Enzymatic synthesis of piceid glycosides by cyclodextrin glucanotransferase

doi:10.1016/j.procbio.2011.11.012

Process Biochemistry

Volume 47, Issue 3, March 2012, Pages 528-532

https://doi.org/10.1016/j.procbio.2011.11.012 Get rights and content

Abstract

Novel glycosides of piceid (3,4′-5-trihydroxy stilbene 3-O-β-d-glucoside) were produced by the transglycosylation reactions of cyclodextrin glucanotransferase (CGTase) from Bacillus macerans, with piceid (PicG₁) and maltodextrin as the acceptor and donor substrates, respectively. The reactions were performed at 40 °C with 2.56 mM piceid (0.1% w/v) and maltodextrin (5% w/v) in 0.02 M citrate phosphate buffer, pH 6.0 containing 5% (v/v) methanol for 6 h. Glucose, maltose, sucrose, maltotriose and α-cyclodextrin (α-CD) were also used to analyze their ability to function as donor substrates, for the glycosylation of piceid. Among the different donor substrates used, the maximum transfer efficiency (TE) of glycosylation of piceid was observed for α-cyclodextrin (78.9%) followed by maltodextrin (72.1%). The partially purified piceid glycoside products (PicG₂ and PicG₃) were identified by mass spectrometry.

Highlights

► CGTase from Bacillus macerans utilizes piceid as glycosyl acceptor to produce piceid glycosides with 2–15 glucose units, by transglycosylation reactions. ► α-Cyclodextrin and maltodextrin are efficient glycosyl donors. ► High transfer efficiencies obtained with α-cyclodextrin (78.9%) and maltodextrin (72.1%), respectively.

Introduction

Stilbenoids are polyphenolic secondary metabolites produced in several plant families. They possess antioxidant [1] and anticancer properties [2]. Grapes and grape products constitute the major source of stilbenoids in the human diet [3].

Piceid (3,4′-5-trihydroxy stilbene 3-O-β-d-glucoside or resveratrol 3-O-β-d-glucoside) is the main component of Polygonum cuspidatum root, and is widely used in Japanese and Chinese folk medicine for the treatment of cardiac ailments such as atherosclerosis and inflammation. Piceid has been shown to inhibit platelet aggregation [4] and lower serum cholesterol levels [5]. Although trans-piceid has been shown to reduce elevated lipid levels, it is less active than its aglycon trans-resveratrol [6].

The absorption of polyphenols from the diet is enhanced by conjugation with glucose [7]. The increased hydrophilicity, resulting from conjugation with glucose is presumed to make the absorption of piceid and its glycosides more efficient than its aglycon, trans-resveratrol. Moreover, piceid can be hydrolyzed in the digestive tract by glucosidases produced by human intestinal bacteria to release the aglycon [8].

Cyclodextrin glucanotransferase from Bacillus macerans is a member of the α-amylase family of glycoside hydrolases. It operates through a double displacement mechanism, retaining the anomeric configuration of the acceptor substrate. CGTase catalyzes four different reactions, namely cyclization, coupling, disproportionation and hydrolysis. Cyclodextrins (CD's) are formed via intramolecular transglycosylation or cyclization reactions and in coupling reactions, the CD's are cleaved and joined to a second sugar molecule or glycoside or sometimes even to a non-carbohydrate acceptor. CGTases can also transfer the covalently bound oligosaccharides to a second sugar molecule by disproportionation or to water by hydrolysis [9], [10]. Torres et al. [11] have recently reported the synthesis of α-glycosyl derivatives of resveratrol by CGTase. The present study deals with the synthesis of novel glycosides of piceid with different degree of polymerization (DP) using CGTase from B. macerans.

