Cyclodextrin Glycosyltransferase Research Articles

Synthesis of alkyl glycosides from cyclodextrin using cyclodextrin glycosyltransferase from Paenibacillus sp. RB01

Alkyl glycosides have potential use as biodegradable detergents due to their high surface activity with low toxicity. Recent progress in the application of enzymes to the preparation of these surface-active compounds demonstrates the advantages to the chemical synthesis. In this work, alkyl glycosides were, for the first time, synthesized from cyclodextrin (CD) and various soluble alcohols by transglycosylation reaction using cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp. RB01. Several alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol and 2-butanol) as glycosyl-acceptor substrates were evaluated. It was found that the reaction products which were analyzed by TLC were maximum for 30% methanol, 20–30% ethanol, 10–20% 1-propanol, 10% 2-propanol, 8% 1-butanol and 5–10% 2-butanol. In addition, the increase in the yield of alkyl glycoside formation was achieved by using methanol as an acceptor. Optimal reaction conditions for methyl glycoside synthesis from CD were to incubate 1.2% (w/v) β-CD and 240 U/mL of CGTase in a water/methanol system containing 30% (v/v) methanol, pH 6.0 and a temperature of 40 °C. At least three main methyl glycoside products were formed having 1–3 monosaccharide units attached to methanol which were in accordance with the results of MS analysis.

Evidence of the Involvement of Asparagine Deamidation in the Formation of Cyclodextrin Glycosyltransferase Isoforms in Paenibacillus sp. RB01

Cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp. RB01 and its recombinant enzyme exhibit three isoforms (I, II, and III) with the same apparent size but different charge. Here, we demonstrate for the first time that the deamidation of labile Asns causes the change in molecular forms of CGTase. The faster increase in number of isoforms was observed upon incubation in deamidation buffer at the more alkaline pH. The increase in levels of isoform II and III over time correlated with the increase in isoaspartate, a unique deamidation product. The predicted labile Asns were individually mutated to Asp, then the selected mutant and wild type isoforms were tryptic digested and labile Asns were investigated by MALDI-TOF. From the results, Asn427 was the most susceptible residue for deamidation, followed by Asn336, Asn415, and Asn567. In addition, Gln389 might also share a role. The advantage of using appropriate CGTase isoform in cyclodextrin production is reported.

Biochemical properties and cyclodextrin production profiles of isoforms of cyclodextrin glycosyltransferase

Cyclodextrin glycosyltransferase (CGTase) catalyzes the conversion of starch to cyclodextrin (CD), an important host molecule for the study of host–guest interactions. CGTase from Paenibacillus sp. RB01 and its recombinant form showed the same isoform pattern. The three isoforms, two major (isoforms I and II) and one minor (isoform III), all had a different net charge but the same molecular mass. The aim of this work was to characterize the three isoforms, and especially to compare their CD production profiles. Isoforms I and II were separated on a FPLC Mono Q column and showed the same optimum pH (pH 5 for dextrinizing and pH 6–7 for cyclization activity) and optimum temperature (65–70 °C for both activities). However, the two isoforms differed in their catalytic efficiency of the coupling reaction with variable concentrations of the β-CD donor in the presence of a fixed amount of cellobiose acceptor, with kcat/Km values of 3.46 × 10−3 and 2.20 × 10−3 mM−1 min−1, for isoforms I and II, respectively. Both isoforms I and II were found to have β-CGTase activity and gave a similar CD6:CD7:CD8 product ratio of 0.2:1.0:0.6, with an increase in the ratio of the small-ring to the large-ring CDs from 1.0:0.5 to 1.0:0.3 from a 6 to 24 h reaction time. However, in terms of maximal CD yields, the two isoforms differed in their optimal reaction temperature and time required, the optimal conditions being at 40 °C for 6 h for isoform I and at 60 °C for 24 h for isoform II.

