Β-cyclodextrin Glycosyltransferase Research Articles

Immobilization of β-cyclodextrin glycosyltransferase on gelatin enhances β-cyclodextrin production

The β-cyclodextrin glycosyltransferase (β-CGTase) from Bacillus circulans STB01 was immobilized on gelatin with an immobilization yield of 100 % and an activity recovery of 23.1 %. Compared with the free β-CGTase, the immobilized exhibited a broader pH optimum with two peaks (pH 5.5 and pH 8.5) as well as significantly enhanced thermostability and pH stability. Immobilized β-CGTase retained 85.5 % of its activity after storage for 50 days at 4 ℃ and 91.9 % of its activity after 20 cycles of enzyme activity assay. β-Cyclodextrin inhibition experiments showed that immobilized β-CGTase was less susceptible to product inhibition than free β-CGTase. As a result, immobilized β-CGTase delivered a 9.7 % increase of product yield in a single use and the total yield after 10 times of use was 9.2-fold of the yield using free enzyme. According to the results, immobilization can greatly improve the utility of β-CGTase as a catalyst for the industrial production of β-cyclodextrin.

Optimization of maltooligosyl fructofuranosidesproduction by recombinant β-cyclodextrin glycosyltransferase

Coupling sugar is a kind of new sweetener which can substitute sucrose. It has a good application prospect in food, medicine and other fields because of its good coloration, water retention and anti caries. The purpose of this study was to find cheap and easily available donor and acceptor, and to optimize the preparation process of coupling sugar by using β-cyclodextrin glycosyltransferase from Bacilluscirculans 251. Using sucrose as acceptor, the factors of preparing coupling sugar was optimized, including enzyme dosage, starch types, temperature, pH, ratio of starch/sucrose, and cooperation of isoamylase and β-CGTase. When 105 g/L potato starch and 95 g/L sucrose was used as substrates, the yield of coupling sugar reached 88.4%, which was catalyzed by 13.5 U/g immobilized β-CGTase and 45.0 U/g isoamylase under the conditions of pH 5.5 and 40 °C for 21 h. In this study, isoamylase and β-CGTase were used to prepare coupling sugar innovatively. This method had obvious advantages in yield and cost, which laid both theoretical and experimental foundation for the industrial enzymatic preparation of coupling sugars.

Biosynthesis of raw starch degrading β-cyclodextrin glycosyltransferase by immobilized cells of Bacillus licheniformis using potato wastewater

The study was sought to enhance the synthesis of thermal stable β-cyclodextrin glycosyltransferase (β-CGTase) using potato wastewater as a low-cost medium and assess the degree to which it is efficient for industrial production of β-cyclodextrin (β-CD) from raw potato starch. Thermophilic bacteria producing β-CGTase was isolated from Saudi Arabia and the promising strain was identified as <i>Bacillus licheniformis</i> using phylogenetic analysis of the 16S rRNA gene. Alginate-encapsulated cultures exhibited twice-fold of β-CGTase production more than free cells. Scanning electron microscopy (SEM) of polymeric capsules indicated the potential for a longer shelf-life, which promotes the restoration of activity in bacterial cells across semi-continuous fermentation of β-CGTase production for 252 h. The optimal conditions for β-CGTase synthesis using potato wastewater medium were at 36 h, pH of 8.0, and 50°C with 0.4% potato starch and 0.6% yeast extract as carbon and nitrogen sources, respectively. The purified enzyme showed a specific activity of 63.90 U/mg with a molecular weight of ∼84.6 kDa as determined by SDS-PAGE analysis. The high enzyme activity was observed up to 60°C, and complete stability was achieved at 75°C. High levels of activity and stability were shown at pH 8.0, and the pH range from 7.0–10.0, respectively. The enzyme has an appreciable affinity for raw potato starch with a Km of 5.7 × 10⁻⁶ M and a Vmax of 87.71 µmoL/mL/min. β-CD production was effective against 25 U/g of raw potato starch. The outcomes demonstrated its feasibility to develop a fermentation process by integrating the cost-effective production of β-CGTase having distinctive properties for β-CD production with ecofriendly utilization of potato wastewater.

Combined Optimization of Codon Usage and Glycine Supplementation Enhances the Extracellular Production of a β-Cyclodextrin Glycosyltransferase from Bacillus sp. NR5 UPM in Escherichia coli.

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.