Section snippets

Materials

CGTase from B. macerans was obtained from Amano Enzyme Europe Ltd. (Milton Keynes, UK). β-Amylase [(1,4)-α-d-glucan maltohydrolase] from soybean was obtained from TCI, Europe (Zwijndrecht, Belgium). Piceid (PicG₁) was procured from Baoji Herbest Co. (Shaanxi, China). α-Cyclodextrin, maltose and maltotriose were obtained from Sigma–Aldrich Co. (Munich, Germany). Maltodextrin [dextrose equivalent (DE) 5–7 and DP ∼15–16] was supplied by Lyckeby Culinar AB (Fjälkinge, Sweden). All the other

Glycosylation reactions of piceid

Chromatographic analysis of the control reaction mixtures showed no product formation in the absence of enzyme. In enzyme-catalyzed reactions, CGTase utilized piceid (PicG₁) as acceptor substrate and maltodextrin as donor substrate, to produce a mixture of piceid glycoside products with 2–14 glucose units (PicG₂–PicG₁₄). Due to higher hydrophilicity, the products (PicG₂–PicG₁₄), eluted earlier than PicG₁ in HPLC (Fig. 1a–c). After partial purification, two of the products were identified as

Conclusion

CGTase from B. macerans has been shown to utilize piceid as an acceptor substrate to produce a mixture of piceid glycosides ranging in DP from 2 to 15. It was possible to achieve product yields up to 78.9% and 72.1% with α-CD and maltodextrin as donor substrates at pH 6 in 6 h. Anomer-selective enzymatic synthesis of piceid glycosides in a one pot reaction under mild conditions provide significant process advantages over conventional organic synthesis, which requires elaborate

Acknowledgements

The first author acknowledges Erasmus Mundus External Co-operation Window 15 for the scholarship and the Department of Biotechnology, Lund University, Sweden for providing the research facilities.

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Quantitative structure-antioxidant activity relationship of trans-resveratrol oligomers, trans-4,4′-dihydroxystilbene dimer, trans-resveratrol-3-O-glucuronide, glucosides: trans-piceid, cis-piceid, trans-astringin and trans-resveratrol-4′-O-β-d-glucopyranoside
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Cyclodextrin glucanotransferase (CGTase) catalyzed synthesis of dodecyl glucooligosides by transglycosylation using α-cyclodextrin or starch
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Engineering of cyclodextrin product specificity and pH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1
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Substrate complexation and aggregation influence the cyclodextrin glycosyltransferase (CGTase) catalysed synthesis of alkyl glycosides
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Anticancer activity of resveratrol on implanted human primary gastric carcinoma cells in nude mice
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F. Orsini et al.

Isolation, synthesis, and antiplatelet aggregation activity of resveratrol 3-O-d-glucopyranoside and related compounds

J Nat Prod

(1997)

