Cyclodextrin Glucanotransferase Research Articles

Valorization of Potato Peels Starch for Efficient β‐Cyclodextrin Production and Purification through an Eco‐Friendly Process

AbstractPotato peels (PP) are agro‐industrial co‐products generated in huge quantities by the potato processing industry. Obtaining value‐added products from PP is a current research challenge. As cyclic oligosaccharides is produced from starch by the action of cyclodextrin glucanotransferase (CGTase), β‐cyclodextrins (β‐CDs) are increasingly used in various industrial applications. In an attempt to develop a low‐cost process for β‐CDs production, starch is firstly extracted from PP. The productions of the US132 CGTase and β‐CDs are then optimized via experimental designs. The US132 CGTase production from the recombinant T7 express Escherichia coli strain is enhanced 20‐fold to reach 59.95 U mL−1. Subsequently, the β‐CDs production by the US132 CGTase is optimized to reach a high level (67.26 g L−1). The β‐CDs are then purified to homogeneity through a simple eco‐friendly crystallization method that provided a purification yield of 20%. This study reveals that β‐CDs production from valorized starch is a promising strategy used to valorize PP and minimize β‐CDs production cost.

Preparation and characterization of a novel 3D polymer support for the immobilization of cyclodextrin glucanotransferase and efficient biocatalytic synthesis of α-arbutin

In macropores of a melamine sponge epoxy resin was cured in ethanol solution. The produced polymer gel could uniformly deposit on the surface of melamine in either porous or nonporous morphology. The composite sponge with porous coating can be used as a large-sized and well-mass transferred support for the immobilization of cyclodextrin glucosyltransferase (CGTase, from Thermoanaerobacter sp.) through the method of adsorption and crosslinking, and a column reactor was made for the preparation of α-arbutin in a sealed circulation way. The porosity and specific surface area were 91.6 % and 46 m2/g, respectively. The loading amount and the specific activity of immobilized CGTase under the optimal immobilization conditions were 42 mg/gsupport and 651 U/gsupport, respectively. 5 mg/ml maltodextrin and 10 mM hydroquinone were used as a glucosyl donor and acceptor for the biosynthesis of α-arbutin, respectively. Under optimized conditions, the α-arbutin yield reached 61 % within 24 h at 40 °C. After 10 cycles, the immobilized CGTase retained 33 % of the α-arbutin yield.

Cyclodextrins produced by cyclodextrin glucanotransferase mask beany off-flavors in plant-based meat analogs.

The widening gap between the supply and demand for meat products has increased the need to produce plant-based meat analogs as protein sources. Meat analogs are principally composed of soy-based textured vegetable proteins. Despite ongoing technical developments, one of the unresolved challenges for plant-based meat analogs is the off-flavor from soy, which limits their consumer acceptability. Among the various methods developed for overcoming this challenge, masking the beany flavors with cyclodextrins (CDs) is an attractive, cost-effective, and safe strategy. However, the current established CD treatment method does not meet the requirement for a clean-label. This study aimed to develop more acceptable off-flavor-masking technologies for plant-based patties for modern clean-label preferences using enzymatic methods. We used the cyclodextrin glucanotransferase (CGT), “Amano,” as a commercially available food-grade CGT. The CGT-catalyzed reaction in plant-based patties yielded 17.1 g/L CD. As CGT could yield sufficient CD in the patties, we investigated whether CDs produced by CGT could mask the off-flavors released from the plant-based patties. The CGT-treated patties had significantly lower volatilization amounts of the known beany off-flavor-generating compounds compared to the non-treated patties. Moreover, CGT treatment improved the texture of the patties and increased their water- and oil-holding capacity. As CGT is rendered inactive after cooking, it would not be considered an additive. These findings indicated that CDs produced by the CGT reaction could effectively mask off-flavors of meat analogs and improve their physical properties while meeting clean-label requirements.

Permeabilized whole-cell biocatalyst containing co-expressed two enzymes facilitates the synthesis of maltoheptaose (G7) from starch

Maltoheptaose (G7) is one of the mixtures of maltodextrin widely used in the food, pharmaceutical, and cosmetics industries. A genetically engineered strain, which simultaneously expressed cyclodextrin glucanotransferase (CGTase) from Gracilibacillus alcaliphilus SK51.001 and cyclomaltodextrinase (CDase) from Bacillus sphaericus E-244, two enzymes, was constructed by cloning the above two genes into a plasmid and transformed into the host Escherichia coli BL21(DE3) (E. coli) strain, resulted in recombinant cells harboring the vector pETDuet-GaCGT/BsCD (pGaBs). These cells were used as whole-cell catalysts for the biotransformation of G7 from the inexpensive substrate (starch). Due to the high molecular weight of starch, the cell membrane prevents the entry of starch into the cellular system. Therefore, the pGaBs cell wall was permeabilized by lysozyme, EDTA, and heat treatment. After reaching the optimized conditions of permeabilized pGaBs cell amount, lysozyme amount, reaction temperature, and metal ion concentration, approximately 4.1 g/L of G7 was produced from 30 g/L starch in 1 h with the addition of Ca2+. This co-expression system offers a one-pot synthesis approach to the production of G7 using an inexpensive substrate, avoiding enzyme purification steps.