Production of Cyclodextrin Glycosyltransferase (CGTase) by Bacillus lehensis S8 using Sago Starch as Carbon Source

Production of Cyclodextrin Glycosyltransferase (CGTase) by Bacillus lehensis S8 using Sago Starch as Carbon Source

Expression and characterization of a fusion protein‐containing cyclodextrin glycosyltransferase from Paenibacillus sp. A11

A recombinant cyclodextrin glycosyltransferase (CGTase) gene fused with thioredoxin (Trx), hexa-histidine (His(6)) and S-protein (S) at the N terminus and a proline-rich peptide (PRP) at the C terminus, was constructed using the wild-type gene from Paenibacillus sp. A11, the pET-32a vector and Escherichia coli BL21(DE3) as the host cell. The expression levels and enzyme characteristics of the Trx-His(6)-CGTase-PRP fusion protein, the recombinant CGTase without fusion peptides, and the wild-type CGTase were compared. The maximum specific activity for the Trx-His(6)-CGTase-PRP fusion enzyme was 2.7 fold higher than that of the non-fusion form at the optimal IPTG concentration. The Trx-His(6)-CGTase-PRP fusion protein was purified to homogeneity by starch adsorption and Ni-NTA affinity chromatography, with a specific activity of 2,268 units/mg protein at a 61% yield. The ease of purification and the higher enzyme yield were obtained with the fusion form when compared to the non-fusion and wild-type enzymes. The fusion enzyme was superior than its wild-type counterpart in terms of stability against high temperature and organic solvents. Moreover, the fusion enzyme could catalyze the synthesis of cyclodextrins in 20% (v/v) dimethylformamide with a higher product yield of CD(7) and CD(8) compared to that of the wild-type enzyme in the same buffer-solvent system.

Kinetic characteristics of β-cyclodextrin production by cyclodextrin glycosyltransferase from newly isolated Bacillus sp. C26

The kinetic characteristics of β-cyclodextrin production by a cyclodextrin glycosyltransferase (CGTase) produced by Bacillus sp. C26, a new isolate from a soil sample was investigated. Considering highest yield and initial production rate of β-cyclodextrin, among the starches examined, soluble starch, tapioca starch, sago starch, corn starch and rice starch, tapioca starch was the best substrate for this CGTase. The optimum temperature for tapioca starch gelatinization prior to its use as a substrate for β-cyclodextrin production was 65oC. The yield and initial production rate of β-cyclodextrin increased with increasing starch concentration up to 6% and an enzyme concentration up to 48 U/g-starch. The kinetic parameters of V max and K m of β-cyclodextrin production from tapioca starch by CGTase were 1.59 mg/mL/h and 22.3 mg/mL, respectively. Considering high initial production rate and high yield of β-cyclodextrin, the optimum reaction temperature was at 50oC. This study provided the necessary kinetic information that may be useful to define the most suitable condition for industrialized production ofβ-cyclodextrin with the high yield and productivity.

Synthesis of cellobiose-containing oligosaccharides by intermolecular transglucosylation of cyclodextrin glycosyltransferase from Paenibacillus sp. A11

Intermolecular transglucosylation of cyclodextrin glycosyltransferase (CGTase) was investigated for its use in oligosaccharide synthesis. From the kinetic parameters of the CGTase-catalyzed transglucosylation reaction, using β-cyclodextrin (β-CD) as the glucosyl donor and various saccharides or derivatives as acceptors, the efficient acceptors of the Paenibacillus sp. A11 enzyme were glucose, sorbose, lactose and cellobiose. Amongst these acceptors, cellobiose showed the highest k cat / K m value. The transglucosylation yields of the reactions for cellobiose, sorbose and glucose acceptors were 78, 57 and 54%, respectively, making cellobiose the most efficient acceptor of the tested saccharides in coupling with β-CD. The optimal condition for the coupling reaction was determined as: 2% (w/v) β-CD and 0.5% (w/v) cellobiose, incubated with 64 U/mL of CGTase at 30 °C for 2 h. Two main transfer products detected by HPLC, PC1 and PC2, with retention times of 3.81 and 4.42 min, respectively, and a product ratio of 3:1, had a molecular mass of 504 and 666 Da, respectively, as analyzed by mass spectrometry. The structures suggested by NMR were a trisaccharide and a novel tetrasaccharide-containing cellobiose of the structures glc (α1 → 4) glc (β1 → 4) glc and glc (α1 → 4) glc (α1 → 4) glc (β1 → 4) glc, respectively. The products were found to be resistant to hydrolysis by α-amylase.