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Resveratrol and cyclodextrins, an easy alliance: Applications in nanomedicine, green chemistry and biotechnology
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Citation Excerpt :
A primary nucleophilic attack of the 4’-hydroxyphenyl group of piceid (picG1) on the Carbon 1 at the reducing end of an opened α-CD, α-maltohexaose, results in an α-(1,4) linkage between piceid and the maltohexaose leading to the release of an initial coupling product called picG7. Following successive disproportionation reactions of picG7, various glucoside derivatives of picG1 like picG2, picG3, etc…are then obtained (Mathew et al., 2012). This mechanism may explain not only the recovery of monoglucosides but also of di and tri-glucosides during resveratrol glucosylation in the presence of CGTases (see below) (Ioannou et al., 2021; Marié et al., 2018; Torres et al., 2011).
Most drugs or the natural substances reputed to display some biological activity are hydrophobic molecules that demonstrate low bioavailability regardless of their mode of absorption. Resveratrol and its derivatives belong to the chemical group of stilbenes; while stilbenes are known to possess very interesting properties, these are limited by their poor aqueous solubility as well as low bioavailability in animals and humans. Among the substances capable of forming nanomolecular inclusion complexes which can be used for drug delivery, cyclodextrins show spectacular physicochemical and biomedical implications in stilbene chemistry for their possible application in nanomedicine. By virtue of their properties, cyclodextrins have also demonstrated their possible use in green chemistry for the synthesis of stilbene glucosylated derivatives with potential applications in dermatology and cosmetics. Compared to chemical synthesis and genetically modified microorganisms, plant cell or tissue systems provide excellent models for obtaining stilbenes in few g/L quantities, making feasible the production of these compounds at a large scale. However, the biosynthesis of stilbenes is only possible in the presence of the so-called elicitor compounds, the most commonly used of which are cyclodextrins. We also report here on the induction of resveratrol production by cyclodextrins or combinatory elicitation with methyljasmonate in plant cell systems as well as the mechanisms by which they are able to trigger a stilbene response. The present article therefore discusses the role of cyclodextrins in stilbene chemistry both at the physico-chemical level as well as the biomedical and biotechnological levels, emphasizing the notion of "easy alliance" between these compounds and stilbenes.
Biosynthesis of methyl glucoside and its antibacterial activity against Staphylococcus aureus and Escherichia coli
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This transglycosylation reaction, transferring from donor to acceptor, proceeds via the major catalytic machinery of enzymes for the synthesis of new glycosidic bonds (Mosi, He, Uitdehaag, Dijkstra, & Withers, 1997). Thus, from this mechanism, CGTase also catalyzes transglycosylation to various compounds other than saccharides, such as piceid (Mathew, Hedström, & Adlercreutz, 2012) and ascorbic acid (Aga, Yoneyama, Sakai, & Yamamoto, 1991). Mathew et al. (2012) have reported the efficient synthesis of piceid glycosides through disproportionation catalyzed by CGTase from Bacillus macerans using maltodextrin as a glycosyl donor and piceid as a glycosyl acceptor.
In this study, the methyl glucosides (MG_n) was synthesized using β-cyclodextrin (β-CD) and methanol through the coupling reaction of recombinant cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans A11. The optimal condition for the synthesis of MG_n consisted of an incubation of 1.5% (w/v) β-CD and 500 U/mL of CGTase in a water/methanol solution containing 30% (v/v) methanol at pH 6.0, 50 °C for 120 h. Upon analysis by TLC, at least three MG products were observed. The molecular weight of the main transferred product was 217.08 Da; this value was in accordance with methyl monoglucoside (MG₁). MG₁ was produced and prepared on a large scale and subsequently purified by preparative TLC. The combined ¹H- and ¹³C-NMR analysis confirmed that the structure of MG₁ was methyl-α-d-glucopyranoside. In addition, MG₁ showed emulsification activity and stability in its formation in water and n-hexadecane. The antibacterial activity of MG₁ was also determined by agar disc diffusion method. The results found that the MG₁ (1, 5 and 10 mg/disc) showed antibacterial activity against E. coli ATCC 25922 only, with inhibition zones of 28, 38 and 40 mm, respectively. The MIC values (mg/mL) of MG₁ against S. aureus ATCC 25923 and E. coli ATCC 25922 were found to be 20.00 and 0.63, while MBC values (mg/mL) were 40.00 and 0.63, respectively.
Characterization of cyclodextrin glycosyltransferases (CGTases) and their application for synthesis of alkyl glycosides with oligomeric head group
