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The ability of γ-cyclodextrin(γ-CD)to form inclusion complexes with various guest compounds, together with its relatively high solubility, gives γ-CD advantages over both α-cyclodextrin(α-CD) and β-cyclodextrin(β-CD). The thermostability of γ-cyclodextrin glycosyltransferases (γ-CGTase) which can produce γ-CD discovered so far is poor, which is one of the problems limiting its industrial application. To enhance the thermostability of γ-CGTase from Bacillus clarkii 7364, potential disulfide bond mutants were predicted by Disulfide by Design. Five pairs of mutants were constructed by analyzing the predicted results. The t 1/2 of S172C-R183C was 2.25 h, which was 3.17 times of WT. Molecular dynamics simulations confirmed that the increase of thermostability of S172C-R183C may be due to the formation of disulfide bond, which leads to the increase of structure rigidity of protein. Then the ability of S172C-R183C mutant to produce cyclodextrins(CDs) was tested. Compared with WT, the maximum CD yield, γ-CD yield and γ-CD specificity of S172C-R183C in the non-complexant catalysed reaction were increased by 6.9 %, 8.8 %, and 2.4 % respectively. In the complexant catalyzed reaction, the CDs yield of S172C-R183C increased by 17.0 %, in which the γ-CD yield increased by 21.7 %, and the γ-CD specificity increased by 3.9 %. Molecular docking results showed that S172C-R183C formed more hydrogen bonds with γ-CD, especially Y186, which was the key central site affecting product specificity. This work involving rational protein engineering provided a new method for adjusting γ-CGTase thermostability. [Display omitted] • The thermostability of γ-CGTase was improved by disulfide bond engineering. • The t 1/2 of S172C-R183C was 3.17 times of WT. • The thermostability improvement mechanism was analyzed using MD simulation. • The yield of CDs and the proportion of γ-CD in CDs of S172C-R183C have increased. [ABSTRACT FROM AUTHOR]

Cyclodextrin glycosyltransferase (CGTase) catalyzes intermolecular transglycosylation through either disproportionation or cyclization‐coupling pathway. Kinetics analysis reveals that the hesperidin glycosylation process catalyzed by a CGTase variant (M1) is primarily accomplished through the disproportionation pathway. The cyclization‐coupling pathway exhibits a lower reaction rate and competitively consumes glycosyl donor and yield byproducts that impair disproportionation. Under the guidance of reaction kinetics, mutagenesis was targeted at residues in the −3, +1, and +2 subsites, known to control the selectivity between disproportionation and cyclization. A quadruple variant was identified with 2.9 times hesperidin glycosylation activity compared to M1, and 20.3 times compared to the wild‐type. Kinetic analysis reveals a fourfold improvement of kcat/KmA for disproportionation and an 85.5% reduction in kcat/Km for cyclization after mutagenesis. Binding free energy analysis further confirms that the mutagenesis favors the binding of hesperidin, and destabilizes the binding of cyclodextrin. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Food & Fermentation Industries is the property of Food & Fermentation Industries and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Large-ring cyclodextrin (LR-CD) is a cyclic α-1,4-glucan consisting of nine to several hundred glucopyranose units. LR-CD is mainly prepared by the cyclization activity of cyclodextrin glucosyltransferase or 4-α-glucanotransferase that catalyzes intramolecular transglycosylation reaction on amylose. Compared with common α-, β-, and γ-CD, LR-CD has a larger cavity size and exhibits unique structural and physicochemical properties, showing potential applications in several fields like food, medicine and biology. However, LR-CD has not been industrially produced or applied due to the high cost and low productivity as well as lack of effective separation and purification methods. In this article, recent progress and challenges on the enzymatic preparation of LR-CD are reviewed from the aspects of optimizing reaction substrates, designing new reaction pathways based on template induction, enzyme modification, and separation and purification of products. We hope this article could provide a reference for the industrial production and application of LR-CD.

Cyclodextrin glycosyltransferase (CGTase) can catalyze the glycosylation of hesperidin, resulting in α-glycosyl hesperidin with significantly improved water solubility. In this study, a rational design of CGTase to improve its hesperidin glycosylation activity was investigated. The strategy we employed involved docking hesperidin in its near-attack conformation and virtually mutating the surrounding residues, followed by calculating the changes in binding energy using Rosetta flex-ddG. The mutations with a stabilization effect were then subjected to an activity assay. Starting from CGTase-Y217F, we obtained three double-point mutants, Y217F/M351F, Y217F/M351L, and Y217F/D393H, with improved hesperidin glycosylation activities after screening twenty variants. The best variant, Y217F/D393H, exhibited a catalytic activity of 1305 U/g, and its kcat/KmA is 2.36 times higher compared to CGTase-Y217F and 15.14 times higher compared to the wild-type CGTase. Molecular dynamic simulations indicated that hesperidin was repulsed by CGTase-Y217F when bound in a near-attack conformation. However, by introducing a second-point mutation with a stabilization effect, the repulsion effect is weakened, resulting in a reduction in the distances between the bond-forming atoms and, thus, favoring the reaction. [ABSTRACT FROM AUTHOR]

In this paper, the cgt gene encoding cyclodextrin glycosyltransferase (CGTase) from Bacillus stearothermophilus was cloned into pWB980 plasmid for extracellular expression in Bacillus subtilis SCK6. Through adding a six-histidine affinity tag fused to the C-terminus, the recombinant CGTase could be purified by nickel ion affinity chromatography, and its molecular weight was approximately 76 kDa on SDS-PAGE. Then, the enzymatic properties were determined, and results were as follows: the optimum temperature and pH were identified as 40 ℃ and pH 5.0, respectively. CGTase had good tolerance to metal ions of Mn2+, Ca2+, and Mg2+. The enzyme activity was activated by Na+, Al3+, Fe3+, and Ni+, and it was remarkably inhibited by Cu2+ and Zn2+. To improve the aqueous solubility of rutin, CGTase was used to catalyze the transglycosylation reaction, and the conversion rate could reach as high as 80.13% under optimal conditions. Furthermore, the reaction mixture was treated with glucoamylase and microporous adsorbent resin. The yield of glycosyl-rutin was 56.1%, and its purity was 74.3%, which further improved the value of the product. [ABSTRACT FROM AUTHOR]

