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Glycosylation is one of the most important post-modification processes of small molecules and enables the parent molecule to have increased solubility, stability, and bioactivity. Enzyme-based glycosylation has achieved significant progress due to advances in protein engineering, DNA recombinant techniques, exploitation of biosynthetic gene cluster...
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... Therefore, the need to modify apigenin has been of interest in recent years. Among the post-modification applications, glucosylation, which is the functional modification of natural flavonoids by the introduction of sugar moieties to the flavonoid's core, has broadly been known to increase the bioactivity, conformation, solubility, half-life, and pharmacokinetic properties [16][17][18]. For instance, apigenin 7-O--glucoside as synthesized by Thuan et al. 2018, appeared to have several additional properties, including higher antioxidant, solubility, and anti-proliferative activities [19]. ...
... Acetonitrile (ACN) and water (0.1% trifluoroacetic acid) were used as the mobile phase. ACN concentrations were as follows: 20%, (0-5 min); 50%, (5-10 min); 70%, (10-15 min); 90%, (15)(16)(17)(18)(19)(20) min; and 10%, (20-25 min); with a flow rate of 1 ml/min. ...
Glucosylation is a well-known approach to improve the solubility, pharmacological, and biological properties of flavonoids, making flavonoid glucosides a target for large-scale biosynthesis. However, the low yield of products coupled with the requirement of expensive UDP-sugars limits the application of enzymatic systems for large-scale. C. glutamicum is a Gram-positive and generally regarded as safe (GRAS) bacteria frequently employed for the large-scale production of amino acids and bio-fuels. Due to the versatility of its cell factory system and its non-endotoxin producing properties, it has become an attractive system for the industrial-scale biosynthesis of alternate products. Here, we explored the cell factory of C. glutamicum for efficient glucosylation of flavonoids using apigenin as a model flavonoid, with the heterologous expression of a promiscuous glycosyltransferase, YdhE from Bacillus licheniformis and the endogenous overexpression of C. glutamicum genes galU1 encoding UDP-glucose pyrophosphorylase and pgm encoding phosphoglucomutase involved in the synthesis of UDP-glucose to create a C. glutamicum cell factory system capable of efficiently glucosylation apigenin with a high yield of glucosides production. Consequently, the production of various apigenin glucosides was controlled under different temperatures yielding almost 4.2 mM of APG1(apigenin-4'-O-β-glucoside) at 25°C, and 0.6 mM of APG2 (apigenin-7-O-β-glucoside), 1.7 mM of APG3 (apigenin-4',7-O-β-diglucoside) and 2.1 mM of APG4 (apigenin-4',5-O-β-diglucoside) after 40 h of incubation with the supplementation of 5 mM of apigenin and 37°C. The cost-effective developed system could be used to modify a wide range of plant secondary metabolites with increased pharmacokinetic activities on a large scale without the use of expensive UDP-sugars.
... Sun, Zhao, et al., 2020). Polyphenol biosynthesis often involves the late-stage attachment of one or more sugar residues to the aglycone core structure Thuan & Sohng, 2013). A distinct natural motif of polyphenol glycosylation is that of the di-C-glycoside. ...
Polyphenolic aglycones featuring two sugars individually attached via C‐glycosidic linkage (di‐C‐glycosides) represent a rare class of plant natural products with unique physicochemical properties and biological activities. Natural scarcity of such di‐C‐glycosides limits their use‐inspired exploration as pharmaceutical ingredients. Here, we show a biocatalytic process technology for reaction‐intensified production of the di‐C‐β‐glucosides of two representative phenol substrates, phloretin (a natural flavonoid) and phenyl‐trihydroxyacetophenone (a phenolic synthon for synthesis), from sucrose. The synthesis proceeds via an iterative two‐fold C‐glycosylation of the respective aglycone, supplied as inclusion complex with 2‐hydroxypropyl β‐cyclodextrin for enhanced water solubility of up to 50 mmol/L, catalyzed by a kumquat di‐C‐glycosyltransferase (di‐CGT), and it uses UDP‐Glc provided in situ from sucrose by a soybean sucrose synthase, with catalytic amounts (≤3 mol%) of UDP added. Time course analysis reveals the second C‐glycosylation as rate‐limiting (0.4–0.5 mmol/L/min) for the di‐C‐glucoside production. With internal supply from sucrose keeping the UDP‐Glc at a constant steady‐state concentration (≥50% of the UDP added) during the reaction, the di‐C‐glycosylation is driven to completion (≥95% yield). Contrary to the mono‐C‐glucoside intermediate which is stable, the di‐C‐glucoside requires the addition of reducing agent (10 mmol/L 2‐mercaptoethanol) to prevent its decomposition during the synthesis. Both di‐C‐glucosides are isolated from the reaction mixtures in excellent purity (≥95%), and their expected structures are confirmed by NMR. Collectively, this study demonstrates efficient glycosyltransferase cascade reaction for flexible use in natural product di‐C‐β‐glucoside synthesis from expedient substrates.