Altered product specificity of a cyclodextrin glycosyltransferase by molecular imprinting with cyclomaltododecaose

Cyclodextrin glycosyltransferases (CGTases), members of glycoside hydrolase family 13, catalyze the conversion of amylose to cyclodextrins (CDs), circular alpha-(1,4)-linked glucopyranose oligosaccharides of different ring sizes. The CD containing 12 alpha-D-glucopyranose residues was preferentially synthesized by molecular imprinting of CGTase from Paenibacillus sp. A11 with cyclomaltododecaose (CD(12)) as the template molecule. The imprinted CGTase was stabilized by cross-linking of the derivatized protein. A high proportion of CD(12) and larger CDs was obtained with the imprinted enzyme in an aqueous medium. The molecular imprinted CGTase showed an increased catalytic efficiency of the CD(12)-forming cyclization reaction, while decreased k(cat)/K(m) values of the reverse ring-opening reaction were observed. The maximum yield of CD(12) was obtained when the imprinted CGTase was reacted with amylose at 40 degrees C for 30 min. Molecular imprinting proved to be an effective means toward increase in the yield of large-ring CDs of a specific size in the biocatalytic production of these interesting novel host compounds for molecular encapsulations.

Mutations of Lysine 47 in Cyclodextrin Glycosyltransferase from Paenibacillus macerans Enhance β-Cyclodextrin Specificity

The nature of amino acid residue 47 shows a clear discrimination between the different groups of cyclodextrin glycosyltransferase (CGTase). The effects of amino acid side chain at position 47 on cyclodextrin product specificity were investigated by replacing Lys47 in the CGTase from Paenibacillus macerans strain JFB05-01 with arginine, histidine, threonine, serine, or leucine. All of the mutations reduced alpha-cyclodextrin-forming activity, whereas significant increases in beta-cyclodextrin-forming activity were achieved. Especially, the mutations of Lys47 into threonine, serine, or leucine converted P. macerans CGTase from alpha-type to beta/alpha-type. As a result, all of the mutants displayed a shift in product specificity toward the production of beta-cyclodextrin. Thus, they were more suitable for the industrial production of beta-cyclodextrin than the wild-type enzyme. The enhancement of beta-cyclodextrin specificity might be due to weakening or removal of hydrogen-bonding interactions between the side chain of residue 47 and the bent intermediate specific for alpha-cyclodextrin formation.

Avaliação de métodos para manutenção e preservação de bactéria esporulada produtora da enzima CGTase

The conservation of cells without morphologic, physiologic or genetic changes is a biotechnology necessity. Bacillus firmus strain 37 is a sporulating bacteria that produces the cyclodextrin glycosyltransferase (CGTase) enzyme, which transforms starch into cyclodextrins (CDs). This study aimed to evaluate the maintenance and preservation of B. firmus strain 37 stored in a solid medium, sterile soil and in glycerol at low temperature (-70oC). In order to evaluate the best bacteria maintenance method, cell immobilization procedures were used on inorganic matrices. The immobilized cells were submitted to the initial biomass effect test and scanning electron microscopy (SEM). Periodic transfer was not an appropriate method, because the strain reduced the production of CGTase. The storage in sterile soil proved effective and the enzyme production remained constant. The conservation in low temperatures was also satisfactory, with cell counts practically the same after 360 days. The immobilization evaluated by SEM did not show any difference in the adsorption of the cells preserved by the different methods. The same happened for the initial biomass effect test, which presented higher beta-CD production when 1.5 g of cells was used.

A novel cyclodextrin glycosyltransferase from Bacillus sphaericus strain 41: Production, characterization and catalytic properties

The alkalophilic Bacillus sphaericus strain 41 was isolated from soybean-soil culture, using a highly alkaline pH medium containing 1% Na 2CO 3. The cyclodextrin glycosyltransferase (CGTase) from this microorganism was purified up to 315-fold with a yield of 31%, by biospecific affinity-column chromatography using Sepharose 6B gel and β-cyclodextrin (β-CD) as the ligand. The molecular weight of the purified enzyme was estimated to be 59 kDa by SDS-PAGE. In addition to the cyclization, the CGTase showed disproportionation and coupling activities. For cyclization activity, the optimal pH was 6.0 and the temperature was 65 °C. The enzyme showed pH stability in the range of 6.0–7.0. Thermal deactivation was noticeable above 70 °C, and the enzyme was highly stable below 65 °C. The activation and deactivation energy for the production of β-CD were 9.4 kcal/mol and 28.0 kcal/mol, respectively. The influence of substrate concentration on the initial rate of CD production was studied, and the kinetic parameters were determined. The K m was 0.0008 mol/L and V max was 0.0631 mol of β-CD/(L h), using maltodextrin as substrate. The CGTase was strongly inhibited by the products, and produced a level of CDs reaching 22 g/L with a β-CD ratio of 54%. This enzyme produced α-, β- and γ-CD in the ratio of 0.40:1:0.45.