Structural and property characterization of corn starch modified by cyclodextrin glycosyltransferase and specific cyclodextrinase

Enzymatic modification is generally applied to enhance desirable attributes of starch. In this study, β-cyclodextrin glycosyltransferase (β-CGTase) together with a specific cyclodextrinase (CDase) was used to modify corn starch. For single β-CGTase treatment, the molecular weight (Mw) was reduced to 36.7 × 105 g/mol and 7.7 × 105 g/mol for 1 h and 6 h reaction, and the debranched samples had a high proportion of chains with degree of polymerization (DP) < 13, indicating an apparent depolymerization effect. CDase could hardly act on starch, while it had a high preference for cyclodextrin hydrolysis and produced mainly maltotetraose with β-CGTase. There was a synergistic effect between β-CGTase and CDase in starch degradation. Interestingly, the utilization of dual-enzyme significantly promoted the retrogradation inhibition property of starch, reducing the retrogradation enthalpy from 5.65 J/g to 1.42 J/g. It was proved that β-CGTase and CDase had a great prospect in the future application.

A Cyclodextrin-Based Controlled Release System in the Simulation of In Vitro Small Intestine.

A novel cyclodextrin (CD)-based controlled release system was developed in the small intestine to control the rate of drug release, on the premise of enteric-coated tablets. The system was designed based on the enzymes exogenous β-cyclodextrin glycosyltransferase (β-CGTase) and endogenous maltase-glucoamylase (MG), wherein MG is secreted in the small intestine and substituted by a congenerous amyloglucosidase (AG). The vanillin-/curcumin-β-CD complexes were prepared and detected by Fourier transform infrared (FT-IR), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC), and host CD degradation was measured based on the glucose yield. The combination of β-CGTase and AG was also functional in the CD complex system. The variations in the concentrations of added β-CGTase, with AG constantly in excess, could effectively alter the rate of host CD degradation and guest release by monitoring glucose production and color disappearance, thus, demonstrating that guest release in the CD complex system could be precisely controlled by changing the amount of β-CGTase used. Thus, the in vitro simulation of the system indicated that a novel controlled release system, based on endogenous MG, could be established in the small intestine. The CD-based controlled release system can be potentially applied in drug delivery and absorption in the small intestine.

Inclusion of tributyrin during enzymatic synthesis of cyclodextrins by β-cyclodextrin glycosyltransferase from Bacillus circulans

Tributyrin is a precursor of butyric acid, which is beneficial to intestinal health. Due to its unpleasant odor and poor solubility, tributyrin is usually used after inclusion complexation with suitable wall materials. In this paper, tributyrin was creatively added to the enzymatic synthesis of cyclodextrins catalyzed by β-cyclodextrin glycosyltransferase from Bacillus circulans STB01. The tributyrin formed inclusion complexes directly during enzymatic cyclodextrins synthesis and increased the yield of cyclodextrins from 25.84% to 44.01%. This process was investigated by determining inclusion complex stability constant and by using molecular modeling and 1H NMR. The inclusion complexes stability constants of tributyrin with α-, β-, and γ-cyclodextrin were 25.11, 58.56 and 6.93 L/mol, respectively. β-Cyclodextrin had the largest stability constant and the most significant increase in yield (72.80%). Molecular modeling of the three-dimensional structures of the tributyrin-cyclodextrin inclusion complexes indicated that the morphology of the complexed groups was related to the size of the cyclodextrin cavity. Finally, the supramolecular structures were verified with 1H NMR through the chemical shift changes of protons at key positions. This study created a new method and a new inclusion wall material, instead of single pure cyclodextrin for complexing guest molecules. These advances are important because they may expand the application of cyclodextrins due to significant cost reduction.

Discovery and Characterization of a Novel Method for Effective Improvement of Cyclodextrin Yield and Product Specificity

Cyclodextrins(CDs) are widely used in food, pharmaceuticals, drug delivery, and chemical industries and in agriculture and environmental engineering. To improve the yield and selectivity of CDs, this work presented a facile, scalable and efficient enzymatic synthesis of β-CD from starch using β-cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) from Bacillus cereus. First, we found that the pretreatment of starch dramatically influenced CDs yield that was related to the structure and molecular weight of the substrate starch. Second, alcohol solvents influenced the yield and product selectivity of CDs; tertiary alcohols enhanced CDs yield(from 54.95% to 68.21%) and secondary alcohols increased the product selectivity(β-CD/γ-CD changed from 6.25 to 8.05). Fluorescence quenching analysis showed that the binding constants and entropy of the solvents influenced the yield and product selectivity, respectively. In conclusion, the results demonstrate that this study provides a promising method for the industrial production of β-CD.