Development of a method for preparing cyclic nigerosylnigerose syrup and investigation of its value as a dietary fiber.

Cyclic nigerosylnigerose (CNN) syrup, containing 76% water-soluble dietary fiber, was prepared from starch on an industrial scale, using isoamylase, 6-α-glucosyltransferase, 3-α-isomaltosyltransferase, and cyclodextrin glucanotransferase. CNN syrup has a unique linkage pattern, consisting mainly of α-1,3 and α-1,6 glucoside linkages, and is characterized by its low weight average molecular weight (807) and moderate sweetness (relative sweetness=25), unlike in well-known dietary fiber materials. The glass transition temperature of CNN is higher than that of the straight chain structures, maltotetraose and maltosyltrehalose. Even when 40% of normally added sucrose was replaced with CNN syrup, sponge cake puffed up sufficiently. The no observed adverse effect level for a single dose of CNN syrup was 0.88 and 0.89 g dry solid/kg body weight for men and women, respectively. The increase in blood glucose and insulin concentrations during consumption of CNN syrup was lower than that of glucose.

Molecular cloning, and optimized production and characterization of recombinant cyclodextrin glucanotransferase from Bacillus sp. T1.

The online version contains supplementary material available at 10.1007/s13205-022-03111-8.

Highlight on mutations affecting the US132 cyclodextrin glucanotransferase binding specificity, thermal stability, and anti-staling activity

We have already reported that the triple mutant (K47E-S382P-N655S of Paenibacillus pabuli US132 cyclodextrin glucanotransferase US132 (CGTase)) altered the CGTase specificity. In the current study, the single (K47E, S382P and N655S) and double (K47E+S382P, K47E+N655S, and S382P+N655S) mutants were constructed to elucidate the synergic or antagonist substitutions effect on the enzyme behavior. For the six generated mutants, an improvement of the dextrinization/cyclization ratio from 4.4 to 6-fold was observed when compared to the wild-type enzyme. The mutations effect on enzyme specificity was not attributed to synergy modulation since the single mutant N655S had the highest ratio enhancement. Moreover, the mutant N655S revealed the highest β-cyclodextrin binding affinity with a high amount of hydrophobic bonds which might be contributed to the apparent decrease in the cyclization activity. On the other hand, mutations N655S, K47E, and (K47E-N655S) showed the same positive effect on thermal activity. The highest stability was attained at 70 °C by N655S to be 3.6-fold higher than the wild-type. The addition of N655S to wheat flour induced a decrease of dough and bread hardness and led to an increase in dough and bread cohesiveness and a rise in bread masticability values compared to the control. This mutant addition also corrected the dough elasticity decrease engendered by the wild-type CGTase indicating that N655S-CGTase could be an alternative anti-staling agent.

PH-Responsive templates modulate the dynamic enzymatic synthesis of cyclodextrins.

Product selection in the dynamic enzymatic synthesis of cyclodextrins can be controlled by changing the pH. Using cyclodextrin glucanotransferase to make labile the glycosidic linkages in cyclodextrins (CDs), we generate a dynamic combinatorial library of interconverting linear and cyclic α-1,4-glucans. Templates can be employed to favour the selective production of specific CDs and, herein, we show that by using ionisable templates, the synthesis of α-CD or β-CD can be favoured by simply changing the pH. Using 4-nitrophenol as the template, β-CD is the preferred product at low pH, while α-CD is the preferred product at high pH. Furthermore, a new methodology is described for the simulation of product distributions in dynamic combinatorial libraries with ionisable templates at any given pH.

