapsulated with three different ratios of sodium alginate: 0.5, 0.7, and 1.0%. The slowly digestible and RS fractions of the encapsulated starch were 57.5 and 97.7%, respectively. After cooking, the proportions of these fractions still ranged from 55.7 to 96.1%, depending on the type of starch and concentration of sodium alginate, whereas the un-encapsulated starch contained between 2.9 and 48.3% slowly digestible and RS after cooking. In vitro release was studied in both uncooked and cooked encapsulated samples. When incubated in simulated gastric fluid, the beads swelled and started to float but did not erode, even after thermal treatment. Incubation in simulated intestinal and colonic fluids caused the beads to release starch in both uncooked and cooked starch-encapsulated samples. Encapsulated AS control and native waxy corn starch samples released much more starch than AS-modified starches. The total amount of starch released did not exceed 20% for uncooked samples and 25% for cooked samples. [ABSTRACT FROM AUTHOR]

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]

The amylosucrase from Neisseria polysaccharea is a transglucosidase from the GH13 family of glycoside-hydrolases that naturally catalyzes the synthesis of α-glucans from the widely available donor sucrose. Interestingly, natural molecular evolution has modeled a dense hydrogen bond network at subsite −1 responsible for the specific recognition of sucrose and conversely, it has loosened interactions at the subsite +1 creating a highly promiscuous subsite +1. The residues forming these subsites are considered to be likely involved in the activity as well as the overall stability of the enzyme. To assess their role, a structure-based approach was followed to reshape the subsite −1. A strategy based on stability change predictions, using the FoldX algorithm, was considered to identify the best candidates for site-directed mutagenesis and guide the construction of a small targeted library. A miniaturized purification protocol was developed and both mutant stability and substrate promiscuity were explored. A range of 8°C between extreme melting temperature values was observed and some variants were able to synthesize series of oligosaccharides with distributions differing from that of the parental enzyme. The crucial role of subsite −1 was thus highlighted and the biocatalysts generated can now be considered as starting points for further engineering purposes. [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]

Incorporating structural elements of thermostable homologs can greatly improve the thermostability of a mesophilic protein. Despite the effectiveness of this method, applying it is often hampered. First, it requires alignment of the target mesophilic protein sequence with those of thermophilic homologs, but not every mesophilic protein has a thermophilic homolog. Second, not all favorable features of a thermophilic protein can be incorporated into the structure of a mesophilic protein. Furthermore, even the most stable native protein is not sufficiently stable for industrial applications. Therefore, creating an industrially applicable protein on the basis of the thermophilic protein could prove advantageous. Amylosucrase (AS) can catalyze the synthesis of an amylose-like polysaccharide composed of only α-1, 4-linkages using sucrose as the lone energy source. However, industrial development of AS has been hampered owing to its low thermostability. To facilitate potential industrial applications, the aim of the current study was to improve the thermostability of Deinococcus geothermalis amylosucrase (DgAS) further; this is the most stable AS discovered to date. By integrating ideas from mesophilic AS with well-established protein design protocols, three useful design protocols are proposed, and several promising substitutions were identified using these protocols. The successful application of this hybrid design method indicates that it is possible to stabilize a thermostable protein further by incorporating structural elements of less-stable homologs. [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]

Extremophiles are nature's tiny warriors as they call inhospitable environments their home. They possess special factor(s) that offer an edge over other life forms susceptible to harsh conditions. One such family of extremophiles under discussion here is Deinococcaceae. The microbes belonging to Deinococcaceae are primarily radiophiles, the world's most radiation resistant bacteria, in addition to having resistance to high temperature, metals, cold etc. in specific species. Gamma rays have always been known to be lethal to living cells as it damages DNA, the blueprint of life. But, Deinococci sustain extremely high doses of gamma radiation, about 3000 times more than the dose humans succumb to. This review brings forth the utility of these special factors of Deinococcaceae for a broad range of biotechnological applications., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author. Published by Elsevier B.V.)

Many drugs and prebiotics derive their activities from sugar substituents. Due to the prevalence and complexity of these biologically active compounds, enzymatic glycodiversification that facilitates easier access to these compounds can make profound contributions to the pharmaceutical, food, and feed industries. Amylosucrases (ASases) are attractive tools for glycodiversification because of their broad acceptor substrate specificity, but the lack of structural information and their poor thermostability limit their industrial applications. Herein, we reported the crystal structure of ASase from Calidithermus timidus, which displays a homotetrameric quaternary organization not previously observed for other ASases. We employed a workflow composed of five common strategies, including interface engineering, folding energy calculations, consensus sequence, hydrophobic effects enhancement, and B-factor analysis, to enhance the thermostability of C. timidus ASase. As a result, we obtained a quadruple-point mutant M31 ASase with a half-life at 65 °C increased from 22.91 h to 52.93 h, which could facilitate biosynthesis of glucans with a degree of polymerization of more than 20 using sucrose as a substrate at 50 °C. In conclusion, this study provides a structural basis for understanding the multifunctional biocatalyst ASase and presents a powerful methodology to effectively and systematically enhance protein thermostability., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Arbutins (α- and β-arbutins) are glycosylated hydroquinones that are commercially used in the cosmetic industry. These compounds have an inhibitory function against tyrosinase, a critical enzyme for generating pigments, which leads to the prevention of melanin formation, resulting in a whitening effect on the skin. Although β-arbutin is found in various plants including bearberry, wheat, and pear, α-arbutin and other arbutin derivatives are synthesized by chemical and enzymatic methods. This article presents a mini-review of recent studies on the production of α-arbutin and other α- and β-arbutin derivatives via enzymatic bioconversion methods. In addition, the structures of α- and β-arbutin derivatives and their biological activities are discussed. The catalytic characteristics of various enzymes used in the biosynthesis of arbutin derivatives are also reviewed. [ABSTRACT FROM AUTHOR]