Exploring unfolding pathway of CGTase: insight from molecular dynamics simulation.

Thermostability of proteins in general and especially thermophilic proteins has been subject of a wide variety of studies based on theoretical and experimental investigation. Thermostability seems to be a property obtained through many minor structural modifications. In order to gain better understanding of the thermostability mechanisms of proteins, molecular dynamics simulations of the thermal unfolding reaction of Cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) from Bacillus macerans has been carried out for 1.5 ns each at five different temperatures. Distortion of the enzyme is initiated simultaneously in the N- terminal and domain D, while the thermal unfolding of the outer domains of CGTase is faster than that of the catalytic core domain. The catalytic center was well protected by the (a/b)8 TIM-barrel at simulation temperatures up to 600 K. In addition, the unfolding of the 8 b-sheets obeyed the random ordered mechanism, in which the b-sheets 8, 1 and 6 unfolded more rapidly than the others. At the same time, the influences of non-bonded interactions, such as hydrogen bonds and salt bridges, to protein stabilities were also analyzed in detail. The results show clearly that the stability of the protein is not evenly distributed over the whole structure. Our study provides insight into the structure-stability relationship of CGTase, which may help to enlighten our knowledge of protein structural properties, non-bonded interactions that stabilize secondary peptide structures or promote folding and also to better understand their action.

Immobilization and stabilization of cyclodextrin glycosyltransferase using reversible and irreversible attachments on agarose matrix

Immobilization and stabilization of cyclodextrin glycosyltransferase using reversible and irreversible attachments on agarose matrix

Efficient synthesis of a long carbohydrate chain alkyl glycoside catalyzed by cyclodextrin glycosyltransferase (CGTase)

Alkyl glycosides with long carbohydrate groups are surfactants with attractive properties but they are very difficult to synthesize. Here, a method for extension of the carbohydrate group of commercially available dodecyl-beta-d-maltoside (DDM) is presented. DDM was converted to dodecyl-beta-d-maltooctaoside (DDMO) in a single step by using a CGTase as catalyst and alpha-cyclodextrin (alpha-CD) as glycosyl donor. The coupling reaction is under kinetic control and the maximum yield depends on the selectivity of the enzyme. The Bacillus macerans CGTase favored the coupling reaction while the Thermoanaerobacter enzyme also catalyzed disproportionation reactions leading to a broader product range. A high ratio alpha-CD/DDM favored a high yield of DDMO and yields up to 80% were obtained using the B. macerans enzyme as catalyst.

Cellulose Nanocrystal/Gold Nanoparticle Composite as a Matrix for Enzyme Immobilization

A novel nanocomposite consisting of cellulose nanocrystals (CNCs) functionalized with gold nanoparticles (AuNPs) serving as an excellent support for enzyme immobilization with phenomenally high loading is presented in this work. As testing models, cyclodextrin glycosyl transferase (CGTase) and alcohol oxidase were conjugated on an activated CNC/AuNP matrix. This catalytic platform exhibits significant biocatalytic activity with excellent enzyme stability and without apparent loss of the original activity. The recovered specific activities were approximately 70% and 95% for CGTase and alcohol oxidase, respectively. This novel and inexpensive material is anticipated to extend to other enzymes, enhancing the enzyme loading and activity as well as the stability in both operation and storage.

Mutations at subsite −3 in cyclodextrin glycosyltransferase from Paenibacillus macerans enhancing α-cyclodextrin specificity

A major disadvantage of cyclodextrin production is the limited cyclodextrin product specificity of cyclodextrin glycosyltransferase (CGTase). Here, we described mutations of Asp372 and Tyr89 at subsite -3 in the CGTase from Paenibacillus macerans strain JFB05-01. The results showed that Asp372 and Tyr89 played important roles in cyclodextrin product specificity of CGTase. The replacement of Asp372 by lysine and Tyr89 by aspartic acid, asparagine, lysine, and arginine resulted in a shift in specificity towards the production of alpha-cyclodextrin, which was most apparent for the mutants D372K and Y89R. Furthermore, the changes in cyclodextrin product specificity for the single mutants D372K and Y89R could be combined in the double mutant D372K/Y89R, which displayed a 1.5-fold increase in the production of alpha-cyclodextrin, with a concomitant 43% decrease in the production of beta-cyclodextrin when compared to the wild-type CGTase. Thus, the D372K and Y89R single and double mutants were much more suitable for the industrial production of alpha-cyclodextrin than the wild-type enzyme. The enhanced alpha-cyclodextrin specificity of these mutants might be a result of stabilizing the bent conformation of the intermediate in the cyclization reaction.

