High-Yield Synthesis of Transglycosylated Mogrosides Improves the Flavor Profile of Monk Fruit Extract Sweeteners.

Luo Han Guo fruit extract (Siraitia grosvenorii), mainly composed of mogroside V (50%), could be considered a suitable alternative to free sugars; however, its commercial applications are limited by its unpleasant off-notes. In the present work, a central composite design method was employed to optimize the transglycosylation of a mogroside extract using cyclodextrin glucosyltransferases (CGTases) from three different bacteriological sources (Paenibacillus macerans, Geobacillus sp., and Thermoanaerobacter sp.) considering various experimental parameters such as maltodextrin and mogroside concentration, temperature, time of reaction, enzymatic activity, and pH. Product structures were determined by liquid chromatography coupled to a diode-array detector (LC-DAD), liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Sensory analysis of glucosylated mogrosides showed an improvement in flavor attributes relevant to licorice flavor and aftereffect. Consequently, an optimum methodology was developed to produce new modified mogrosides more suitable when formulating food products as free sugar substitutes.

Bioconversion of Stevioside to Rebaudioside E Using Glycosyltransferase UGTSL2.

Rebaudioside E, one of the minor components of steviol glycosides, was first isolated and identified from Stevia rebaudiana in 1977. It is a high-intensity sweetener that tastes about 150-200 times sweeter than sucrose and is also a precursor for biosynthesis of rebaudioside D and rebaudioside M, the next-generation Stevia sweeteners. In this work, new unknown steviol glycosides were enzymatically synthesized from stevioside by coupling UDP-glucosyltransferase UGTSL2 from Solanum lycopersicum and sucrose synthase StSUS1 from Solanum tuberosum. Rebaudioside E was speculated to be the main product of glucosylation of the Glc(ß1→C-19) residue of stevioside along with the formation of a (ß1→2) linkage based on the analysis of the regioselectivity and stereoselectivity of UGTSL2, and verified afterwards by LC-MS/MS with standard. In a 20-ml bioconversion reaction of 20 g/l stevioside by UGTSL2 and StSUS1, 15.92 g/l rebaudioside E was produced for 24 h.

A Novel Reversibly Glycosylated Polypeptide-2 of Bee Pollen from Rape (Brassica napus L.): Purification and Characterization.

BACKGROUND: Reversibly glycosylated polypeptide (RGP), a kind of hydrosoluble and plasmodesmal-associated protein found in plants, plays a crucial role in the development of pollen. OBJECTIVE: A novel RGP 2 was isolated and identified from rape (Brassica napus L.) bee pollen. METHODS: RGP2 was isolated and purified by ion-exchange column and gel filtration chromatography, and characterized by MALDI-TOF-MS, LC-MS, immunological histological chemistry, and transmission electron microscope. RESULTS: Our results indicated that the RGP2 is an acidic protein (pI=5.46) with the molecular weight 42388 Da. It contained 17 kinds of amino acids, among which aspartic acid had the highest amount (71.56 mg/g). Homologous alignment of amino acid sequence results showed that RGP2 was 80.33%, 85.02%, 86.06%, and 88.93% identical to Arabidopsis thaliana RGP2 (AtRGP2), Oryza sativa RGP (OsRGP), Triticum aestivum RGP (TaRGP), and Zea maize RGP (ZmRGP), respectively. The localization results showed that RGP2 in rape anther existed in exine and intine of anther cells of rape flower by immunological histological chemistry and the subcellular localization identified that RGP2 appeared around the Golgi apparatus in cytoplasm by transmission electron microscope. CONCLUSION: RGP2 has a highly conserved sequence of amino acid residues and potential glycosylation sites.

NoPv1: a synthetic antimicrobial peptide aptamer targeting the causal agents of grapevine downy mildew and potato late blight.