Improved extracellular secretion of β-cyclodextrin glycosyltransferase from Escherichia coli by glycine supplementation without apparent cell lysis

The use of an effective inducer feeding strategy without causing cell lysis presents significant advantage to enhance the secretion of an enzyme to the culture medium of Escherichia coli. The cgt gene encoding β-cyclodextrin glycosyltransferase (β-CGTase) was cloned into pQE30xa as an N-terminal His-tagged protein and transformed into E. coli. The induction strategy was applied towards enhancing the extracellular secretion of the recombinant β-CGTase by increasing permeability of the outer membrane of E. coli. The supplementation of 1.2 mM glycine following 2 h of fermentation at 37°C enhanced the activity of β-CGTase to 38.295 U/mL, which was approximately 1.3-fold higher than the control (without induction). Further flow cytometry analysis was adopted as a rapid and highly reproducible approach to determine the effect of glycine supplementation on the viability of E. coli cells. The supplementation of glycine did not contribute to apparent cell lysis, with no adverse effects on cell viability, hence indicating the effectiveness of glycine in enhancing the extracellular secretion of β-CGTase. The recombinant β-CGTase was then purified through a combination of diafiltration and Ni-NTA affinity chromatography with 18.4-fold increase in purity. An effective glycine feeding strategy could enhance the extracellular secretion of β-CGTase without adverse effects on cell viability. This strategy could be applied potentially to enhance the secretion of a recombinant protein to the culture medium from E. coli cells without having cell lysis.

Leu600 mutations decrease product inhibition of the β-cyclodextrin glycosyltransferase from Bacillus circulans STB01

The limits that cyclodextrin products impose on their industrial production from starch by cyclodextrin glycosyltransferases (CGTases) are a severe problem. In this paper, mutants at residue Leu600 of the β-CGTase from Bacillus circulans STB01 were constructed in an effort to decrease the product inhibition exhibited by β-cyclodextrin. A kinetic analysis of the inhibition of the wild-type and mutant β-CGTases by β-cyclodextrin revealed that the mutations do not alter the inhibition mode, which is mixed-type. However, the values of the inhibition constants (Ki and Ki′) of the mutants L600I, L600E and L600R are higher than those of the wild-type enzyme, weakening the product inhibition. The mutant L600Y only exhibited a decrease in noncompetitive inhibition, with the value of Ki′ increasing by 40%. Comparison of the Km′ values and the 3D model structures of the wild-type and mutant CGTases suggests that this decrease in product inhibition is related to a decrease in binding affinity between the product cyclodextrin and the enzyme.

Optimization of the fermentation conditions for the mutant strain of β-cyclodextrin glycosyltransferase H167C to produce cyclodextrins.

The cyclodextrin glycosyltransferase (CGTase) was used to catalyze the conversion of starch into cyclodextrins (CD) in industry. Improving the activity of CGTase to produce more CD with relative low cost is intensely interesting and has drawn wide attention. Amino acid mutation of His167 into Cys significantly enhanced β-CGTase activity; however, optimization of culture conditions for β-CGTase-H167C remains unclear. To determine this, the medium and culture conditions for β-CGTase-H167C were optimized with response surface methodology. Maximum activity of β-CGTase-H167C was obtained with the medium containing 1.1% corn starch, 4.4% corn steep liquor, 1.1% peptone, 0.02% MgSO4·7H2O and 0.1% K2HPO4·3H2O that were cultured with the initial pH 8.4, incubation temperature at 37.4°C, with 5% inoculation size and shaking speed at 202r/min. Under the optimal conditions, the activity of β-CGTase-H167C was up to 4355U/mL, which is 1.93-fold in comparison with the initial activity. Our results established the promising culture strategy for the production of cyclodextrins by β-CGTase-H167C.