The food enzyme cyclomaltodextrin glucanotransferase ((1‐4)‐α‐d‐glucan:(1‐4)‐α‐d‐glucan 4‐α‐d‐[(1‐4)‐α‐d‐glucano]‐transferase; EC 2.4.1.19) is produced with the non‐genetically modified bacteria Anoxybacillus caldiproteolyticus strain TCM3‐539 by Hayashibara Co., Ltd. It is free from viable cells of the production strain. The food enzyme is intended to be used for the manufacture of glucosyl hesperidin and ascorbic acid 2‐glucoside. Since residual amounts of total organic solids are removed by filtration, adsorption, chromatography and crystallisation, dietary exposure estimation was considered not necessary. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and one match with a respiratory allergen was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that the food enzyme does not give rise to safety concerns under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Abstract The food enzyme cyclomaltodextrin glucanotransferase ((1‐4)‐α‐d‐glucan:(1‐4)‐α‐d‐glucan 4‐α‐d‐[(1‐4)‐α‐d‐glucano]‐transferase; EC 2.4.1.19) is produced with the non‐genetically modified bacteria Anoxybacillus caldiproteolyticus strain TCM3‐539 by Hayashibara Co., Ltd. It is free from viable cells of the production strain. The food enzyme is intended to be used for the manufacture of glucosyl hesperidin and ascorbic acid 2‐glucoside. Since residual amounts of total organic solids are removed by filtration, adsorption, chromatography and crystallisation, dietary exposure estimation was considered not necessary. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and one match with a respiratory allergen was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that the food enzyme does not give rise to safety concerns under the intended conditions of use.

Cinnamaldehyde, a yellowish essential oil, has potential applications because of its antibacterial ability but is limited by its instability. In this study, we prepared the microencapsulated cinnamaldehyde emulsion co-stabilized by cyclodextrin glycosyltransferase-catalyzed (CGTase-catalyzed) starch products and Tween 80. Microscopic structure analysis by transmission electron microscopy, inverted fluorescence microscopy, and size distribution determination revealed that additional CGTase-catalyzed starch products could increase their homogeneity and decrease their particle sizes by 67.8%. Fluorescence spectra analysis showed the interactions between cinnamaldehyde and the wall materials with a decrease of 29.2% in fluorescence intensity. The cinnamaldehyde emulsion had good temperature, ionic strength, pH, and storage stability. Time-kill curve and inhibition zone showed increased antibacterial activity. Mechanism analysis by thermal gravimetric analysis and differential scanning calorimetry showed that the emulsion had a dual structure of gel network and cyclodextrin inclusion, contributing to its stability. This study could give new insights into the cinnamaldehyde emulsion and provide a good guidance for its application. [Display omitted] • Cinnamaldehyde emulsion was stabilized by CGTase-catalyzed products. • CGTase-catalyzed products increased the homogeneity and decreased particle sizes. • The emulsion had better environmental stress stability and storage stability. • The emulsion had better antibacterial activity. • Gel network structure and cyclodextrin inclusion greatly enhanced the stability. [ABSTRACT FROM AUTHOR]

[Display omitted] • First study to evaluate the US effect on CGTase activity (activation up to 93 %). • Greater improvements were observed for activity measured at sub-optimal temperatures. • Sonication at high temperature (80 °C) and long time (120 min) reduced CGTase activity. • Increases in activity remained constant after 24 h of enzyme solution storage. • Sonication increased fluorescence (≤11 %) and reduced CGTase particle size (≤85 %). This work explored the impact of ultrasound (US) on the activity, stability, and macrostructural conformation of cyclodextrin glycosyltransferase (CGTase) and how these changes could maximize the production of β-cyclodextrins (β-CDs). The results showed that ultrasonic pretreatment (20 kHz and 38 W/L) at pH 6.0 promoted increased enzymatic activity. Specifically, after sonication at 25 °C/ 30 min, there was a maximum activity increase of 93 % and 68 % when biocatalysis was carried out at 25 and 55 °C, respectively. For activity measured at 80 °C, maximum increase (31 %) was observed after sonication at 25 °C/ 60 min. Comparatively, US pretreatment at low pH (pH = 4.0) resulted in a lower activity increase (max. 28 %). These activation levels were maintained after 24 h of storage at 8 °C, suggesting that changes on CGTase after ultrasonic pretreatment were not transitory. These pretreatments altered the conformational structure of CGTase, revealed by an up to 11 % increase in intrinsic fluorescence intensity, and resulted in macrostructural modifications, such as a decrease in particle size and polydispersion index (up to 85 % and 45.8 %, respectively). Therefore, the sonication of CGTase under specific conditions of pH, time, and temperature (especially at pH 6.0/ 30 min/ 25 °C) promotes macrostructural changes in CGTase that induce enzyme activation and, consequently, higher production of β-CDs. [ABSTRACT FROM AUTHOR]

Hesperidin, a flavonoid enriched in citrus peel, can be enzymatically glycosylated using CGTase with significantly improved water solubility. However, the reaction catalyzed by wild-type CGTase is rather inefficient, reflected in the poor production rate and yield. By focusing on the aglycon attacking step, seven residues were selected for mutagenesis in order to improve the transglycosylation efficiency. Due to the lack of high-throughput screening technology regarding to the studied reaction, we developed a size/polarity guided triple-code strategy in order to reduce the library size. The selected residues were replaced by three rationally chosen amino acids with either changed size or polarity, leading to an extremely condensed library with only 32 mutants to be screened. Twenty-five percent of the constructed mutants were proved to be positive, suggesting the high quality of the constructed library. Specific transglycosylation activity of the best mutant Y217F was assayed to be 935.7 U/g, and its kcat/KmA is 6.43 times greater than that of the wild type. Homology modeling and docking computation suggest the source of notably enhanced catalytic efficiency is resulted from the combination of ligand transfer and binding effect. [ABSTRACT FROM AUTHOR]

Cyclodextrin glycosyltransferase (CGTase) can catalyze the glycosylation of hesperidin, resulting in α-glycosyl hesperidin with significantly improved water solubility. In this study, a rational design of CGTase to improve its hesperidin glycosylation activity was investigated. The strategy we employed involved docking hesperidin in its near-attack conformation and virtually mutating the surrounding residues, followed by calculating the changes in binding energy using Rosetta flex-ddG. The mutations with a stabilization effect were then subjected to an activity assay. Starting from CGTase-Y217F, we obtained three double-point mutants, Y217F/M351F, Y217F/M351L, and Y217F/D393H, with improved hesperidin glycosylation activities after screening twenty variants. The best variant, Y217F/D393H, exhibited a catalytic activity of 1305 U/g, and its kcat/KmA is 2.36 times higher compared to CGTase-Y217F and 15.14 times higher compared to the wild-type CGTase. Molecular dynamic simulations indicated that hesperidin was repulsed by CGTase-Y217F when bound in a near-attack conformation. However, by introducing a second-point mutation with a stabilization effect, the repulsion effect is weakened, resulting in a reduction in the distances between the bond-forming atoms and, thus, favoring the reaction.