Grapevine (Vitis vinifera L.) is a crop of major economic importance. However, grapevine yield is guaranteed by the massive use of pesticides to counteract pathogen infections. Under temperate-humid climate conditions, downy mildew is a primary threat for viticulture. Downy mildew is caused by the biotrophic oomycete Plasmopara viticola Berl. & de Toni, which can attack grapevine green tissues. In lack of treatments and with favourable weather conditions, downy mildew can devastate up to 75% of grape cultivation in one season and weaken newly born shoots, causing serious economic losses. Nevertheless, the repeated and massive use of some fungicides can lead to environmental pollution, negative impact on non-targeted organisms, development of resistance, residual toxicity and can foster human health concerns. In this manuscript, we provide an innovative approach to obtain specific pathogen protection for plants. By using the yeast two-hybrid approach and the P. viticola cellulose synthase 2 (PvCesA2), as target enzyme, we screened a combinatorial 8 amino acid peptide library with the aim to identify interacting peptides, potentially able to inhibit PvCesa2. Here, we demonstrate that the NoPv1 peptide aptamer prevents P. viticola germ tube formation and grapevine leaf infection without affecting the growth of non-target organisms and without being toxic for human cells. Furthermore, NoPv1 is also able to counteract Phytophthora infestans growth, the causal agent of late blight in potato and tomato, possibly as a consequence of the high amino acid sequence similarity between P. viticola and P. infestans cellulose synthase enzymes.

Amylosucrase-modified waxy potato starches recrystallized with amylose: The role of amylopectin chain length in formation of low-digestible fractions.

This study examined the contribution of amylose to the organization of each starch fraction in recrystallized starch. Amylosucrase (AS)-modified waxy potato starches with different branch chain lengths were completely solubilized with amylose (3:1 ratio) and recrystallized at 4 °C for 48 h. The content of rapidly digestible starch and resistant starch (RS) showed linear change with degree of AS modification, while slowly digestible starch (SDS) did not. The changes in structural characteristics were tracked according to serial removal of each fraction. Results from iodine binding property, branch chain length, X-ray diffraction, and thermal property analysis indicated that branch chain length of amylopectin determined the length of the amylose-amylopectin double helix and the mobility of amylose and that formation of SDS or RS could be induced by controlling the length of amylopectin chains. These findings could be used for production of customized starches with specific digestive properties for health benefits.

Design and study of anticaries effect of different medicinal plants against S.mutans glucosyltransferase.

BACKGROUND: The present study was aimed to evaluate the molecular level anticaries effect of different medicinal plants against Streptococcus mutans (S.mutans) glucosyltransferases (gtf). METHODS: A total of six natural sources named as Terminalia chebula (T.chebula), Psidium guajava (P.guajava), Azadirachta indica (A.indica) and Pongamia pinnata (P.pinnata); two essential oils, clove (Syzygium aromaticum) and peppermint oil (Mentha piperita) were selected as test samples. Hydroalcoholic plant extracts and essential oils were examined for their inhibitory potential on gtf isolated from S.mutans. Polyherbal mouth wash was prepared and its effect on gtf activity was compared with commercial chlorhexidine mouth wash (5%w/v). Enzyme kinetic study was carried out in order to explore the molecular mechanism of enzyme action. RESULTS: Out of six natural sources tested, A.indica has shown maximum inhibitory effect of 91.647% on gtf and T.chebula has shown IC50 of 1.091 mg/ml which is significant when compared to standard chlorhexidine. From the final result of kinetic analysis it was found that T.chebula, P.guajava and P.pinnata have show uncompetitive inhibition where as A.indica has shown non-competitive inhibition. Surprisingly, both essential oils have shown allosteric inhibition (sigmoidal response). The polyherbal moutwash has shown significant inhibitory potential on gtf (95.936%) when compared to commercial chlorhexidine mouthwash (p < 0.05). CONCLUSION: All the tested samples have shown considerable gtf inhibitory action. Moreover polyherbal mouth wash has shown promising noncompetitive inhibitory activity against gtf and it could be the future formulation to combat dental caries.

Alginate-pectin co-encapsulation of dextransucrase and dextranase for oligosaccharide production from sucrose feedstocks.