Improved activity of \u03b2-cyclodextrin glycosyltransferase from Bacillus sp. N-227 via mutagenesis of the conserved residues

β-Cyclodextrin glycosyltransferase (β-CGTase) belongs to the α-amylase family of enzymes and converts starch to cyclic oligosaccharides called β-cyclodextrins (β-CD). The β-CGTase from alkalophilic Bacillus sp. N-227 was separately mutagenized to give three site-directed β-CGTase mutants, Y127F, R254F and D355R, that showed enhanced cyclization activity towards a starch substrate from 1.64 to 2.1-folds. Kinetic studies indicate that the mutants had higher affinity towards the substrate than the wild type β-CGTase. The Y127F mutant had the highest affinity which was indicated by the lowest Km of 15.30 mM and the highest catalytic activity. Increasing hydrophobicity around the catalytic center appeared to favor the cyclization activity of the mutants. The β-CGTase and the three mutants showed optimal enzyme activity at 60 °C and pH 6.0. All the enzymes were stable for at least 60 min across a wide pH range (5.0–7.0).

Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system.

Bacillus subtilis ATCC 6051a is an undomesticated strain used in the industrial production of enzymes. Because it is poorly transformable, genetic manipulation in this strain requires a highly efficient genome editing method. In this study, a Streptococcus pyogenes CRISPR/Cas9 system consisting of an all-in-one knockout plasmid containing a target-specific guide RNA, cas9, and a homologous repair template was established for highly efficient gene disruption in B. subtilis ATCC 6051a. With an efficiency of 33% to 53%, this system was used to disrupt the srfC, spoIIAC, nprE, aprE and amyE genes of B. subtilis ATCC 6051a, which hamper its use in industrial fermentation. Compared with B. subtilis ATCC 6051a, the final mutant, BS5 (ΔsrfC, ΔspoIIAC, ΔnprE, ΔaprE, ΔamyE), produces much less foam during fermentation, displays greater resistant to spore formation, and secretes 2.5-fold more β-cyclodextrin glycosyltransferase into the fermentation medium. Thus, the CRISPR/Cas9 system proved to be a powerful tool for targeted genome editing in an industrially relevant, poorly transformable strain.

Polyethylene glycols enhance the thermostability of β-cyclodextrin glycosyltransferase from Bacillus circulans

We investigated the ability of six polyethylene glycols (PEGs), with molecular weights ranging from 400 to 20,000Da, to enhance the thermostability of β-cyclodextrin glycosyltransferase (β-CGTase) from Bacillus circulans. We found that PEGs with different molecular weights could activate and stabilize this β-CGTase, but to different degrees. The most significant increase (about 20%) in β-cyclodextrin-forming activity was achieved by adding 10–15% PEG 400. PEGs with low molecular weights also significantly enhanced the thermostability of β-CGTase; 15% PEG 1000 prolonged its half-life at 60°C by 6.5-fold, compared to a control. Fluorescence spectroscopy and circular dichroism analysis indicated that PEGs helped protect the tertiary and secondary structure of β-CGTase, respectively. This study provides an effective approach for improving the thermostability of CGTases and related enzymes.

Nanosilica Sol Leads to Further Increase in Polyethylene Glycol (PEG) 1000-Enhanced Thermostability of β-Cyclodextrin Glycosyltransferase from Bacillus circulans

A major disadvantage of cyclodextrin production is the limited thermostability of cyclodextrin glycosyltransferase. The ability of combinations of nanosilica sol with polyethylene glycol (PEG) 1000 to enhance the thermostability of the β-cyclodextrin glycosyltransferase from Bacillus circulans was investigated. It was found that 10% PEG 1000 combined with 0.05% nanosilica sol could activate the β-cyclodextrin glycosyltransferase by 17.2%. Furthermore, 0.05% nanosilica sol leads to further increase in PEG 1000-enhanced thermostability of β-cyclodextrin glycosyltransferase. With the simultaneous addition of 10% PEG 1000 and 0.05% nanosilica into the enzyme solution, which was allowed to incubate for 60 min at 60 °C, 61.3% of β-cyclodextrin-forming activity could be retained, which was much higher than that with only 10% PEG 1000 added. Atomic force microscopy, fluorescence spectroscopy, and circular dichroism analysis indicated that silica nanoparticles helped PEG 1000 further protect the tertiary and secondary structures of β-cyclodextrin glycosyltransferase. This study provides an effective approach for improving the thermostability of cyclodextrin glycosyltransferase and related enzymes.