Fisetin is a flavonoid that exhibits high antioxidant activity and is widely employed in the pharmacological industries. However, the application of fisetin is limited due to its low water solubility. In this study, glycoside derivatives of fisetin were synthesized by an enzymatic reaction using cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp. RB01 in order to improve the water solubility of fisetin. Under optimal conditions, CGTase was able to convert more than 400 mg/L of fisetin to its glycoside derivatives, which is significantly higher than the previous biosynthesis using engineered E. coli. Product characterization by HPLC and LC-MS/MS revealed that the transglycosylated products consisted of at least five fisetin glycoside derivatives, including fisetin mono-, di- and triglucosides, as well as their isomers. Enzymatic analysis by glucoamylase and α-glucosidase showed that these fisetin glycosides were formed by a-1,4-glycosidic linkages. Molecular docking demonstrated that there are two possible binding modes of fisetin in the enzyme active site containing CGTaseglysosyl intermediate, in which O7 and O4' atoms of fisetin positioned close to the C1 of glycoside donor, corresponding to the isomers of the obtained fisetin monoglucosides. In addition, the water solubility and the antioxidant activity of the fisetin monoglucosides were tested. It was found that their water solubility was increased at least 800 times when compared to that of their parent molecule while still maintaining the antioxidant activity. This study revealed the potential application of CGTase to improve the solubility of flavonoids. [ABSTRACT FROM AUTHOR]

γ-Cyclodextrin (γ-CD) is an attractive material among the natural cyclodextrins owing to its excellent properties. γ-CD is primarily produced from starch by γ-cyclodextrin glycosyltransferase (γ-CGTase) in a controlled system. However, difficulty in separation and low conversion rate leads to high production costs for γ-CD. In this study, γ-CGTase from Bacillus sp. G-825-6 STB17 was used in γ-CD production from cassava starch. With the introduction of sodium tetraphenylborate (NaBPh 4), the total conversion rate was promoted from an initial 18.07 % to 50.49 % and the γ-CD ratio reached 78.81 % with a yield of 39.79 g/L. Furthermore, the mechanism was conducted via the determination of binding constant, which indicated that γ-CD exhibited much stronger binding strength with NaBPh 4 than β-CD. The reformation of water molecules and the chaotropic effect might be the main driving forces for the interaction. Additionally, the conformations of CD complexes were depicted by NMR and molecular docking. The results further verified different binding patterns between CDs and tetraphenylborate ions, which might be the primary reason for the specific binding. This system not only guides γ-CD production with an efficient and easy-to-remove production aid but also offers a new perspective on the selection of complexing agents in CD production. [Display omitted] [ABSTRACT FROM AUTHOR]

Actinomyces viscous (A. viscous) is well documented as a major cariogenic bacterium in the oral cavity and needs to be inhibited and removed timely. Essential oils (EOs) are recognized as secure antibacterial agents for treating oral diseases, but their volatility and insolubility limit their application. In this study, cinnamaldehyde was screened as the optimum EO for inhibiting the A. viscous growth by a micro-agar dilution method and microencapsulated by cyclodextrin glycosyltransferase (CGTase)-catalyzed products. The antibacterial effects against A. viscous were investigated and compared with the free cinnamaldehyde. Antibacterial diameter, antibacterial efficiency and stability, and time-kill curve results revealed that the cinnamaldehyde emulsion had better antibacterial properties. 1 MIC of the cinnamaldehyde emulsion had an inhibitory zone of 9.92 nm, a 100 % inhibition rate when acting for 2 min or 5 min, and still maintained the same inhibitory effect for 2 years. The extracellular environment showed more pH decrease, conductivity increase, and protein leakage, suggesting damage to the cell membrane. Microstructure and flow cytometric analysis further revealed that the CGTase-catalyzed products induced more changes in the A. viscous membrane integrity. Based on the results, CGTase-catalyzed products can be used as a potential substance for encapsulating EOs for treating oral bacteria. • Cinnamaldehyde was microencapsulated as an emulsion by CGTase-catalyzed products. • The cinnamaldehyde emulsion had better antibacterial activity against A. viscous. • The emulsion caused more pH decrease, conductivity increase, and protein leakage. • The CGTase-catalyzed products induced more changes in the A. viscous membrane integrity. [ABSTRACT FROM AUTHOR]

Fisetin is a flavonoid that exhibits high antioxidant activity and is widely employed in the pharmacological industries. However, the application of fisetin is limited due to its low water solubility. In this study, glycoside derivatives of fisetin were synthesized by an enzymatic reaction using cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp. RB01 in order to improve the water solubility of fisetin. Under optimal conditions, CGTase was able to convert more than 400 mg/L of fisetin to its glycoside derivatives, which is significantly higher than the previous biosynthesis using engineered E. coli. Product characterization by HPLC and LC-MS/MS revealed that the transglycosylated products consisted of at least five fisetin glycoside derivatives, including fisetin mono-, di- and triglucosides, as well as their isomers. Enzymatic analysis by glucoamylase and α-glucosidase showed that these fisetin glycosides were formed by α-1,4-glycosidic linkages. Molecular docking demonstrated that there are two possible binding modes of fisetin in the enzyme active site containing CGTase-glysosyl intermediate, in which O7 and O4’ atoms of fisetin positioned close to the C1 of glycoside donor, corresponding to the isomers of the obtained fisetin monoglucosides. In addition, the water solubility and the antioxidant activity of the fisetin monoglucosides were tested. It was found that their water solubility was increased at least 800 times when compared to that of their parent molecule while still maintaining the antioxidant activity. This study revealed the potential application of CGTase to improve the solubility of flavonoids.

This study examined the effectiveness of a hierarchical copper-based metal–organic framework (H–Cu-BTC) in comparison with its microporous counterpart (Cu-BTC) for the immobilization of cyclodextrin glycosyltransferase (CGTase) for use in cyclodextrin production. The adsorption capacity, conformational changes, and operational stability of the immobilized enzymes were examined. The presence of both macropores and micropores in the proposed H–Cu-BTC resulted in an enhanced maximum adsorption capacity of 49.5 mg/g for CGTase as compared to 30.6 mg/g for Cu-BTC, which contains only micropores. The presence of macropores in H–Cu-BTC was also shown to more favorably affect the secondary structure of the immobilized enzyme. Using H–Cu-BTC, the proportion of β-sheets, which form the major structure in the protein and are responsible for the enzyme's stability, was shown to increase from 56% in free CGTase to 76.1% after immobilization. However, when using the microporous Cu-BTC, the proportion of β-sheets decreased to 44.1%. The favorable surface attachment of CGTase to H–Cu-BTC reflected its better reusability, wherein the activity was preserved up to 87% of the original CGTase activity after ten repeated cycles of reuse, compared to only 70% using Cu-BTC. The successful immobilization of CGTase on H–Cu-BTC demonstrated that it could be used as a robust biocatalyst for the conversion of starchy waste into cyclodextrins. • H–Cu-BTC exhibits enhanced adsorption capacity (49.5 mg/g) over Cu-BTC (30.6 mg/g). • Macropores in H–Cu-BTC positively influence immobilized CGTase secondary structure. • H–Cu-BTC preserves 87% of CGTase activity after ten cycles, improving reusability. • Successful immobilization on H–Cu-BTC establishes it as a robust biocatalyst. • H–Cu-BTC offers a solution for converting starchy waste into cyclodextrins. [ABSTRACT FROM AUTHOR]