The genes for dextransucrase and dextranase were cloned from the genomic regions of Leuconostoc mesenteroides MTCC 10508 and Streptococcus mutans MTCC 497, respectively. Heterologous expression of genes was performed in Escherichia coli. The purified enzyme fractions were entrapped in the alginate-pectin beads. A high immobilization yield of dextransucrase (~ 96%), and dextranase (~ 85%) was achieved. Alginate-pectin immobilization did not affect the optimum temperature and pH of the enzymes; rather, the thermal tolerance and storage stability of the enzymes was improved. The repetitive batch experiments suggested substantially good operational stability of the co-immobilized enzyme system. The synergistic catalytic reactions of alginate-pectin co-entrapped enzyme system were able to produce 7-10 g L-1 oligosaccharides of a high degree of polymerization (DP 3-9) from sucrose (~ 20 g L-1) containing feedstocks, e.g., table sugar and cane molasses. The alginate-pectin-based co-immobilized enzyme system is a useful catalytic tool to bioprocess the agro-industrial bio-resource for the production of prebiotic biomolecules.

A specific UDP-glucosyltransferase catalyzes the formation of triptophenolide glucoside from Tripterygium wilfordii Hook. f.

Tripterygium wilfordii Hook. f. is a perennial woody vine member of the Celastraceae family. As a traditional Chinese medicine, it contains complex chemical components and exerts various pharmacological activities. In the present study, we identified a glucosyltransferase, TwUGT1, that can catalyze the synthesis of an abietane-type diterpene glucoside, namely, triptophenolide14-O-beta-D-glucopyranoside, and investigated the pharmacological activity of triptophenolide glucoside in diverse cancer cells. Triptophenolide glucoside exhibited significant inhibitory effects on U87-MG, U251, C6, MCF-7, HeLa, K562, and RBL-2H3 cells as determined by pharmacological analysis. The triptophenolide glucoside content of T. wilfordii was analyzed using Agilent Technologies 6490 Triple Quad LC/MS. The glucosyltransferase TwUGT1 belongs to subfamily 88 and group E in family 1. Molecular docking and site-directed mutagenesis of TwUGT1 revealed that the His30, Asp132, Phe134, Thr154, Ala370, Leu376, Gly382, His387, Glu395 and Gln412 residues play crucial roles in the catalytic activity of triptophenolide 14-O-glucosyltransferase. In addition, TwUGT1 was also capable of glucosylating phenolic hydroxyl groups, such as those in liquiritigenin, pinocembrin, 4-methylumbelliferone, phloretin, and rhapontigenin.

Modulation of photosynthetic parameters, sugar metabolism, polyamine and ion contents by silicon amendments in wheat (Triticum aestivum L.) seedlings exposed to arsenic.

The objective of the present investigation was to consider the effectiveness of exogenous silicate supplementation in reviving the arsenate imposed alterations on pigment content, Hill activity, photosynthetic parameters, sugar metabolism, polyamine, and ion contents in wheat (Triticum aestivum L. cv. PBW-343) seedlings. Experiments were conducted under different levels of arsenate (0, 25 µM, 50 µM, and 100 µM) in combination with silicate (0, 5 mM) in a hydroponic environment with modified Hoagland's solution for 21 days to determine the ameliorative role of silicon (Si). Arsenate exposure led to a decline in chlorophyll content by 28% and Hill activity by 30% on an average along with photosynthetic parameters. Activity of starch phosphorylase increased causing a subsequent decrease in starch contents by 26%. Degradation of starch enhanced sugar contents by 61% in the test cultivar. Dose-dependant increments in the activities of carbohydrate metabolizing enzymes viz., sucrose synthase, sucrose phosphate synthase, and acid invertase were also noted. Putrescine content was significantly enhanced along with a consequent decline in spermidine and spermine contents. The macro- and micronutrient contents declined proportionally with arsenate imposition. Conversely, silicate amendments irrespective of all arsenate concentrations brought about considerable alterations in all parameters tested with respect to arsenate treatment alone. Marked improvement in pigment content and Hill activity also improved the gas exchange parameters. Soluble sugar contents decreased and starch contents were enhanced. Increase in polyamine contents improved the ionic balance in the test cultivar as well. This study highlights the potentiality of silicon in ameliorating the ecotoxicological risks associated with arsenic pollution and the probable ability of silicon to offer an approach in mitigating arsenate-induced stress leading to restoration of growth and metabolism in wheat seedlings.