α-Arbutin (α-Ab) is a powerful skin whitening agent that blocks epidermal melanin biosynthesis by inhibiting the enzymatic oxidation of tyrosine and l-3,4-dihydroxyphenylalanine ( l-DOPA). α-Ab was effectively synthesized from hydroquinone (HQ) by enzymatic biotransformation using amylosucrase (ASase). The ASase gene from Deinococcus geothermalis (DGAS) was expressed and efficiently purified from Escherichia coli using a constitutive expression system. The expressed DGAS was functional and performed a glycosyltransferase reaction using sucrose as a donor and HQ as an acceptor. The presence of a single HQ bioconversion product was confirmed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The HQ bioconversion product was isolated by silica gel open column chromatography and its chemical structure determined by H and C nuclear magnetic resonance (NMR). The product was determined to be hydroquinone- O-α- d-glucopyranoside with a glucose molecule linked to HQ through an α-glycosidic bond. However, the production yield of the transfer reaction was significantly low (1.3%) due to the instability of HQ in the reaction mixture. The instability of HQ was considerably improved by antioxidant agents, particularly ascorbic acid, implying that HQ is labile to oxidation. A maximum yield of HQ transfer product of 90% was obtained at a 10:1 molar ratio of donor (sucrose) and acceptor (HQ) molecules in the presence of 0.2 mM ascorbic acid. [ABSTRACT FROM AUTHOR]

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]

Trehalose (α- d-glucopyranosyl α- d-glucopyranoside) is an important non-reducing disaccharide used in the food industry due to its mild sweetness (45% that of sucrose), low cariogenicity, high glass transition temperature, low hygroscopicity, and protein protection properties. In this study, we accomplished the production of trehalose from sucrose as a sole substrate using a novel dual-enzyme system, in which amylosucrase (ASase) and maltooligosyltrehalose synthase-trehalohydrolase (MTSH) fusion enzyme were employed. The biotransformation of sucrose to trehalose was confirmed by high-performance anion-exchange chromatography (HPAEC) analysis. Trehalose was successfully produced by both simultaneous and sequential methods by using ASase and MTSH. A higher trehalose production yield (3.15 ± 0.83 mM trehalose/20 mM sucrose) was observed in the sequential method than the simultaneous method (1.43 ± 0.14 mM trehalose/20 mM sucrose), indicating that the production of maltooligosaccharides from sucrose by ASase was an important step in the biosynthesis of trehalose. [ABSTRACT FROM AUTHOR]

Abstract: Amylosucrase (ASase, EC 2.4.1.4) is a glucosyltransferase that hydrolyzes sucrose into glucose and fructose and produces amylose-like glucan polymers from the released glucose. (+)-Catechin is a plant polyphenolic metabolite having skin-whitening and antioxidant activities. In this study, the ASase gene from Deinococcus geothermalis (dgas) was expressed in Escherichia coli, while the recombinant DGAS enzyme was purified using a glutathione S-transferase fusion system. The (+)-catechin glycoside derivatives were synthesized from (+)-catechin using DGAS transglycosylation activity. We confirmed the presence of two major transglycosylation products using TLC. The (+)-catechin transglycosylation products were isolated using silica gel open column chromatography and recycling-HPLC. Two (+)-catechin major transfer products were determined through 1H and 13C NMR to be (+)-catechin-3′-O-α-d-glucopyranoside with a glucose molecule linked to (+)-catechin and (+)-catechin-3′-O-α-D-maltoside with a maltose linked to (+)-catechin. The presence of (+)-catechin maltooligosaccharides in the DGAS reaction was also confirmed via recycling-HPLC and enzymatic analysis. The effects of various reaction conditions (temperature, enzyme concentration, and molar ratio of acceptor and donor) on the yield and type of (+)-catechin glycosides were investigated. [Copyright &y& Elsevier]

Abstract: In an effort to use a biocatalyst repeatedly with acceptable stability, amylosucrase from Neisseria polysaccharea (NpAS) was non-covalently immobilized on a pH-dependent autoprecipitating polymer, Eudragit L100. About 87% of the enzyme activity and 96% of the protein were recovered after the immobilization process. The immobilized NpAS showed significantly improved thermostability, whereas no difference was observed in the temperature and pH profiles of activity between the native and immobilized NpAS. To evaluate the reusability of the immobilized NpAS, repeated production of linear α-(1,4)-glucans was carried out at 35°C with 0.1M sucrose as substrate. Although only 71–74% of the protein was recovered after each reaction cycle, high stability index was observed from 0.93 to 0.96. Reusability of the immobilized NpAS was further confirmed by comparing its linear α-(1,4)-glucan products with that produced by native one. Almost identical production yield and molecular size distribution of the linear α-(1,4)-glucans were obtained between them. [Copyright &y& Elsevier]