4α-Glucanotransferase (4α-GTase) is unique in its ability to form cyclic oligosaccharides, some of which are of industrial importance. Generally, low amount of enzymes is produced by or isolated from their natural sources: animals, plants, and microorganisms. Heterologous expressions of these enzymes, in an attempt to increase their production for applicable uses, have been widely studied since 1980s; however, the expressions are mostly performed in the prokaryotic bacteria, mostly Escherichia coli. Site-directed mutagenesis has added more value to these expressed enzymes to display the desired properties beneficial for their applications. The search for further suitable properties for food application leads to an extended research in expression by another group of host organism, the generally-recognized as safe host including the Bacillus and the eukaryotic yeast systems. Herein, our review focuses on two types of 4α-GTase: the cyclodextrin glycosyltransferase and amylomaltase. The updated studies on the general structure and properties of the two enzymes with emphasis on heterologous expression, mutagenesis for property improvement, and their industrial applications are provided. [ABSTRACT FROM AUTHOR]

The food enzyme cyclomaltodextrin glucanotransferase ((1→4)‐α‐d‐glucan 4‐α‐d‐[(1→4)‐α‐d‐glucano]‐transferase (cyclising), EC 2.4.1.19) is produced with Anoxybacillus caldiproteolyticus strain St‐88 by PureCircle USA. It is intended to be used in the manufacture of glycosylated steviol glycosides. Residual amounts of total organic solids are removed by the purification steps applied during the production of the modified steviol glycosides; consequently, dietary exposure was not calculated. For the same reason, toxicological studies other than assessment of allergenicity were not considered necessary. Similarity of the amino acid sequence of the food enzyme to those of known allergens was searched and four matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Abstract The food enzyme cyclomaltodextrin glucanotransferase ((1→4)‐α‐d‐glucan 4‐α‐d‐[(1→4)‐α‐d‐glucano]‐transferase (cyclising), EC 2.4.1.19) is produced with Anoxybacillus caldiproteolyticus strain St‐88 by PureCircle USA. It is intended to be used in the manufacture of glycosylated steviol glycosides. Residual amounts of total organic solids are removed by the purification steps applied during the production of the modified steviol glycosides; consequently, dietary exposure was not calculated. For the same reason, toxicological studies other than assessment of allergenicity were not considered necessary. Similarity of the amino acid sequence of the food enzyme to those of known allergens was searched and four matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Alkyl glycoside surfactants with elongated carbohydrate chains are useful in different applications due to their improved biocompatibility. Cyclodextrin glucanotransferases can catalyze the elongation process through the coupling reaction. However, due to the presence of a hydrophobic tail, the interaction between an alkyl glycoside acceptor and the active site residues is weaker than the interaction with maltooligosaccharides at the corresponding site. Here we report the mutations of F197, G263 and E266 near the acceptor subsites in the CGTase C spCGT13 from Carboxydocella sp. The results showed that substitutions of both F197 and G263 were important for the binding of acceptor substrate dodecyl maltoside during coupling reaction. The double mutant F197Y/G263A showed enhanced coupling activity and displayed a 2-fold increase of the primary coupling product using γ-cyclodextrin as donor when compared to wildtype C spCGT13. Disproportionation activity was also reduced, which was also the case for another double mutant (F197Y/E266A) that however not showed the corresponding increase in coupling. A triple mutant F197Y/G263A/E266A maintained the increase in primary coupling product (1.8-fold increase) using dodecyl maltoside as acceptor, but disproportionation was approximately at the same level as in the double mutants. In addition, hydrolysis of starch was slightly increased by the F197Y and G263A substitutions, indicating that interactions at both positions influenced the selectivity between glycosyl and alkyl moieties. [ABSTRACT FROM AUTHOR]

Abstract Background Cyclodextrin glycosyltransferases (CGTases) catalyze the synthesis of cyclodextrins, which are circular α-(1,4)-linked glucans used in many applications in the industries related to food, pharmaceuticals, cosmetics, chemicals, and agriculture, among others. Economic use of these CGTases, particularly γ-CGTase, requires their efficient production. In this study, the effects of chemical chaperones, temperature and inducers on cell growth and the production of soluble γ-CGTase by Escherichia coli were investigated. Results The yield of soluble γ-CGTase in shake-flask culture approximately doubled when β-cyclodextrin was added to the culture medium as a chemical chaperone. When a modified two-stage feeding strategy incorporating 7.5 mM β-cyclodextrin was used in a 3-L fermenter, a dry cell weight of 70.3 g·L− 1 was achieved. Using this cultivation approach, the total yield of γ-CGTase activity (50.29 U·mL− 1) was 1.71-fold greater than that observed in the absence of β-cyclodextrin (29.33 U·mL− 1). Conclusions Since β-cyclodextrin is inexpensive and nontoxic to microbes, these results suggest its universal application during recombinant protein production. The higher expression of soluble γ-CGTase in a semi-synthetic medium showed the potential of the proposed process for the economical production of many enzymes on an industrial scale.