Characterization of a heat responsive UDP: Flavonoid glucosyltransferase gene in tea plant (Camellia sinensis).

Tea plant (Camellia sinensis) accumulates abundant flavonoid glycosides that are the major bioactive ingredients in tea. Biosynthesis of flavonoid glycosides are catalyzed by UDP-glucosyltransferases (UGTs) that are widely present in plants. Among one hundred and seventy-eight UGTs genes that we have previously identified in tea plant, few of them have been functionally characterized. In the present study, we further identified UGT73A17 gene that is responsible for the biosynthesis of a broad range of flavonoid glycosides. Sequence analysis revealed that the deduced UGT73A17 protein showed high identity with 7-O-glycosyltransferases at amino acid level and it was clustered into the clade containing several 7-O-glycosyltransferases from other plant species. Enzymatic assays revealed that the recombinant UGT73A17 protein (rUGT73A17) exhibited activity toward flavonols (kaempferol, quercetin, and myricetin), flavones (apigenin, luteolin, and tricetin), flavanone (naringenin), isoflavones (genistein) and epicatechin gallate, yielding 7-O-glucosides as the major in vitro products. In particular, rUGT73A17 displayed higher activity at high temperatures (eg. 50°C) than at low temperatures, which was consistent with its relatively high expression level at high temperatures. Two amino acid substitutions at I296L and V466A improved the enzymatic activity of rUGT73A17. Our study demonstrated that UGT73A17 is responsible for the biosynthesis of a broad range of flavonoid glucosides, which is also involved in heat response and quality of tea plant.

Characterization of the Asiatic Acid Glucosyltransferase, UGT73AH1, Involved in Asiaticoside Biosynthesis in Centella asiatica (L.) Urban.

Centella asiatica (L.) Urban contains two ursane-type triterpene saponins, asiaticoside and madecassoside, as major secondary metabolites. In order to select candidate genes encoding UDP-glucosyltransferases (UGTs) involved in asiaticoside biosynthesis, we performed transcriptomic analysis of leaves elicited by methyl jasmonate (MeJA). Among the unigenes, 120 isotigs and 13 singletons of unique sequences were annotated as UGTs, including 37 putative full-length cDNAs, and 15 of the putative UGT genes were named according to the UGT committee nomenclature protocols. One of them, UGT73AH1, was characterized by heterologous expression in Escherichia coli BL21 (DE3) cells. After induction with IPTG, a total protein extract was assayed with UDP-glucose and asiatic acid. UPLC-QTOF/MS analysis showed that UGT73AH1 catalyzes the glycosylation of asiatic acid to its monoglucoside. It remains unclear whether glycosylation occurs on the triterpene C-2α, C-3ß, C-23, or C-28 position. However, it is very likely that UGT73AH1 glucosylates the C-28 position, because only C-28 bears a glucose moiety in the final pathway product of asiatic acid, while C-2α, C-3ß, and C-23 remain un-conjugated.

Improved soluble bacterial expression and properties of the recombinant flavonoid glucosyltransferase UGT73G1 from Allium cepa.

Glycosylation of quercetin using flavonol-specific glycosyltransferases offers an alternate method for isoquercitrin production. Obtaining sufficient quantities of bioactive enzymes is an important prerequisite for highly effective biocatalysis and biotransformation. In this study, a codon-optimized gene for the flavonoid glucosyltransferase UGT73G1 from Allium cepa was heterologously expressed in the preferred prokaryotic expression host Escherichia coli. By combining expression as a fusion protein with 6-histidine tags with coexpression with molecular chaperones, increased soluble expression of UGT73G1 was achieved in E. coli. Two-terminal 6-histidine tags contributed more to the expression than molecular chaperones, as demonstrated by comparison of specific activities in crude extracts obtained from the recombinant E. coli strains. Studies of the catalytic properties of purified UGT73G1 indicated that its activity was significantly promoted by Mn2+ and Mg2+, while it was strongly inhibited by Cu2+. These expression strategies enhanced the solubility and activity of the overexpressed protein and enabled characterization of this plant-derived glucosyltransferase expressed in a prokaryotic host.