Polyglucosylation of Rutin Catalyzed by Cyclodextrin Glucanotransferase from Geobacillus sp.: Optimization and Chemical Characterization of Products

Despite the presence of a rutinosyl group at 3-OH, the aqueous solubility of the flavonoid rutin is even lower than that of its aglycon quercetin. In this work, we describe a fast, simple, and easily scalable process for polyglucosylation of rutin to enhance aqueous solubility, catalyzed by a cyclodextrin glucanotransferase (CGTase). Several reaction parameters (source of enzyme, rutin/starch weight ratio, cosolvent, pH, and temperature) were assessed to optimize the transglucosylation yield. Under the best conditions (6 mg/mL rutin, 30 mg/mL soluble starch as glucosyl donor, 20% (v/v) acetonitrile, pH 9.2, 3.3 U/mL CGTase from Geobacillus sp., 60 °C), the total glucosides reached a maximum concentration of 6 mM (60% conversion yield). The glucosylated products were chemically characterized by MALDI-TOF mass spectrometry and 2D nuclear magnetic resonance. The glucosylation takes place with an α-configuration at the 4-OH position of the β-Glc moiety. A series of maltooligosyl derivatives with 1–6 residues of glucose linked by α(1 → 4) bonds was obtained. The yield of monoglucosylated product was increased 2-fold by treatment with amyloglucosidase STA1 from S. cerevisiae.

Enzymatic Synthesis of α-Tocopherol Derivative Glycoside, Daidzein Glycoside, Daidzein Oligosaccharide, Resveratrol Oligosaccharide, and Curcumin Oligosaccharides and Their Anti-Allergic Activity and Neuroprotective Activity

Enzymatic glycosylations of an α-tocopherol derivative, daidzein, resveratrol, and curcumin were investigated. The plant polyphenol, resveratrol, was incubated with glucosyltransferase from Phytolacca americana. The resveratrol glycoside obtained was then incubated with cyclodextrin glucanotransferase to obtain resveratrol oligosaccharide. Daidzein and curcumin were also converted into daidzein glycoside, daidzein oligosaccharide, and curcumin oligosaccharides. Also, α-tocopherol derivative, that is, 2, 5,7,8-tetramethyl-2-(4,8-dimethylnonyl)chroman-6-ol, was glycosylated. The glycosides and oligosaccharides had strong anti-allergic activity such as suppression of IgE formation, inhibition of histamine release, and inhibition of O2 - generation. In addition, the glycosides and oligosaccharides showed efficient neuroprotective activity by inhibition of phosphodiesterase.

Development of Freeze-Thaw Stable Starch through Enzymatic Modification.

The use of unmodified starch in frozen foods can cause extremely undesirable textural changes after the freeze-thaw process. In this study, using cyclodextrin glucanotransferase (CGTase) and branching enzymes, an amylopectin cluster with high freeze-thaw stability was produced, and was named CBAC. It was found to have a water solubility seven times higher, and a molecular weight 77 times lower, than corn starch. According to the results of a differential scanning calorimetry (DSC) analysis, dough containing 5% CBAC lost 19% less water than a control dough after three freeze-thaw cycles. During storage for 7 days at 4 °C, bread produced using CBAC-treated dough exhibited a 14% smaller retrogradation peak and 37% less hardness than a control dough, suggesting that CBAC could be a potential candidate for clean label starch, providing high-level food stability under repeated freeze-thaw conditions.

Effect of process parameters on immobilization of cyclodextrin glucanotranferase (CGTase) from Bacillus licheniformis on pineapple waste

Cyclodextrin glucanotransferase (CGTase) is capable of degrading starch to produce cyclodextrin (CD). CD production has increased over the years due to its applications in numerous industries like environmental engineering, food industries, and pharmaceutical industries. The enzyme immobilization approach was commonly implemented to overcome low CD production during the reaction process due to the instability and denaturation of CGTase. The objective of this study is to immobilize CGTase produced by Bacillus licheniformis on pineapple peel. The influence of process parameters on the CGTase immobilization process such as pH (5, 6, 7, 8, and 9), contact time (4, 8, 12, 16, 20, 24, and 28 hr), and temperature (20, 25, 30, 35 and 40 °C) was studied. pH 7 was the best pH for CGTase immobilization, with a 75.97% of immobilization yield. In addition, the optimal temperature that produced the high immobilization yield (76.80%) was at 25 ℃. Meanwhile, the optimum contact time was detected at 24 hr, with 75.53% of immobilized yield. In conclusion, the immobilization process could increase the enzyme stability and increase CD production, valuable for application in industries.