The efficiency of enzymatic cyclodextrin production using cyclodextrin glycosyltransferases (CGTases) is limited by product inhibition. In this study, maltose binding site 2 (MBS2) of the β-CGTase from Bacillus circulans STB01 was modified to decrease product inhibition. First, two point mutants were prepared at position 599 (A599V and A599N). Then, two double mutants incorporating alanine at position 633 (A599N/Y633A and A599V/Y633A) were prepared. Finally, the entire MBS2 region was replaced by that of the α-CGTase from Paenibacillus macerans JFB05-01 to form multipoint mutant MBS2 β → α. All five mutants exhibited mixed-type product inhibition, although both the competitive and uncompetitive components of this inhibition were decreased. The total cyclization activities of A599N, A599V and A599V/Y633A were 15.6%, 76.8% and 70.9% lower than that of the wild-type, respectively, while that of A599N/Y633A was 22.4% higher. Among the mutants, only MBS2 β → α showed catalytic efficiency (k cat / K m) comparable with that of the wild-type. Moreover, A599N, A599N/Y633A and MBS2 β → α produced cyclodextrin yields 13.1%, 15.8% and 19.7% greater than that of the wild-type, respectively. These results suggest that A599N, A599N/Y633A and MBS2 β → α may be more suitable than the wild-type for cyclodextrin production. [ABSTRACT FROM AUTHOR]

Efficient production of cyclodextrins (CDs) has always been challenging. CDs are primarily produced from starch via cyclodextrin glycosyltransferase (CGTase), which acts on α-1,4 glucosidic bonds; however, α-1,6 glucosidic bonds in starch suppress the enzymatic production of CDs. In this study, a glycogen debranching enzyme from Saccharolobus solfataricus STB09 (SsGDE) was utilized to promote the production of β-CD by hydrolyzing α-1,6 glucosidic bonds. The addition of SsGDE (750 U/g of starch) at the liquefaction stage remarkably improved the β-CD yield, with a 43.9% increase. Further mechanism exploration revealed that SsGDE addition could hydrolyze specific branches with less generation of byproducts, thereby promoting CD production. The chain segments of a degree of polymerization ≥13 produced by SsGDE debranching could also be utilized by β-CGTase to convert into CDs. Overall, these findings proposed a new approach of combining SsGDE with β-CGTase to enhance the CD yield.

Germinated brown rice (GBR) consists of bioactive compounds (BCs) that are very useful for diabetes treatment. Modified GBR‐based flour (MGBRF) was produced by modifying the starch in GBR with 0, 299.19, 598.38, and 897.57 U/ml of cyclodextrin glycosyltransferase (CGTase) for 1 hr and then spray‐dried to examine its antidiabetic and cytotoxic effects. The results showed that the slowly digestible starch and resistant starch by modifying the starch in GBR with 598.38 U/ml of CGTase were 55.8% and 5.92% corresponding to the increase of γ‐amino butyric acid (GABA) and ferulic acid (FA) with 4.31 ± 0.68 mg/ml and 3.10 ± 0.02 mg/ml, respectively. The extract from MGBRF showed strong cytotoxic capacity against HepG2. Furthermore, the in vivo study revealed the stability of the glycemic index (GI) by consuming MGBRF with significant impacts on diabetes. These results suggest that MGBRF through the action of CGTase plays a major role in antidiabetes and HepG2 cell product value addition. Practical applications: GBR consists of BCs that are useful for diabetes and cancer treatment. However, when using this or GBR‐based products, it is difficult to evaluate the effect of functional properties, especially for diabetes and/or cancer diseases due to high starch content. Therefore, the modification of starch to limit digestible starch, increase SDS and RS as well as to enhance the effect of BCs on diabetes and cytotoxic activity on cancer cell should be studied before producing various based products from GBR. The results in this study indicated that CGTase increased BCs without any glycosides BCs in the extract. The MGBRF changed to higher RS and SDS while increasing the BCs. The extract of MGBRF showed strong cytotoxic activity against HepG2 cell and a positive effect on type 2‐diabetic mice. Hence, this study produces new information for effective use of GBR‐based food as a functional food. [ABSTRACT FROM AUTHOR]

Abstract The food enzyme cyclomaltodextrin glucanotransferase ((1→4)‐α‐d‐glucan:(1→4)‐α‐d‐glucan 4‐α‐d‐[(1→4)‐α‐d‐glucano]‐transferase; EC 2.4.1.19) is produced with the non‐genetically modified Paenibacillus illinoisenis strain 107 by Hayashibara Co., Ltd. The cyclomaltodextrin glucanotransferase food enzyme is intended to be used solely by the applicant in‐house in starch processing for trehalose production. Since residual amounts of total organic solids (TOS) are removed by the purification steps applied during the production of trehalose, dietary exposure was not calculated. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no‐observed‐adverse‐effect level (NOAEL) of 39 mg TOS/kg body weight per day, the highest dose tested. Similarity of the amino acid sequence to those of known allergens was searched and one match was found. The Panel considered that, under the intended condition of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure cannot be excluded, but the likelihood for such reactions to occur is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Gracilibacillus dipsosauri is a moderately-halophilic Gram-positive bacterium which forms an extracellular α-amylase that is induced by starch, repressed by d-glucose, and active in 2.0 M KCl. Previous studies showed that while enzyme activity could be measured with the synthetic substrate 2-chloro-4-nitrophenyl-α-d-maltotrioside (CNPG3), other assays were inconsistent and the protein showed aberrant mobility during nondenaturing gel electrophoresis. To clarify the properties of this enzyme, the genome of G. dipsosauri was sequenced and was found to be 4.19 Mb in size with an overall G+C content of 36.9%. A gene encoding an α-amylase composed of 691 amino acids was identified. The protein was a member of the glycosyl hydrolase 13 family, which had a molecular mass of 77,396 daltons and a pI of 4.39 due to an unusually large number of aspartate and glutamate residues (95/691 or 13.7%). BLAST analysis of the amino acid sequence revealed significant matches to other proteins with cyclodextrin glycosyltransferase activity. Partial purification of the protein from G. dipsosauri showed that fractions catalyzing the hydrolysis of CNPG3 and p-nitrophenyl-d-maltoheptoside also catalyzed the formation of β-cyclodextrin but not α-cyclodextrin or γ-cyclodextrin. Formation of β-cyclodextrin was not stimulated by high salt concentrations but did occur with rice, potato, wheat, and corn starches and amylopectin. These studies explain the unusual features of the α-amylase from G. dipsosauri and indicate it should be classified as EC 2.4.1.19. The availability of the complete genomic sequence of G. dipsosauri will provide the basis for studies on other enzymes from this halophile which may be useful for biotechnology. [ABSTRACT FROM AUTHOR]