Knockdown of a cellulose synthase gene BoiCesA affects the leaf anatomy, cellulose content and salt tolerance in broccoli.

Cellulose is the major component of cell wall materials. A 300 bp specific fragment from the cDNA fragment was chosen to insert into vector pFGC1008 at forward and reverse orientations to construct the recombinant RNAi vector. Knockdown of BoiCesA caused "dwarf" phenotype with smaller leaves and a loss of the content of cellulose. Moreover, RT-PCR analysis confirmed that the expression of the RNAi apparatus could repress expression of the CesA gene. Meanwhile, examination of the leaves from the T3 of RNAi transformants indicated reduction of cell expansion in vascular bundles, particularly on their abaxial surface. The proline and soluble sugar content increased contrarily. Under the salt stress, the T3 of RNAi plants showed significant higher resistance. The expression levels of some salt tolerance related genes (BoiProH, BoiPIP2;2, BoiPIP2;3) were significantly changed in T3 of RNAi plants. The results showed that the hairpin structure of CesA specific fragment inhibited the endogenous gene expression and it was proved that the cDNA fragment was relevant to the cellulose biosynthesis. Moreover, modulation cellulose synthesis probably was an important influencing factor in polysaccharide metabolism and adaptations of plants to stresses. This will provide technological possibilities for the further study of modulation of the cellulose content of crops.

From Plant Extract to a cDNA Encoding a Glucosyltransferase Candidate: Proteomics and Transcriptomics as Tools to Help Elucidate Saponin Biosynthesis in Centella asiatica.

Centella asiatica (L.) Urban (Apiaceae), a small annual plant that grows in India, Sri Lanka, Malaysia, and other parts of Asia, is well-known as a medicinal herb with a long history of therapeutic uses. The bioactive compounds present in C. asiatica leaves include ursane-type triterpene sapogenins and saponins-asiatic acid, madecassic acid, asiaticoside, and madecassoside. Various bioactivities have been shown for these compounds, although most of the steps in the biosynthesis of triterpene saponins, including glycosylation, remain uncharacterized at the molecular level. This chapter describes an approach that integrates partial enzyme purification, proteomics methods, and transcriptomics, with the aim of reducing the number of cDNA candidates encoding for a glucosyltransferase involved in saponin biosynthesis and facilitating the elucidation of the pathway in this medicinal plant.

Optical Resolution of (RS)-Denopamine to (R)-Denopamine P-D- Glucoside by Glucosyltransferase from Phytolacca americana Expressed in Recombinant Escherichia coli.

The optical resolution of racemic compounds by stereoselective glucosylation was investigated using plant glucosyltransferase from Phytolacca americana expressed in recombinant Escherichia coli. The glucosyltransferase glucosylated chemoselectively the phenolic hydroxyl group of phenol compounds. The (R)-stereoselective glucosylation of (RS)-denopamine by glucosyltransferase occurred to give (R)-denopamine ß-D-glucoside.

Enzymatic Synthesis of Quercetin Monoglucopyranoside and Maltooligosaccharides.

Quercetin 3-O-ß-monoglucopyranoside and quercetin 3-O-ß-maltooligosaccharide were synthesized from quercetin using glucosyltransferase-3 from Phytolacca americana and cyclodextrin glucanotransferase.

Synthesis of Resveratrol Glycosides by Plant Glucosyltransferase and Cyclodextrin Glucanotransferase and Their Neuroprotective Activity.