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Cyclodextrin glycosyltransferases (CGTases) from Paenibacillus macerans, Thermoanaerobacter sp. ATCC 53627, Bacillus stearothermophilus and a Carboxydocella sp. (phylogenetically identified from genomic DNA) were characterized with respect to their catalytic activity in different reactions, with emphasis on reactions useful for the elongation of the carbohydrate group of alkyl glycosides. All CGTases had activities between 95 and 115 U/mg in the coupling reaction between α-cyclodextrin (α-CD) as glucosyl donor and β-dodecyl maltoside as glucosyl acceptor, but differed very much in the competing hydrolysis of α-CD. The α-CD hydrolysis activity ranged from 0.13 U/mg for P. macerans CGTase to 10.5 U/mg for the Carboxydocella sp. (CspCGT13). Furthermore, the disproportionation activity was much lower for the Paenibacillus CGTase compared to the other CGTases, and consequently this enzyme produced the highest yield of the primary coupling product β-dodecyl maltooctaoside, which is a valuable surfactant. For production of a polydisperse alkyl glycoside product, disproportionation reactions are useful and the other three CGTases of the current study are efficient catalysts. The newly discovered Carboxydocella sp. (CspCGT13) CGTase has the special feature to produce more of products with even longer carbohydrate groups than the primary coupling product.
Novel insight into the secretory expression of recombinant enzymes in Escherichia coli
2014, Process Biochemistry
The secretory expression of recombinant enzymes in Escherichia coli has generally been a challenging task. In the present study, we investigated the expression of the extracellular enzyme cyclodextrin glycosyltransferase in E. coli. Our results indicated that when the overexpressed pre-proteins were not translocated across the inner membrane in a timely manner, they aggregated near the inner side of the E. coli inner membrane, resulting in the formation of insoluble inclusion bodies, which eventually blocked the pre-protein translocation channels and subsequently impeded further protein secretion. This mechanism suggests that for the efficient production of extracellular enzymes in E. coli, it is very important to maintain a balance between the rate of pre-protein synthesis and translocation, which can be achieved by altering the cultivation process. Our findings provide novel insight into the secretory expression of extracellular enzymes and may shed light on the further development of new strategies for extracellular protein production in E. coli.
Regioselective glycosylation of hydroquinone to α-arbutin by cyclodextrin glucanotransferase from Thermoanaerobacter sp.
2013, Biochemical Engineering Journal
Citation Excerpt :
At higher donor concentrations longer glycosides are formed more. Similar results have been reported in our previous works [23,24]. A high maltodextrin/HQ ratio favors maximum formation of HQGn and in turn other disproportionation products like HQG1–HQG7.
Hydroquinone glycosides were produced by transglycosylation reactions catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. (Toruzyme^® 3.0L). The reactions were carried out in an aqueous system containing hydroquinone (HQ) and maltodextrin as acceptor and donor substrate molecules respectively. The conditions for the synthesis of hydroquinone glucoside (α-arbutin) were 9 mM hydroquinone, maltodextrin (5%, w/v) in 20 mM citrate phosphate buffer, pH 5.5 and 0.025 mg/ml toruzyme at 40 °C for 24 h. The transfer efficiency of hydroquinone glycosylation was 31.8% and 29.2% respectively, when α-cyclodextrin and maltodextrin were employed as donor substrates. The major glycoside product was identified as hydroquinone-1-O-α-d-glucopyranoside (α-arbutin) on the basis of mass spectrometric, nuclear magnetic resonance analysis and component analysis of its enzymatic hydrolysates. The highest molar yield of α-arbutin (21.2%) was obtained when α-cyclodextrin was used as the donor substrate. A two step enzymatic reaction system comprising of CGTase and amyloglucosidase helped to attain a molar yield of 30% for α-arbutin. At room temperature the solubility of α-arbutin in water was determined to be 12.8 g/100 ml which is approximately 1.8 fold higher than that of hydroquinone.
Metabolic Engineering And In Vitro Biosynthesis Of Phytochemicals And Non-Natural Analogues
2013, Plant Science
Citation Excerpt :
However, further glycosylation reactions of piceid can take place at higher yields. The glycosylation of piceid using glucose, maltose, sucrose, maltotriose and α-cyclodextrin as donor substrates and obtained high yields with α-cyclodextrin (79%) and maltodextrin (72.1%) [123]. Piceid and its glycosides are valuable compounds for the treatment of cardiac ailments such as atherosclerosis and inflammation.
Over the years, natural products from plants and their non-natural derivatives have shown to be active against different types of chronic diseases. However, isolation of such natural products can be limited due to their low bioavailability, and environmental restrictions. To address these issues, in vivo and in vitro reconstruction of plant metabolic pathways and the metabolic engineering of microbes and plants have been used to generate libraries of compounds. Significant advances have been made through metabolic engineering of microbes and plant cells to generate a variety of compounds (e.g. isoprenoids, flavonoids, or stilbenes) using a diverse array of methods to optimize these processes (e.g. host selection, operational variables, precursor selection, gene modifications). These approaches have been used also to generate non-natural analogues with different bioactivities. In vitro biosynthesis allows the synthesis of intermediates as well as final products avoiding post-translational limitations. Moreover, this strategy allows the use of substrates and the production of metabolites that could be toxic for cells, or expand the biosynthesis into non-conventional media (e.g. organic solvents, supercritical fluids). A perspective is also provided on the challenges for generating novel chemical structures and the potential of combining metabolic engineering and in vitro biocatalysis to produce metabolites with more potent biological activities.