... The sweetness of mogrosides differ depending on the number of glycosylation and glycosylation sites 21 . The great advantage of mogroside V is its superb flavour compared with that of steviosides, rubusoside and glycyrrhizin, which normally are slightly bitter tasting sweet 22,23 . Interestingly, this natural sweetener with antiglycation effects 24 has a high sweetness, has a low-calorie content and is nontoxic; its sweetness is approximately 300 times higher than that of sucrose 25,26 ; and its history of medicinal use is >300 years 27 . ...
Mogrosides are widely used as high-value natural zero-calorie sweeteners that exhibit an array of biological activities and allow for vegetable flavour breeding by modern molecular biotechnology. In this study, we developed an In-fusion based gene stacking strategy for transgene stacking and a multi-gene vector harbouring 6 mogrosides biosynthesis genes and transformed it into Cucumis sativus and Lycopersicon esculentum. Here we show that transgenic cucumber can produce mogroside V and siamenoside I at 587 ng/g FW and 113 ng/g FW, respectively, and cultivated transgenic tomato with mogroside III. This study provides a strategy for vegetable flavour improvement, paving the way for heterologous biosynthesis of mogrosides.
... In our study, protein WP_023856884.1 has the catalytic domain of the Six-hairpin glycosidase superfamily. To use this class of enzymes in different industrial conditions several enzymes functional in alkaline/ acidic pH and/or at high temperatures have been discovered from various microorganisms [103][104][105]. In several studies, bacterial glycosidases were characterized to improve human health and the treatment of different diseases [106,107]. ...
Members of the Bacillus genus are industrial cell factories due to their capacity to secrete significant quantities of biomolecules with industrial applications. The Bacillus paralicheni-formis strain Bac84 was isolated from the Red Sea and it shares a close evolutionary relationship with Bacillus licheniformis. However, a significant number of proteins in its genome are annotated as functionally uncharacterized hypothetical proteins. Investigating these pro-teins' functions may help us better understand how bacteria survive extreme environmental conditions and to find novel targets for biotechnological applications. Therefore, the purpose of our research was to functionally annotate the hypothetical proteins from the genome of B. paralicheniformis strain Bac84. We employed a structured in-silico approach incorporating numerous bioinformatics tools and databases for functional annotation, physicochemical characterization, subcellular localization, protein-protein interactions, and three-dimensional structure determination. Sequences of 414 hypothetical proteins were evaluated and we were able to successfully attribute a function to 37 hypothetical proteins. Moreover, we performed receiver operating characteristic analysis to assess the performance of various tools used in this present study. We identified 12 proteins having significant adaptational roles to unfavorable environments such as sporulation, formation of biofilm, motility, regulation of transcription, etc. Additionally, 8 proteins were predicted with biotechnological potentials such as coenzyme A biosynthesis, phenylalanine biosynthesis, rare-sugars biosynthesis, antibiotic biosynthesis, bioremediation, and others. Evaluation of the performance of the tools showed an accuracy of 98% which represented the rationality of the tools used. This work shows that this annotation strategy will make the functional characterization of unknown proteins easier and can find the target for further investigation. The knowledge of these hypothetical proteins' potential functions aids B. paralicheniformis strain Bac84 in effectively creating a new biotechnological target. In addition, the results may also facilitate a better understanding of the survival mechanisms in harsh environmental conditions.
... The mechanisms of glycosyl hydrolase catalysis of two amino acid residues are classified into two categories: a) inverting mechanism and b) retaining mechanism (McCarter & Stephen Withers, 1994). In the former, β to α anomeric transition occurs through a single displacement, while anomeric carbon remains in the same position due to double displacement in the case of the retaining mechanism (Thuan & Sohng, 2013). However, the position of the proton donor in both mechanisms remains unchanged. ...
