Catalytic properties of β-cyclodextrin glucanotransferase from alkalophilic<Emphasis Type="Italic">Bacillus</Emphasis> sp. BL-12 and intermolecular transglycosylation of stevioside

The catalytic properties of β-cyclodextrin glucanotransferase (β-CGTase) from alkalophilicBacillus sp. BL-12 specific for the intermolecular transglycosylation of stevioside were investigated. The molecular mass of purified β-CGTase by ultra-filtration and β-cyclodextrin polymer affinity chromatography was estimated to be 90 kDa, which is high compared to other known bacterial CGTases. The optimal pH and temperature were 9.0 and 50°C, respectively, and thermal stability at 40°C was elevated 10-fold in the presence of 1% maltodextrin. The kinetic parameters of the new β-CGTase from alkalophilicBacillus sp. BL-12 indicate that it is more suitable for transglycosylation than the cyclization reaction. Maltodextrin was the most suitable glycosyl donor for transglycosylation of stevioside. The transglycosylation of stevioside was carried out using 60 units of CGTase per gram of maltodextrin, 20 g/L stevioside as the glycosyl acceptor, and 50 g/L maltodextrin as the gycosyl donor at 40°C for 6 h, and a conversion yield of stevioside as high as 76% was obtained.     

Site-saturation engineering of lysine 47 in cyclodextrin glycosyltransferase from Paenibacillus macerans to enhance substrate specificity towards maltodextrin for enzymatic synthesis of 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G)

In this work, the site-saturation engineering of lysine 47 in cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans was conducted to improve the specificity of CGTase towards maltodextrin, which can be used as a cheap and easily soluble glycosyl donor for the enzymatic synthesis of 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G) by CGTase. When using maltodextrin as glycosyl donor, four mutants K47F (lysine→ phenylalanine), K47L (lysine→ leucine), K47V (lysine→ valine) and K47W (lysine→ tryptophan) showed higher AA-2G yield as compared with that produced by the wild-type CGTase. The transformation conditions (temperature, pH and the mass ratio of l-ascorbic acid to maltodextrin) were optimized and the highest titer of AA-2G produced by the mutant K47L could reach 1.97 g/l, which was 64.2 % higher than that (1.20 g/l) produced by the wild-type CGTase. The reaction kinetics analysis confirmed the enhanced maltodextrin specificity, and it was also found that compared with the wild-type CGTase, the four mutants had relatively lower cyclization activities and higher disproportionation activities, which was favorable for AA-2G synthesis. The mechanism responsible for the enhanced substrate specificity was further explored by structure modeling and it was indicated that the enhancement of maltodextrin specificity may be due to the short residue chain and the removal of hydrogen bonding interactions between the side chain of residue 47 and the sugar at ?3 subsite. Here the obtained mutant CGTases, especially the K47L, has a great potential in the production of AA-2G with maltodextrin as a cheap and easily soluble substrate.     

Systems Engineering of Tyrosine 195, Tyrosine 260, and Glutamine 265 in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Enhance Maltodextrin Specificity for 2-O-d-Glucopyranosyl-l-Ascorbic Acid Synthesis

In this work, the site saturation mutagenesis of tyrosine 195, tyrosine 260 and glutamine 265 in the cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans was conducted to improve the specificity of CGTase for maltodextrin, which can be used as a cheap and easily soluble glycosyl donor for the synthesis of 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G). Specifically, the site-saturation mutagenesis of three sites—tyrosine 195, tyrosine 260, and glutamine 265—was performed, and it was found that the resulting mutants (containing the mutations Y195S [tyrosine → serine], Y260R [tyrosine → arginine], and Q265K [glutamine → lysine]) produced higher AA-2G yields than the wild type and the other mutant CGTases when maltodextrin was used as the glycosyl donor. Furthermore, double and triple mutations were introduced, and four mutants (containing Y195S/Y260R, Y195S/Q265K, Y260R/Q265K, and Y260R/Q265K/Y195S) were obtained and evaluated for the capacity to produce AA-2G. The Y260R/Q265K/Y195S triple mutant produced the highest titer of AA-2G at 1.92 g/liter, which was 60% higher than that (1.20 g/liter) produced by the wild-type CGTase. The kinetics analysis of AA-2G synthesis by the mutant CGTases confirmed the enhanced maltodextrin specificity, and it was also found that compared with the wild-type CGTase, all seven mutants had lower cyclization activities and higher hydrolysis and disproportionation activities. Finally, the mechanism responsible for the enhanced substrate specificity was explored by structure modeling, which indicated that the enhancement of maltodextrin specificity may be related to the changes of hydrogen bonding interactions between the side chain of residue at the three positions (195, 260, and 265) and the substrate sugars. This work adds to our understanding of the synthesis of AA-2G and makes the Y260R/Q265K/Y195S mutant a good starting point for further development by protein engineering.     