The food enzyme cyclomaltodextrin glucanotransferase ((1→4)‐α‐d‐glucan:(1→4)‐α‐d‐glucan 4‐α‐d‐[(1→4)‐α‐d‐glucano]‐transferase; EC 2.4.1.19) is produced with the non‐genetically modified Paenibacillus illinoisenis strain 107 by Hayashibara Co., Ltd. The cyclomaltodextrin glucanotransferase food enzyme is intended to be used solely by the applicant in‐house in starch processing for trehalose production. Since residual amounts of total organic solids (TOS) are removed by the purification steps applied during the production of trehalose, dietary exposure was not calculated. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no‐observed‐adverse‐effect level (NOAEL) of 39 mg TOS/kg body weight per day, the highest dose tested. Similarity of the amino acid sequence to those of known allergens was searched and one match was found. The Panel considered that, under the intended condition of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure cannot be excluded, but the likelihood for such reactions to occur is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Sophoricoside glycosylated derivatives, especially long-chain glycosylated sophoricosides (LCGS), have greatly improved water solubility compared with sophoricoside. Here, cyclodextrin glycosyltransferase from Paenibacillus macerans (PmCGTase) was employed for sophoricoside glycosylation. Saturation mutagenesis of alanine 156, alanine 166, glycine 173, and leucine 174 was performed due to their nonconservative properties among α-, β-, and γ-CGTases with different product specificities. Variants L174P, A156V/L174P, and A156V/L174P/A166Y greatly improved the product specificity for LCGS. pH significantly affected the extent of glycosylation catalyzed by the variants. Further investigations revealed that the pH-regulated mechanism for LCGS synthesis mainly depends on a disproportionation route at a lower pH (pH 4) and a cyclization-coupling route at a higher pH (pH 8) and equivalent effects of cyclization-coupling and disproportionation routes at pH 5. Whereas short-chain glycosylated sophoricosides (SCGS) are primarily produced via disproportionation of maltodextrin at pH 4 and secondary disproportionation of LCGS at pH 8. At pH 5, SCGS synthesis mainly depends on a hydrolysis route by the wild type (WT) and a secondary disproportionation route by variant A156V/L174P/A166Y. Kinetics analysis showed a decreased Km value of variant A156V/L174P/A166Y. Dynamics simulation results demonstrated that the improved LCGS specificity of the variant is possibly attributed to the enhanced affinity to long-chain substrates, which may be caused by the changes of hydrogen bond interactions at the -5, -6, and -7 subsites. Our results reveal a pH-regulated mechanism for product specificity of CGTase and provide guidance for engineering CGTase toward products with different sugar chain lengths. [ABSTRACT FROM AUTHOR]

Cyclodextrin glycosyltransferases (CGTases) are widely used in starch deep processing, so reducing their cost by improving their production is of significant industrial interest. The CGTase from Bacillus stearothermophilus NO2 possesses excellent catalytic properties but suffers from low production in E. coli. In this study, directed evolution was used to create three point mutants (I631T, I641T and K647E) that were produced in E. coli with shake-flask yields 1.7-, 2.1-, and 2.2-fold higher than that of wild-type, respectively. The wild-type and K647E were then produced in a 3-L fermenter. The CGTase activity of the K647E (1904 U mL-1) was 2.0-fold higher than that of the wild-type. The K647E fermentation supernatant could be used directly to prepare 2-O-α-d-glucopyranosyl-l-ascorbic acid, reducing the costs associated with its production. Structural modeling of the three mutants suggested that hydrophilicity, hydrogen bonding, and negative charge may be responsible for their improved production. Since K647 is conserved in the CGTase family, the corresponding residues in the CGTases from Bacillus circulans 251, Paenibacillus macerans, and Anaerobranca gottschalkii were changed to glutamic acid. Productions of the resulting K647E mutants were 2.0-, 1.5-, and 1.0-fold higher than those of their respective wild-types. Electrostatic protein surface analysis suggested that mutations occurring at low negative surface charge may increase CGTase production. [ABSTRACT FROM AUTHOR]

Cinnamaldehyde is popular due to its cinnamon flavor and antimicrobial activity, but is constrained by its instability. In this study, the cyclodextrin glycosyltransferase-catalyzed (CGTase-catalyzed) products from corn, tapioca, or potato starch were used for a new wall material for microencapsulating cinnamaldehyde emulsion. The sedimentation and dilution stability revealed that the emulsion prepared by the CGTase-catalyzed products from 1.5% of tapioca starch displayed the most uniform and stable properties, with a particle size of 195.18 nm and encapsulation efficiency of 92.5%. The fine structure analysis of these products revealed that no retrogradation, high cyclodextrin yield and conversation rate, and a high proportion of medium chains (the degree of polymerization 13–24) were conducive to the stability of the emulsion. Finally, the correlation between the emulsion's stability and the products' molecular structure was analyzed using the Person correlation coefficient. The results showed that the medium chains exhibited the highest correlation. This study combined the inclusion of the CGTase-catalyzed products with the emulsion embedding for a highly stable and low-cost microencapsulated cinnamaldehyde emulsion, which would provide a guidance for the microencapsulation of other essential oils. [Display omitted] • CGTase-catalyzed products were used for wall materials for cinnamaldehyde emulsion. • The emulsion with 1.5% tapioca starch enzymatic products had the best stability. • The medium chains (DP 13–24) exhibited the highest correlation with the stability. • Gel network structure and cyclodextrin inclusion greatly enhanced the stability. [ABSTRACT FROM AUTHOR]

• Semi-rational engineering on surface residues of CGTase for enhanced OSs resistance. • A variant G539I/R146F/D147N with enhanced OSs resistance and thermostability was obtained. • Strengthened structural compactness and rigidity of CGTase is conducive to its OSs resistance. Cyclodextrin glycosyltransferase (CGTase) is a preferable biocatalyst for production of cyclodextrin and phenols glycosylated derivatives due to its cost-effective donors and broad range of acceptors. Organic solvents (OSs) are often beneficial for solubilizing hydrophobic substrates and product specificity, but often harmful towards CGTases. Herein, using Paenibacillus macerans CGTase (Pm CGTase) as a template and DMSO as a screening solvent, semi-rational engineering was performed at 18 surface residues by a developed high throughput screening method. Using site saturation mutagenesis and iterative saturation mutagenesis, 5 OSs-resistant variants (G539I, G539V, G539I/R146F, G539V/R146A, G539I/R146F/D147N) were obtained. Compared with WT, the best variant G539I/R146F/D147N showed a 79.3 % enhanced residual activity at 30 % DMSO and a 60 % increased t 1/2 value at both 40 and 50 °C. In addition, a decreased K m value and an increased k cat value led to 41.9 % higher catalytic efficiency of G539I/R146F/D147N than WT in 15 % DMSO. This variant was also applied to glycosylation of sophoricoside in 20 % DMSO, leading to 26.2 % increased conversion. Molecular dynamics demonstrates that its enhanced OSs resistance may be attributed to the strengthened structural compactness and rigidity. Our results provide guidance for engineering OSs resistance of Pm CGTase, which would further improve its application potential. [Display omitted] [ABSTRACT FROM AUTHOR]