Highly efficient and low-cost process for synthesis of 2-O-α-d-glucopyranosyl-6-O-(2-propylpentanoyl)-l-ascorbic acid

2-O-α-d-Glucopyranosyl-6-O-(2-propylpentanoyl)-l-ascorbic acid (6-bOcta-AA-2G) has shown a remarkable antitumor effect in an in vivo study. However, an efficient and low-cost process for synthesis of 6-bOcta-AA-2G has not been established yet. The process used for synthesis of 6-bOcta-AA-2G in this study consisted of three steps. The first step was acylation of l-ascorbic acid (AA) at the C-6 position with 2-propylpentanoic acid in concentrated H2SO4 at room temperature for 24 h to obtain 6-O-(2-propylpentanoyl)-l-ascorbic acid (6-bOcta-AA). Next, specific glycosylation of 6-bOcta-AA at the C-2 position by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. was carried out using soluble starch as a low-cost glycosyl donor in acetate buffer (pH 6.0) at 40 °C for 24 h. In this glycosylation reaction by CGTase, oligoglucosylated 6-bOcta-AAs seemed to be generated, and thus these oligoglucosides were further hydrolyzed by amyloglucosidase from Rhizopus sp. at 40 °C for 1 h in the reaction mixture containing CGTase to efficiently obtain 6-bOcta-AA-2G. The overall yield of 6-bOcta-AA-2G was 19.4 %. The presented synthetic process can be used for mass production of 6-bOcta-AA-2G as an antitumor agent.

Chaotropic and Kosmotropic Anions Regulate the Outcome of Enzyme-Mediated Dynamic Combinatorial Libraries of Cyclodextrins in Two Different Ways.

We demonstrate how different anions from across the Hofmeister series can influence the behavior of enzyme-mediated dynamic combinatorial libraries of cyclodextrins (CDs). Using cyclodextrin glucanotransferase to catalyze reversible transglycosylation, dynamic mixtures of interconverting cyclodextrins can be formed wherein the relative concentrations of α-CD, β-CD and γ-CD is determined by their intrinsic stabilities and any stabilizing influences of added template (guest) molecules. Here, we find that addition of high concentrations of kosmotropic anions can be used to enhance the effects of added hydrophobic templates, while chaotropic anions can themselves act as templates, causing predictable and significant changes in the cyclodextrin composition due to weak, but specific, binding interactions with α-CD.

Dynamic enzymatic synthesis of γ-cyclodextrin using a photoremovable hydrazone template

Dynamic enzymatic synthesis of γ-cyclodextrin using a photoremovable hydrazone template

Combine strategy of treated activated charcoal and cell surface protein curli induction for enhanced performance in Escherichia coli immobilization

Immobilization of Escherichia coli (E. coli) on commercial activated charcoal was enhanced by mild chemical treatment coupled with curli production from E. coli. The chemical used to treat the activated charcoal were sodium hydroxide, hydrochloric acid, ammonium hydroxide, and acetic acid while nickel (II) chloride was used to promote the production of curli. Characteristics of the activated charcoal before and after chemical treatments were analyzed including its surface properties, pore size, and crystalline structure. The immobilization of E. coli was enhanced greatly after sodium hydroxide treatment which gave rise to more than 120 % cell immobilized compared to the untreated activated charcoal which was mainly attributed to the larger size of macropore, surface area, and pore volume. Curli were produced by the induction of nickel (II) chloride and further enhanced the cell immobilization by at least 50 %. Overall, the combine strategy enhanced cell immobilization by more than 160 %. The resulting biocatalyst from the enhanced cell immobilization managed to be reused up to 10 cycles for the enzyme cyclodextrin glucanotransferase expression while retaining up to 60 % of the enzyme’s initial activity.

Synthesis of novel oligomeric anionic alkyl glycosides using laccase/TEMPO oxidation and cyclodextrin glucanotransferase (CGTase)-catalyzed transglycosylation.

Modification of alkyl glycosides, to alter their properties and widen the scope of potential applications, is of considerable interest. Here, we report the synthesis of new anionic alkyl glycosides with long carbohydrate chains, using two different approaches: laccase/2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidation of a long-carbohydrate-chain alkyl glycosideand cyclodextrin glucanotransferase (CGTase)-catalyzed elongation of anionic alkyl glycosides. The laccase/TEMPO oxidation of dodecyl β- d-maltooctaoside proceeded efficiently with the formation of aldehyde and acid products. However, depolymerization occurred to a large extent, limiting the product yield and purity. On the other hand, CGTase-catalyzed coupling/disproportionation reactions with α-cyclodextrin and dodecyl β- d-maltoside diuronic acid (DDM-2COOH) or octyl β- d-glucuronic acid (OG-COOH) as substrates gave high conversions, especially when the CGTase Toruzyme was used. It was found that pH had a strong influence on both the enzyme activity and the acceptor specificity. With non-ionic substrates (dodecyl β- d-maltoside and octyl β- d-glucoside), Toruzyme exhibited high catalytic activity at pH 5-6, but for the acidic substrates (DDM-2COOH and OG-COOH) the activity was highest at pH 4. This is most likely due to the enzyme favoring the protonated forms of DDM-2COOH and OG-COOH, which exist at lower pH (pKa about 3).