... In the process of inverting mechanism, single displacement takes place by shifting the anomeric position from β to α, catalytic base is placed at a certain distance from the anomeric position to hold the molecules of water that are found between the base and molecule of sugar without changing proton donor's position. Intermediates such as epoxides and oxacarbenium ions are observed while the mechanism occurs (Sobala et al., 2020) whereas in the retaining process, double-displacement mechanism involves retaining the anomeric carbons in the same position without any change in proton donor's position within a distance in which it can form hydrogen bond as it involves no water molecules resulting in retaining in the surrounding region of the anomeric position of the sugar molecule (Thuan and Sohng, 2013) (Henrissat and Davies, 1997). Cellulase is classified as endo-1,4β -D-glucanases, cellobiohydrolase and β-glucosidases. ...
Every year, 180 billion tonnes of cellulose are produced by plants as waste biomass after the cultivation of the desired product. One of the smart and effective ways to utilize this biomass rather than burn it is to utilize the biomass to adequately meet the energy needs with the help of microbial cellulase that can catalytically convert the cellulose into simple sugar units. Marine actinobacteria is one of the plentiful gram-positive bacteria known for its industrial application as it can produce multienzyme cellulase with high thermal tolerance, pH stability and high resistant towards metal ions and salt concentration, along with other antimicrobial properties. Highly stable cellulase obtained from marine actinobacteria will convert the cellulose biomass into glucose, which is the precursor for biofuel production. This review will provide a comprehensive outlook of various strategic applications of cellulase from marine actinobacteria which can facilitate the breakdown of lignocellulosic biomass to bioenergy with respect to its characteristics based on the location/environment that the organism was collected and its screening strategies followed by adopted methodologies to mine the novel cellulase genome and enhance the production, thereby increasing the activity of cellulase continued by effective immobilization on novel substrates for the multiple usage of cellulase along with the industrial applications.
... To date, two widely accepted types of phenolic GTs are O-and C-GTs, which attach a glycosidic moiety to the oxygen (O) and carbon (C) of aglycone, respectively. 12,33,36 Different types of taxifolin glycosides have been isolated from plants ( Figure 2). ...
Taxifolin (dihydroquercetin) and its derivatives are medicinally important flavanonols with wide distribution in plants. These compounds have been isolated from various plants, such as milk thistle, onions, french maritime, and tamarind. In general, they are commercially generated in semi‐synthetic forms. Taxifolin and related compounds are biosynthesized via phenylpropanoid pathway, and most of the biosynthetic steps have been functionally characterized. The knowledge gained through the detailed investigation of their biosynthesis has provided the foundation for the reconstruction of biosynthetic pathways. Plant and microbial based platforms are utilized for the expression of such pathways for generating taxifolin‐related compounds, either by whole cell biotransformation, or through reconfiguration of the genetic circuits. In this review, we summarize recent advances in the biotechnological production of taxifolin and its derivatives. This article is protected by copyright. All rights reserved
... Glycosylation represents one of the critical tailoring reactions in nature for modification of small organic molecules, generating structural diversity of natural products like antibiotics, alkaloids and plant polyphenols (Thibodeaux, Melancon, and Liu 2007;Thuan and Sohng 2013). Importantly, the glycosylation of natural products may improve their physicochemical and biological activities (Griffith, Langenhan, and Thorson 2005;Kren and Martinkova 2001). ...
... The chemical synthesis of polyphenol glycosides involves multiple activation and protection steps and generally shows poor stereoselectivity, resulting in low yields (Desmet et al. 2012). Enzymatic glycosylation using biocatalysts shows good stereoselectivity and catalytic efficiency under mild conditions, and requires no toxic chemicals (Moulis et al. 2021;Thuan and Sohng 2013). In nature, Leloir-glycosyltransferases (EC 2.4.1) with high stereoselectivity and broad product specificity catalyze the glucosylation of polyphenols (De Bruyn et al. 2015). ...
... However, these can be overcome by engineering microorganisms for in situ production of activated sugar-nucleotides and for the expression of Leloir-glycosyltransferases, and thus for polyphenol glucoside production, as reviewed in several recent papers (De Bruyn et al. 2015;Kim et al. 2015). At high substrate concentrations, some glycoside hydrolases such as α-amylase, α-glucosidase, α-galactosidase, β-glucosidase and cyclodextrin glucanotransferase, can also catalyze the in vitro glucosylation of polyphenols (Thuan and Sohng 2013;Slamova, Kapesova, and Valentova 2018). However, glycoside hydrolases suffer from their high hydrolysis activity, resulting in low yields. ...