This study was designed to investigate the structure of synthesized hesperidin glycosides (HGs) and evaluate their antibacterial and α-glucosidase inhibitory activities. The preliminary structure of HGs was confirmed by glucoamylase treatment and analyzed on thin layer chromatography (TLC). The LC-MS/MS profiles of HGs showed the important fragments at m/z ratios of 345.21 (added glucose to glucose of rutinose in HG1) and 687.28 (added maltose to glucose of rutinose in HG2), confirming that the structures of HG1 and HG2 were α-glucosyl hesperidin and α-maltosyl hesperidin, respectively. In addition, 1H and 13C-NMR of hesperidin derivatives were performed to identify their α-1,4-glycosidic bonds. The MIC and MBC studies showed that transglycosylated HG1 and HG2 had better antibacterial and bactericidal activities than hesperidin and diosmin, and were more active against Staphylococcus aureus than Escherichia coli. Hesperidin, HG1, HG2, and diosmin inhibited α-glucosidase with IC50 values of 2.75 ± 1.57, 2.48 ± 1.61, 2.36 ± 1.48, and 2.99 ± 1.23 mg/mL, respectively. The inhibition kinetics of HG2 shown by a Lineweaver–Burk plot confirmed HG2 was an α-glucosidase competitive inhibitor with an inhibitor constant, Ki, of 2.20 ± 0.10 mM. Thus, HGs have the potential to be developed into antibacterial drugs and treatments for treating α-glucosidase-associated type 2 diabetes.

Inhibition of cyclodextrin glycosyltransferases (CGTases) by their product cyclodextrins limits the efficiency of cyclodextrin production. In an effort to produce variants with good activity but reduced product inhibition, six mutants were constructed at position 603 of the CGTase from Bacillus circulans STB01, which exhibits mixed-type product inhibition. In a kinetic analysis, N603I showed reduced noncompetitive inhibition while N603K, N603H and N603R showed increased noncompetitive inhibition. Unexpectedly, N603E and N603D exhibited reductions in both competitive and noncompetitive product inhibition. Noncompetitive product inhibition is closely related to the interaction between the cyclodextrin and the enzyme in maltose binding site 2 (MBS2). Structural models led to the suggestion that there is increased interaction between maltose binding sites 1 and 2 in mutants N603E and N603D, which may have led to the unexpected results. N603D exhibited a 23.9% greater cyclodextrin yield per gram of enzyme than the wild-type, suggesting it has potential for industrial use. Further reductions in product inhibition may be gained through studies of maltose binding site interactions. Unlabelled Image • N603I showed reduced noncompetitive inhibition, while N603K, N603H and N603R showed increased noncompetitive inhibition. • N603E and N603D exhibited reductions in both competitive and noncompetitive product inhibition unexpectedly. • N603D, exhibiting a 23.9% greater cyclodextrin yield per gram of enzyme, has potential for industrial use. [ABSTRACT FROM AUTHOR]

Enzymatic synthesis of pinoresinol-α-glucosides (PGs) was performed through the transglycosylation reaction catalyzed by the recombinant cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans A11 using β-cyclodextrin as the glycosyl donor and pinoresinol as the acceptor molecule. Incubation of 0.5% (wt/vol) β-cyclodextrin with 1.5% (wt/vol) pinoresinol (P) and 80.0 U/mL of CGTase in 20 mM of Tris–HCl buffer (pH 9.0) at 50oC for 60 h was optimal for PGs synthesis. Under these conditions, two PG transfer products with molecular weights of 544 and 682 Da corresponding to pinoresinol monoglucoside (PG1-I and PG1-II) and pinoresinol diglucoside (PG2), respectively, were detected by TLC and MS. The structures of PG1 and PG2 were confirmed as pinoresinol-α-D-glucopyranoside and pinoresinol-α-D-diglucopyranoside, respectively, by 1H-NMR analysis. The free-radical scavenging and anti-inflammatory activities of PG1 and PG2 were reduced as compared with the original P using α,α-diphenyl-β-picrylhydrazyl radical scavenging reactions and the β-glucuronidase inhibition assay. The loss of antioxidant and anti-inflammatory activities might result from the addition of glucose in the position 4-OH of P that may have disturbed its electron rotation. In vivo, PGs are converted to Ps before they are absorbed and so loss of activity may be minimal. Interestingly, the α-glycosylated compounds which showed the change of physicochemical properties such as increase of solubility and sweetness could promote a positive effect on the bioavailability of original P. [ABSTRACT FROM AUTHOR]

BACKGROUND: High‐purity α‐, β‐, or γ‐cyclodextrin (CD) production using cyclodextrin glycosyltransferase (CGTase) as biocatalyst to cyclize starch is advantageous owing to its low cost of purification and product specificity. Several approaches have been investigated to enhance product specificity of CGTase, such as new CGTase isolation, CGTase gene (cgt) mutation, and chemical addition. RESULTS: Recombinant CGTase was cross‐linked by glutaraldehyde to obtain cross‐linked enzyme aggregates of recombinant CGTase (CLEA‐CGTase) at 85 °C because free CGTase showed high product specificity at 85 °C. The CLEA‐CGTase was prepared under the conditions 75 U mL−1 CGTase with 0.1% (v/v) glutaraldehyde for 10 min at 85 °C after optimization first, and produced a high proportion of β‐CD from soluble starch at 50 °C in the conversion system. The conversion process was carried out with 10 to 200 U mL−1 CLEA‐CGTase, resulting in 100% β‐CD from soluble starch after 420 min conversion of the potato starch solution at 50 °C. And the proportion of β‐CD was still higher than 90% when 8000 U mL−1 CLEA‐CGTase was added. CONCLUSIONS: CLEA technology maintained CGTase conformation of 85 °C, and produced a high proportion of β‐CD at 50 °C. This research showed the CLEA technology kept the conformation of enzyme for specific product production. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) is one of the most important L-ascorbic acid derivatives because of its resistance to reduction and oxidation and its easy degradation by α-glucosidase to release L-ascorbic acid and glucose. Thus, AA-2G has commercial uses in food, medicines and cosmetics. This article presents a review of recent studies on the enzymatic production of AA-2G using cyclodextrin glycosyltransferase. Reaction mechanisms with different donor substrates are discussed. Protein engineering, physical and biological studies of cyclodextrin glycosyltransferase are introduced from the viewpoint of effective AA-2G production. Future prospects for the production of AA-2G using cyclodextrin glycosyltransferase are reviewed. [ABSTRACT FROM AUTHOR]

Cyclodextrins (CDs) and their derivatives have attracted significant attention in the pharmaceutical, food, and textile industries, which has led to an increased demand for their production. CD is typically produced by the action of cyclodextrin glycosyltransferase (CGTase) on starch. Owing to the relatively high cost of enzymes, the economic feasibility of the entire process strongly depends on the effective retention and recycling of CGTase in the reaction system, while maintaining its stability. CGTase enzymes immobilized on various supports such as porous glass beads or glyoxyl-agarose have been previously used to achieve this objective. Nevertheless, the attachment of biocatalysts on conventional supports is associated with numerous drawbacks, including enzyme leaching prominent in physical adsorption, reduced activity as a result of chemisorption, and increased mass transfer limitations. Recent reports on the successful utilization of metal–organic frameworks (MOFs) as supports for various enzymes suggest that CGTase could be immobilized for enhanced production of CDs. The three-dimensional microenvironment of MOFs could maintain the stability of CGTase while posing minimal diffusional limitations. Moreover, the presence of different functional groups on the surfaces of MOFs could provide multiple points for attachment of CGTase, thereby reducing enzyme loss through leaching. The present review focuses on the advantages MOFs can offer as support for CGTase immobilization as well as their potential for application in CD production.