Addition of polar organic solvents can improve the product selectivity of cyclodextrin glycosyltransferase. Solvent effects on cgtase

Cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) is an enzyme that produces cyclodextrins from starch via an intramolecular transglycosylation reaction. Addition of small amounts (10% v/v) of polar organic solvents can affect both the overall production yield and the type of cyclodextrin produced from a maltodextrin substrate under simulated industrial process conditions. Using CGTase from Thermoanaerobacter sp. all solvents produced an increase in cyclodextrin yield when compared with a control, the greatest increase being obtained with addition of ethanol (26%). In addition product selectivity was affected by the nature of the organic solvent used: beta-cyclodextrin was favoured in the absence of any solvent and on the addition of dimethylsulphoxide, t-butanol and dimethylformanide while alpha-cyclodextrin was favoured by addition of acetonitrile, ethanol and tetrahydrofuran. With CGTase from Bacillus circulans strain 251 relatively smaller increases in overall cyclodextrin production were achieved (between 5-10%). Addition of t-butanol to a B. circulans catalysed reaction however did produce the largest selectivity for beta-cyclodextrin of any solvent-enzyme combination (82%). The effect of solvent addition was shown not to be related to the product inhibition of CGTase, but may be related to reduced competition from the intermolecular transglycosylation reaction that causes degradation of cyclodextrin products. This rate of this reaction was shown to be dependent on the nature of the organic solvent used.     

Transglycosylation of L-ascorbic acid

Cyclodextrin glucanotransferases (CGTase, EC 2.4.1.19) produced by mesophilic, thermophilic, and halophilic bacilli, as well as maltase (EC 3.2.1.20) produced by various strains of Saccharomyces cerevisiae have been applied for transglycosylation of L-ascorbic acid using starch, maltodextrin, gamma-cyclodextrin, and maltose as donors of glucosyl residue. The CGTases produced by thermophilic strains are the most efficient. The degree of transglucosylation is more than 60%.     

环糊精葡萄糖基转移酶的结构特征与催化机理

随着环糊精在食品、医药等领域的应用越来越广,生产环糊精所必需的环糊精葡萄糖基转移酶(CGT酶)已经成为当今研究的热点。特别是近二十年来,国外对该酶进行了比较深入的研究。首先介绍了CGT酶的功能特性与结构特征。CGT酶是一种多功能型酶,能催化三种转糖基反应(歧化、环化和耦合反应)和水解反应,其中,能将淀粉转化为环糊精的环化反应是特征反应;作为α-淀粉酶家族的成员,CGT酶除了具有与α-淀粉酶相同的A、B、C结构域外,还存在D和E结构域。另外,对CGT酶的催化机理包括底物结合方式、转糖苷反应机理以及环化机理等进行了详细的讨论。     

Iterative Saturation Mutagenesis of ?6 Subsite Residues in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Improve Maltodextrin Specificity for 2-O-d-Glucopyranosyl-l-Ascorbic Acid Synthesis

2-O-d-Glucopyranosyl-l-ascorbic acid (AA-2G), a stable l-ascorbic acid derivative, is usually synthesized by cyclodextrin glycosyltransferase (CGTase), which contains nine substrate-binding subsites (from +2 to −7). In this study, iterative saturation mutagenesis (ISM) was performed on the −6 subsite residues (Y167, G179, G180, and N193) in the CGTase from Paenibacillus macerans to improve its specificity for maltodextrin, which is a cheap and easily soluble glycosyl donor for AA-2G synthesis. Site saturation mutagenesis of four sites—Y167, G179, G180, and N193—was first performed and revealed that four mutants—Y167S, G179R, N193R, and G180R—produced AA-2G yields higher than those of other mutant and wild-type CGTases. ISM was then conducted with the best positive mutant as a template. Under optimal conditions, mutant Y167S/G179K/N193R/G180R produced the highest AA-2G titer of 2.12 g/liter, which was 84% higher than that (1.15 g/liter) produced by the wild-type CGTase. Kinetics analysis of AA-2G synthesis using mutant CGTases confirmed the enhanced maltodextrin specificity and showed that compared to the wild-type CGTase, the mutants had no cyclization activity but high hydrolysis and disproportionation activities. A possible mechanism for the enhanced substrate specificity was also analyzed through structure modeling of the mutant and wild-type CGTases. These results indicated that the −6 subsite played crucial roles in the substrate binding and catalytic reactions of CGTase and that the obtained CGTase mutants, especially Y167S/G179K/N193R/G180R, are promising starting points for further development through protein engineering.     