A review on immobilization strategy of cyclodextrin glucanotransferase producing Escherichia coli as whole cell biocatalyst

This paper reviewed the immobilization strategy on cyclodextrin glucanotransferase (CGTase) producing Escherichia coli as whole cell biocatalyst. Biotransformation of insoluble substrate such as starch and lignocellulosic biomass with whole cell leads to the application of biological steps to chemocatalysis, which makes the whole process more effective. In this whole cell biocatalysis process, the excretion of the enzymes into the extracellular space by the cell is crucial. However, the use of free whole cell biocatalysts especially Escherichia coli often face with problem related to the cell lysis and stability. Due to that, the biotransformation process becomes inefficient. Thus, in this study the immobilization strategy was investigated to overcome the bottle necks mentioned. First, this study began with a focus in Escherichia coli, immobilization techniques, cyclodextrin, cyclodextrin glucanotransferase and recombinant Escherichia coli, in which can give a better understanding and a bigger picture in order to overcome the bottle necks mentioned. The different immobilization strategy and condition identified expected to improve the whole cell stability and reusability which lead to efficient biotransformation process. The output of the research also will elucidate the mechanism on how different immobilization material, strategy and cultural conditions improve the overall cell performance.

One-Pot Bi-Enzymatic Cascade Synthesis of Novel Ganoderma Triterpenoid Saponins

Ganoderma lucidum is a medicinal fungus whose numerous triterpenoids are its main bioactive constituents. Although hundreds of Ganoderma triterpenoids have been identified, Ganoderma triterpenoid glycosides, also named triterpenoid saponins, have been rarely found. Ganoderic acid A (GAA), a major Ganoderma triterpenoid, was synthetically cascaded to form GAA-15-O-β-glucopyranoside (GAA-15-G) by glycosyltransferase (BtGT_16345) from Bacillus thuringiensis GA A07 and subsequently biotransformed into a series of GAA glucosides by cyclodextrin glucanotransferase (Toruzyme® 3.0 L) from Thermoanaerobacter sp. The optimal reaction conditions for the second-step biotransformation of GAA-15-G were found to be 20% of maltose; pH 5; 60 °C. A series of GAA glucosides (GAA-G2, GAA-G3, and GAA-G4) could be purified with preparative high-performance liquid chromatography (HPLC) and identified by mass and nucleic magnetic resonance (NMR) spectral analysis. The major product, GAA-15-O-[α-glucopyranosyl-(1→4)-β-glucopyranoside] (GAA-G2), showed over 4554-fold higher aqueous solubility than GAA. The present study demonstrated that multiple Ganoderma triterpenoid saponins could be produced by sequential actions of BtGT_16345 and Toruzyme®, and the synthetic strategy that we proposed might be applied to many other Ganoderma triterpenoids to produce numerous novel Ganoderma triterpenoid saponins in the future.

Synergistic action of cyclodextrin glucanotransferase and maltogenic amylase improves the bioconversion of starch to malto-oligosaccharides

Overwhelming consumer consciousness for healthier food has led to high market demand for functional oligosaccharides. These functional sugars can be extracted from various sources, especially starch, through enzymatic approaches. Maltogenic amylases are able to synthesise malto-oligosaccharides (MOS) from starch. However, the use of maltogenic amylase alone presents several limitations such as low specificity towards starch and low productivity of MOS. Cyclodextrin glucanotransferase (CGTase) acts on starch to produce cyclodextrins. Herein, asynchronous and synchronous conversion strategies were used to enhance the MOS yield from soluble starch by exploiting maltogenic amylase and CGTase reactions. Factors including pH, temperature, enzyme loading and enzymes ratio that affect MOS production were investigated. The combination of CGTase and maltogenic amylase (MAG1) from Bacillus lehensis G1 produced the highest MOS yield (307.9 mg.g−1). The synchronous strategy exhibited a 2.1-fold higher MOS production compared to the asynchronous approach (146.8 mg.g−1). Kinetic studies revealed that the catalytic efficiency of the synchronous approach was ∼800-fold (1746.1 mL.g−1. h−1) higher than that of MAG1 alone (18.5 mL.g−1. h−1). The high production of MOS and catalytic efficiency of the reaction demonstrate that the combinatorial catalysis of MAG1 and CGTase is very promising and provides a foundation for industrial-scale production of MOS.