Purification and characterization of an amylase from Opuntiaficus‐indica seeds

In Tunisia, prickly pear fruit grow spontaneously; it is consumed as fresh fruit, juice or jam. When the fruit is used for juice production, the seeds are discarded and go to waste. Our study aimed to extract biomolecules from seeds by producing value-added products from the fruits. An amylase from Opuntia ficus-indica seeds was extracted and purified to homogeneity. An increase in specific activity of 113-fold was observed. The apparent molecular mass of the enzyme is 64 kDa. The optimum pH and temperature for enzyme activity were pH 5 and 60 °C, respectively. Under these conditions, the specific activity is 245.5 U mg(-1) . The enzyme was activated by Co(2+) and Mg(2+) (relative activity 117% and 113% respectively) at lower ion concentrations. It was strongly inhibited by Mn(2+) and Fe(2+) . Cu(2+) inhibited totally the activity of this enzyme, but Ca(2+) has an inhibitory effect which increases with ion concentration. The extracted enzyme belongs to the exo type of amylases and is classified as a β-cyclodextrin glycosyltransferase since it generates mainly β-cyclodextrin from starch. It exhibits high thermal stability and a broad range of pH stability, making it a promising prospect for industrial and food applications.

Synthesis of epicatechin glucosides by a β-cyclodextrin glycosyltransferase

Enzymatic synthesis of (−)-epicatechin (EC) glucosides was performed through the transglucosylation reaction catalyzed by the cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp. RB01. The enzyme showed the same product specificity for the three donor substrates, starch, β-cyclodextrin and maltoheptaose (G7). Using β-cyclodextrin as the glucosyl donor, several EC glucoside products were obtained at an overall minimal yield of 18.1%. The structures of the four main products were elucidated by MS and NMR techniques as (−)-EC-3′-O-α-d-glucopyranoside (EC3A), (−)-EC-3′-O-α-d-diglucopyranoside (EC3B), (−)-EC-3′-O-α-d-triglucopyranoside (EC3C) and (−)-EC-4′-O-α-d-glucopyranoside (EC4A). Of these, EC3A was the major product while EC4A, unique for this CGTase, was formed in the lowest amount. The water solubility and stability against UV irradiation of EC3A were significantly higher than that of EC. Although the antioxidant activity was 1.5-fold lower, the advantage of the enhanced solubility and stability makes the EC3A glucoside more beneficial as food ingredient than its parent EC.

Production of a Thermoactive Β-cyclodextrin Glycosyltransferase with a High Starch Hydrolytic Activity from an Alkalitolerant Bacillus Licheniformis Sk 13.002 Strain

Production of a Thermoactive Β-cyclodextrin Glycosyltransferase with a High Starch Hydrolytic Activity from an Alkalitolerant Bacillus Licheniformis Sk 13.002 Strain

A β-cyclodextrin glycosyltransferase from a newly isolated Paenibacillus pabuli US132 strain: Purification, properties and potential use in bread-making

A bacterial strain designed US132, isolated from a Tunisian soil was selected for its production of a potent cyclodextrin glycosyltransferase (CGTase) activity. This strain was identified as Paenibacillus pabuli by sequencing of the 16S rDNA and the 16S-23S internal transcribed spacer (ITS). The US132 CGTase, purified to homogeneity by hydrophobic interaction chromatography and starch adsorption, is a monomer of approximately 70 kDa. This enzyme exhibited a maximal activity at 65 °C, in presence of 10 mM calcium, and was most active at pH range 5.5–9 with an optimum at 6.5. Using 10% (w/v) of potato starch, this CGTase produced a high level of cyclodextrins reaching 42 g/l with a β-cyclodextrin ratio of 63%. Furthermore, this enzyme can be used in the bread-baking process since its addition in the dough mix improved significantly the loaf volume and decreased the firmness of bread during storage.

Assay for Simultaneous Determination of β-Cyclodextrin Glycosyltransferase (β-CGTase) and Amylase

A new procedure was developed for the simultaneous determination of β-cyclodextrin glycosyltransferase (β-CGTase) and amylase activities in Bacillus macerans cultures. The new amylase assay branched off from the β-CGTase activity measurement according to Kaneko et al. (J. Jpn. Soc. Starch Sci. 34 (1987(45-48), included water-soluble starch as substrate and was based on the well-characterized K 3 [Fe(CN) 6 l reducing sugar analysis procedure. The assay had excellent within-run and between-run CVs, and also had an excellent correlation with the widely used 3,5-dinitrosalicylic acid method of Bernfeld (Meth. Enzymol. 1 [19551, 149-158) using Bacillus licheniformis α-amylase as a model enzyme at 50°C. Neither high cyclodextrin glycosyltransferase activities nor the self-degradation of starch interfered with the new amylase assay.