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Process Biochemistry

Abstract

Highlights

Introduction

Section snippets

Materials

Glycosylation reactions of piceid

Conclusion

Acknowledgements

References (23)

Quantitative structure-antioxidant activity relationship of trans-resveratrol oligomers, trans-4,4′-dihydroxystilbene dimer, trans-resveratrol-3-O-glucuronide, glucosides: trans-piceid, cis-piceid, trans-astringin and trans-resveratrol-4′-O-β-d-glucopyranoside

Eur J Med Chem

Lipid lowering effects of polydatin from Polygonum cuspidatum in hyperlipidemic hamsters

Phytomedicine

The identification of flavonoids as glycosides in human plasma

FEBS Lett

Cyclodextrin glucanotransferase (CGTase) catalyzed synthesis of dodecyl glucooligosides by transglycosylation using α-cyclodextrin or starch

Carbohydr Polym

Engineering of cyclodextrin product specificity and pH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1

J Biol Chem

Substrate complexation and aggregation influence the cyclodextrin glycosyltransferase (CGTase) catalysed synthesis of alkyl glycosides

J Biotechnol

The transglycosylation reaction of cyclodextrin glucanotransferase is operated by a ping-pong mechanism

FEBS Lett

Characteristics of transglycosylation reaction of cyclodextrin glucanotransferase in the heterogenous enzyme reaction system using extrusion starch as a glucosyl donor

Enzyme Microb Technol

Anticancer activity of resveratrol on implanted human primary gastric carcinoma cells in nude mice

World J Gastroenterol

Analysis and quantification of trans resveratrol in wines from Alentejo region (Portugal)

Food Technol Biotechnol

Isolation, synthesis, and antiplatelet aggregation activity of resveratrol 3-O-d-glucopyranoside and related compounds

J Nat Prod

Cited by (18)

Resveratrol and cyclodextrins, an easy alliance: Applications in nanomedicine, green chemistry and biotechnology

Biosynthesis of methyl glucoside and its antibacterial activity against Staphylococcus aureus and Escherichia coli

Characterization of cyclodextrin glycosyltransferases (CGTases) and their application for synthesis of alkyl glycosides with oligomeric head group

Novel insight into the secretory expression of recombinant enzymes in Escherichia coli

Regioselective glycosylation of hydroquinone to α-arbutin by cyclodextrin glucanotransferase from Thermoanaerobacter sp.

Metabolic Engineering And In Vitro Biosynthesis Of Phytochemicals And Non-Natural Analogues

Short communicationEnzymatic synthesis of piceid glycosides by cyclodextrin glucanotransferase

Abstract

Highlights

Introduction

Section snippets

Materials

Glycosylation reactions of piceid

Conclusion

Acknowledgements

Eur J Med Chem

Phytomedicine

FEBS Lett

Carbohydr Polym

J Biol Chem

J Biotechnol

FEBS Lett

Enzyme Microb Technol

Anticancer activity of resveratrol on implanted human primary gastric carcinoma cells in nude mice

World J Gastroenterol

Analysis and quantification of trans resveratrol in wines from Alentejo region (Portugal)

Food Technol Biotechnol

Isolation, synthesis, and antiplatelet aggregation activity of resveratrol 3-O-d-glucopyranoside and related compounds

J Nat Prod

Short communication
Enzymatic synthesis of piceid glycosides by cyclodextrin glucanotransferase