Cyclodextrin glycosyltransferase from Bacillus circulans DF 9R: Activity and kinetic studies

Activity characteristics and kinetic aspects of a cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans DF 9R were studied. A mixture of α-, β- and γ-cyclodextrins (CDs), glucose, maltose and negligible amounts of longer linear dextrins were produced from gelatinized amylose, amylopectin and starch from different sources. In the coupling reaction, CDs were the substrates in the presence of acceptors such as maltose and/or longer oligosaccharides. From oligosaccharides formed by three or more glucose units, this enzyme produced linear chains of several lengths which were then cyclized. CGTase catalytic efficiency was compared employing an analytical grade starch and cassava starch for food use. Since the results obtained were similar for both starches, the use of an economic starch is an advantage. CGTase was inhibited by the substrate and its own products. Starch concentrations over 20 mg/mL inhibited the cyclizing activity. CDs behaved as competitive inhibitors and maltose as an uncompetitive inhibitor while maltotriose showed a mixed inhibition pattern. Limit dextrins showed a scarce inhibitory effect on enzyme activity. CD production could be improved with an ultrafiltration membrane reactor for continuous removal of the products; the starch concentration should be maintained below an inhibitory concentration and limit dextrins would remain in the reactor without affecting enzyme activity.

The Influence of Oxygen Volumetric Mass Transfer Rates on Cyclodextrin Glycosyltransferase Production by Alkaliphilic Bacillus circulans in Batch and Fed-Batch Cultivations

In this work, the influence of oxygen mass transfer rates on the production of cyclodextrin glycosyltransferase (CGTase) by the alkaliphilic bacterium Bacillus circulans ATCC 21783 was investigated. Experimental design and response surface methodology were applied to optimize agitation speed and air flow rate in batch cultivations, in order to identify their significant effects and interactions with the synthesis of CGTase. Results were expressed as the volumetric mass transfer rates of oxygen (kla, [per hour]). The maximal CGTase productivity of 155 U mL−1 h−1 was achieved with kla of 48 h−1. CGTase production was also studied in fed-batch cultures using the optimized parameters obtained in the batch experiments. The maximal CGTase productivity on fed-batch cultivations was 137 U mL−1 h−1 with feeding rates of starch at 0.17 g L−1 h−1.

Influence of starch pretreatment on yield of cyclodextrins and performance of ultrafiltration membranes

The effect of starch pretreatment temperature and exposure time of raw tapioca starch on cyclodextrins (CDs) production and membrane performance using Thermoanaerobacter cyclodextrin glycosyltransferase (CGTase) were investigated in this study. Response surface methodology (RSM) was used to identify the optimum amount of CDs produced. Results indicated that the optimal exposure time and pretreatment temperature for optimum CDs production were 1 h and 70ºC, respectively. Employment of this critical condition (70ºC) as the starch pretreatment temperature, promoted high CDs production and better preservation on the starch granule structure and increased as such as 93% and 94% of swelling power and leached amylose, respectively. In the case of starch pretreatment temperature that is above the critical value (70ºC), such application would apparently increase the solution viscosity and hydraulic resistance that subsequently decrease the membrane flux, despite increasing the CDs yield, suggesting that temperature is a crucial factor in determining the efficiency of CDs production, in particularly in the continuous system.

Enzymatic route to alkyl glycosides having oligomeric head groups

Cyclodextrin glycosyl transferase (CGTase) from Bacillus macerans was used to catalyse the coupling of α-cyclodextrin to alkyl β-glycosides. The acceptor substrate dodecyl β-maltoside was thus converted to dodecyl β-D-maltooctaoside. Further coupling steps and disproportionation reactions occurred, but by optimisation of the reaction time, a yield of 50% of the primary coupling product was obtained. The method worked well for a range of acceptors with different length of the carbohydrate part (1–3 glucose residues) and the hydrocarbon chain (10–14 carbon atoms). With respect to the principles of green chemistry, the method is superior to previously used methods involving protection/deprotection reactions.