Polyphenols exhibit various beneficial biological activities and represent very promising candidates as active compounds for food industry. However, the low solubility, poor stability and low bioavailability of polyphenols have severely limited their industrial applications. Enzymatic glycosylation is an effective way to improve the physicochemical properties of polyphenols. As efficient transglucosidases, glycoside hydrolase family 70 (GH70) glucansucrases naturally catalyze the synthesis of polysaccharides and oligosaccharides from sucrose. Notably, GH70 glucansucrases show broad acceptor substrate promiscuity and catalyze the glucosylation of a wide range of non-carbohydrate hydroxyl group-containing molecules, including benzenediol, phenolic acids, flavonoids and steviol glycosides. Branching sucrase enzymes, a newly established subfamily of GH70, are shown to possess a broader acceptor substrate binding pocket that acts efficiently for glucosylation of larger size polyphenols such as flavonoids. Here we present a comprehensive review of glucosylation of polyphenols using GH70 glucansucrase and branching sucrases. Their catalytic efficiency, the regioselectivity of glucosylation and the structure of generated products are described for these reactions. Moreover, enzyme engineering is effective for improving their catalytic efficiency and product specificity. The combined information provides novel insights on the glucosylation of polyphenols by GH70 glucansucrases and branching sucrases, and may promote their applications.
... Nature has already solved this issue, as resveratrol is mainly found in vegetal matrix in its β-Oglycoside form, also known as piceid. O-Glycosylation not only allows an increase in resveratrol water solubility and bioavailability, but it also prevents its oxidation and oligomerization [9,10]. Resveratrol O-glycoside derivatives can be obtained through chemical synthesis or enzymatic reaction. ...
The O-glycosylation of resveratrol increases both its solubility in water and its bioavailability while preventing its oxidation, allowing a more efficient use of this molecule as a bioactive ingredient in pharmaceutical and cosmetic applications. Resveratrol O-glycosides can be obtained by enzymatic reactions. Recent developments have made it possible to selectively obtain resveratrol α-glycosides from the β-cyclodextrin–resveratrol complex in water with a yield of 35%. However, this yield is limited by the partial hydrolysis of the resveratrol glycosides produced during the reaction. In this study, we propose to intensify this enzymatic reaction by coupling the enzymatic reactor to a membrane process. Firstly, membrane screening was carried out at the laboratory scale and led to the choice of a GE polymeric membrane with a cut-off of 1 kDa. This membrane allowed the retention of 65% of the β-cyclodextrin–resveratrol complex in the reaction medium and the transfer of 70% of the resveratrol α-O-glycosides in the permeate. In a second step, this membrane was used in an enzymatic membrane reactor and improved the yield of the enzymatic glycosylation up to 50%.
... Tailored glycosylation is an attractive method to enhance the bioavailability of polyphenolic compounds like flavonoids by increasing their aqueous solubility and protection from oxidation [6][7][8][9][10]. Glycosylation of polyphenolic compounds can be performed via chemical or enzymatic synthesis [9,11]. The chemical process requires glycosyl activation and multiple steps of protection/deprotection of hydroxyl groups to control the regio-selectivity; it is hazardous and generates toxic wastes [8,9]. ...
Phlorizin is a low soluble dihydrochalcone with relevant pharmacological properties. In this study, enzymatic fructosylation was approached to enhance the water solubility of phlorizin, and consequently its bioavailability. Three enzymes were assayed for phlorizin fructosylation in aqueous reactions using sucrose as fructosyl donor. Levansucrase (EC 2.4.1.10) from Gluconacetobacter diazotrophicus (Gd_LsdA) was 6.5–fold more efficient than invertase (EC 3.2.1.26) from Rhodotorula mucilaginosa (Rh_Inv), while sucrose:sucrose 1-fructosyltransferase (EC 2.4.1.99) from Schedonorus arundinaceus (Sa_1-SST) failed to modify the non-sugar acceptor. Gd_LsdA synthesized series of phlorizin mono- di- and tri-fructosides with maximal conversion efficiency of 73 %. The three most abundant products were identified by ESI-MS and NMR analysis as β-D-fructofuranosyl-(2→6)-phlorizin (P1a), phlorizin-4'-O-β-D-fructofuranosyl-(2→6)-D-fructofuranoside (P2c) and phlorizin-4-O-monofructofuranoside (P1b), respectively. Purified P1a was 16 times (30.57 g L⁻¹ at 25 °C) more soluble in water than natural phlorizin (1.93 g L⁻¹ at 25 °C) and exhibited 44.56 % free radical scavenging activity. Gd_LsdA is an attractive candidate enzyme for the scaled synthesis of phlorizin fructosides in the absence of co-solvent.