Resveratrol was converted by glucosyltransferase from Phytolacca americana into its 3- and 4'-O-ß-D-glucosides. On the other hand, further glycosylation of resveratrol 4'-O-ß-D-glucoside by cyclodextrin glucanotransferase gave the 4'-O-ß-maltoside, 4'-O-ß-maltotrioside, 4'-O-ß-maltotetraoside, and 4'-O-ß- maltopentaoside of resveratrol. The six resveratrol glycosides synthesized here showed higher phosphodiesterase inhibitory activity than resveratrol.

Synthesis of ε-Viniferin Glycosides by Glucosyltransferase from Phytolacca americana and their Inhibitory Activity on Histamine Release from Rat Peritoneal Mast Cells.

Glycosylation of (+)-ε-viniferin was investigated using glucosyltransferase from Phytolacca americana (PaGT3) as a biocatalyst. (+)-ε-Viniferin was converted by PaGT3 into its 4b- and 13b-ß-D-glucosides, the inhibitory activities on histamine release from rat peritoneal mast cells of which were higher than that of (+)-ε-viniferin.

Unidirectional movement of cellulose synthase complexes in Arabidopsis seed coat epidermal cells deposit cellulose involved in mucilage extrusion, adherence, and ray formation.

Cellulose synthase5 (CESA5) synthesizes cellulose necessary for seed mucilage adherence to seed coat epidermal cells of Arabidopsis (Arabidopsis thaliana). The involvement of additional CESA proteins in this process and details concerning the manner in which cellulose is deposited in the mucilage pocket are unknown. Here, we show that both CESA3 and CESA10 are highly expressed in this cell type at the time of mucilage synthesis and localize to the plasma membrane adjacent to the mucilage pocket. The isoxaben resistant1-1 and isoxaben resistant1-2 mutants affecting CESA3 show defects consistent with altered mucilage cellulose biosynthesis. CESA3 can interact with CESA5 in vitro, and green fluorescent protein-tagged CESA5, CESA3, and CESA10 proteins move in a linear, unidirectional fashion around the cytoplasmic column of the cell, parallel with the surface of the seed, in a pattern similar to that of cortical microtubules. Consistent with this movement, cytological evidence suggests that the mucilage is coiled around the columella and unwinds during mucilage extrusion to form a linear ray. Mutations in CESA5 and CESA3 affect the speed of mucilage extrusion and mucilage adherence. These findings imply that cellulose fibrils are synthesized in an ordered helical array around the columella, providing a distinct structure to the mucilage that is important for both mucilage extrusion and adherence.

Functionalized anodic aluminum oxide membrane-electrode system for enzyme immobilization.

A nanoporous membrane system with directed flow carrying reagents to sequentially attached enzymes to mimic nature's enzyme complex system was demonstrated. Genetically modified glycosylation enzyme, OleD Loki variant, was immobilized onto nanometer-scale electrodes at the pore entrances/exits of anodic aluminum oxide membranes through His6-tag affinity binding. The enzyme activity was assessed in two reactions­a one-step "reverse" sugar nucleotide formation reaction (UDP-Glc) and a two-step sequential sugar nucleotide formation and sugar nucleotide-based glycosylation reaction. For the one-step reaction, enzyme specific activity of 6­20 min(­1) on membrane supports was seen to be comparable to solution enzyme specific activity of 10 min(­1). UDP-Glc production efficiencies as high as 98% were observed at a flow rate of 0.5 mL/min, at which the substrate residence time over the electrode length down pore entrances was matched to the enzyme activity rate. This flow geometry also prevented an unwanted secondary product hydrolysis reaction, as observed in the test homogeneous solution. Enzyme utilization increased by a factor of 280 compared to test homogeneous conditions due to the continuous flow of fresh substrate over the enzyme. To mimic enzyme complex systems, a two-step sequential reaction using OleD Loki enzyme was performed at membrane pore entrances then exits. After UDP-Glc formation at the entrance electrode, aglycon 4-methylumbelliferone was supplied at the exit face of the reactor, affording overall 80% glycosylation efficiency. The membrane platform showed the ability to be regenerated with purified enzyme as well as directly from expression crude, thus demonstrating a single-step immobilization and purification process.