The superiority of a metal organic framework, namely, MIL-101 (Cr) over conventional support, zeolite Y, as immobilization support for Cyclodextrin glycosyltransferase (CGTase) was investigated. The adsorption capacity, stability, and secondary structures of CGTase upon immobilization were investigated. CGTase adsorption on MIL-101 and zeolite Y was best represented by the Langmuir isotherm with optimum adsorption capacities of 37.5 and 6.1 mg/g on two supports, respectively. The deconvolution of the amide I band of the FTIR spectrum indicated that free CGTase molecules predominantly contain β-sheets, which increased with the immobilization on MIL-101 and zeolite Y from 56% to 84.1% and 69.7%, respectively, suggesting the existence of CGTase agglomerates on the supports. The CGTase immobilized on MIL-101 showed relative activity of about 50% at a substrate concentration of up to 4 g/L (compared with free enzymes) and above 3 times higher selectivity towards the more valuable α-CD. A diffusion–reaction model was used to predict the behavior of the immobilized system, and reusability studies revealed that immobilized CGTase may be utilized for numerous reaction cycles, with possible improvements via covalent attachment using glutaraldehyde. Overall, the obtained results in this study provide insights into the usefulness of MIL-101 as a suitable support for CGTase. [Display omitted] • Cyclodextrin glycosyltransferase was immobilized on MIL-101 and zeolite. • MIL-101 showed higher adsorption compared to zeolite-Y. • Immobilization altered secondary structures of CGTase on MIL-101. • α-Cyclodextrin is favored at lower substrate concentration. [ABSTRACT FROM AUTHOR]

Two optimization strategies, codon usage modification and glycine supplementation, were adopted to improve the extracellular production of Bacillus sp. NR5 UPM β-cyclodextrin glycosyltransferase (CGT-BS) in recombinant Escherichia coli. Several rare codons were eliminated and replaced with the ones favored by E. coli cells, resulting in an increased codon adaptation index (CAI) from 0.67 to 0.78. The cultivation of the codon modified recombinant E. coli following optimization of glycine supplementation enhanced the secretion of β-CGTase activity up to 2.2-fold at 12 h of cultivation as compared to the control. β-CGTase secreted into the culture medium by the transformant reached 65.524 U/mL at post-induction temperature of 37 °C with addition of 1.2 mM glycine and induced at 2 h of cultivation. A 20.1-fold purity of the recombinant β-CGTase was obtained when purified through a combination of diafiltration and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. This combined strategy doubled the extracellular β-CGTase production when compared to the single approach, hence offering the potential of enhancing the expression of extracellular enzymes, particularly β-CGTase by the recombinant E. coli.

In comparison with natural α- and β-cyclodextrin (CD), γ-CD has attracted much attention due to their large hydrophobic cavities, high water solubility, and bioavailability. However, the production of γ-CD is still rather expensive and time-consuming. To overcome the high cost and long induction time, pUC119 was selected as the gene expression vector, and the recombinant enzyme production time was reduced to 8 h from 72 h. Furthermore, for the first time, we have successfully produced γ-CD using β-CD by cyclodextrin opening reactions through the recombinant CGTase in the presence of maltose. The kinetic mechanism of the coupling reaction was investigated. Moreover, the production of γ-CD could be affected by several key parameters, such as solvent type, reaction time, pH, and temperature. A maximum γ-CD yield of 32.9% was achieved by recombinant CGTase in the presence of 5-cyclohexadecen-1-one. This could be a promising method for the industrial production of γ-CD. [ABSTRACT FROM AUTHOR]

Cyclodextrin glycosyltransferase (CGTase) catalyzes hydrolysis, cyclization, coupling, and disproportionation reactions and is widely used in the starch processing industry. In this work, the expression of CGTase from Geobacillus stearothermophilus in Escherichia coli BL21 (DE3) was significantly improved by promoter engineering and downstream box evolution. Firstly, the effects of the promoter type (P T7 , P trp , P lacUV5 , and the hybrid promoters P tacI and P tacII ) and spacer sequence on the expression of CGTase were examined. P tacI demonstrated the highest rate of transcriptional activity, which was 4.4-, 7.1-, 3.3-, and 1.5-fold greater than that of P T7 , P trp , P lacUV5 , and P tacII , respectively. The spacer sequence of the promoter was optimized using a degenerate base library, and the GC content of the spacer was found to be inversely proportional to CGTase expression. In addition, CGTase expression was higher when TG:CA and TA:TA dimers were present in the spacer sequence. Under the control of the P tacI promoter with an optimized spacer sequence, extracellular CGTase activity reached 170.6 U/mL, which was seven times higher than that of the original strain (25.2 U/mL). Directed evolution of the downstream box sequence was then performed by randomization of the sequence using degenerate codons, similarly as for the optimization of the spacer sequence. After optimizing the downstream box sequence, CGTase activity increased from 170.6 to 214 U/mL. The results obtained here indicate that in addition to promoter type, the spacer sequence of the promoter and the downstream box are important target elements for improved gene expression. [ABSTRACT FROM AUTHOR]

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The properties of a previously unknown enzyme, denominated cyclodextrin glycosyltransferase, produced from Bacillus lehensis, were evaluated using affinity chromatography for protein purification. Enzyme characteristics (optimum pH and temperature; pH and temperature stability), the influence of substances on the enzyme activity, enzyme kinetics, and cyclodextrin production were analysed. Cyclodextrin glycosyltransferase was purified up to 320.74-fold by affinity chromatography using β-cyclodextrin as the binder and it exhibited 8.71% activity recovery. This enzyme is a monomer with a molecular weight of 81.27 kDa, as estimated by SDS-PAGE. Optimum temperature and pH for cyclodextrin glycosyltransferase were 55°C and 8.0, respectively. The Michaelis-Menten constant was 8.62 g/l during maximum velocity of 0.858 g/l.h.