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

Hydroquinone glycosides were produced by transglycosylation reactions catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. (Toruzyme^® 3.0L). The reactions were carried out in an aqueous system containing hydroquinone (HQ) and maltodextrin as acceptor and donor substrate molecules respectively. The conditions for the synthesis of hydroquinone glucoside (α-arbutin) were 9 mM hydroquinone, maltodextrin (5%, w/v) in 20 mM citrate phosphate buffer, pH 5.5 and 0.025 mg/ml toruzyme at 40 °C for 24 h. The transfer efficiency of hydroquinone glycosylation was 31.8% and 29.2% respectively, when α-cyclodextrin and maltodextrin were employed as donor substrates. The major glycoside product was identified as hydroquinone-1-O-α-d-glucopyranoside (α-arbutin) on the basis of mass spectrometric, nuclear magnetic resonance analysis and component analysis of its enzymatic hydrolysates. The highest molar yield of α-arbutin (21.2%) was obtained when α-cyclodextrin was used as the donor substrate. A two step enzymatic reaction system comprising of CGTase and amyloglucosidase helped to attain a molar yield of 30% for α-arbutin. At room temperature the solubility of α-arbutin in water was determined to be 12.8 g/100 ml which is approximately 1.8 fold higher than that of hydroquinone.     

Immobilization of cyclodextrin glucanotransferase on amberlite IRA-900 for biosynthesis of transglycosylated xylitol

Cyclodextrin glucanotransferase (CGTase) fromThermoanaerobacter sp. was adsorbed on the ion exchange resin Amberlite IRA-900. The optimum conditions for the immobilization of the CGTase were pH 6.0 and 600 U CGTase/g resin, and the maximum yield of immobilization was around 63% on the basis of the amount ratio of the adsorbed enzyme to the initial amount in the solution. Immobilization of CGTase shifted the optimum temperature for the enzyme to produce transglycosylated xylitol from 70°C to 90°C and improved the thermal stability of immobilized CGTase, especially after the addition of soluble starch and calcium ions. Transglycosylated xylitol was continuously produced using immobilized CGTase in the column type packed bed reactor, and the operating conditions for maximum yield were 10% (w/v) dextrin (13 of the dextrose equivalent) as the glycosyl donor, 10% (w/v) xylitol as the glycosyl acceptor, 20 mL/h of medium flow rate, and 60°C. The maximum yield of transglycosylated xylitol and productivity were 25% and 7.82 g·L⁻¹·h⁻¹, respectively. The half-life of the immobilized CGTase in a column type packed bed reactor was longer than 30 days.     

Structures of maltohexaose and maltoheptaose bound at the donor sites of cyclodextrin glycosyltransferase give insight into the mechanisms of transglycosylation activity and cyclodextrin size specificity

The enzymes from the alpha-amylase family all share a similar alpha-retaining catalytic mechanism but can have different reaction and product specificities. One family member, cyclodextrin glycosyltransferase (CGTase), has an uncommonly high transglycosylation activity and is able to form cyclodextrins. We have determined the 2.0 and 2.5 A X-ray structures of E257A/D229A CGTase in complex with maltoheptaose and maltohexaose. Both sugars are bound at the donor subsites of the active site and the acceptor subsites are empty. These structures mimic a reaction stage in which a covalent enzyme-sugar intermediate awaits binding of an acceptor molecule. Comparison of these structures with CGTase-substrate and CGTase-product complexes reveals three different conformational states for the CGTase active site that are characterized by different orientations of the centrally located residue Tyr 195. In the maltoheptaose and maltohexaose-complexed conformation, CGTase hinders binding of an acceptor sugar at subsite +1, which suggests an induced-fit mechanism that could explain the transglycosylation activity of CGTase. In addition, the maltoheptaose and maltohexaose complexes give insight into the cyclodextrin size specificity of CGTases, since they precede alpha-cyclodextrin (six glucoses) and beta-cyclodextrin (seven glucoses) formation, respectively. Both ligands show conformational differences at specific sugar binding subsites, suggesting that these determine cyclodextrin product size specificity, which is confirmed by site-directed mutagenesis experiments.     

Conversion of cyclodextrin glycosyltransferase into a starch hydrolase by directed evolution: the role of alanine 230 in acceptor subsite +1

Cyclodextrin glycosyltransferase (CGTase) preferably catalyzes transglycosylation reactions, whereas many other alpha-amylase family enzymes are hydrolases. Despite the availability of three-dimensional structures of several transglycosylases and hydrolases of this family, the factors that determine the hydrolysis and transglycosylation specificity are far from understood. To identify the amino acid residues that are critical for the transglycosylation reaction specificity, we carried out error-prone PCR mutagenesis and screened for Bacillus circulans strain 251 CGTase mutants with increased hydrolytic activity. After three rounds of mutagenesis the hydrolytic activity had increased 90-fold, reaching the highest hydrolytic activity ever reported for a CGTase. The single mutation with the largest effect (A230V) occurred in a residue not studied before. The structure of this A230V mutant suggests that the larger valine side chain hinders substrate binding at acceptor subsite +1, although not to the extent that catalysis is impossible. The much higher hydrolytic than transglycosylation activity of this mutant indicates that the use of sugar acceptors is hindered especially. This observation is in favor of a proposed induced-fit mechanism, in which sugar acceptor binding at acceptor subsite +1 activates the enzyme in transglycosylation [Uitdehaag et al. (2000) Biochemistry 39, 7772-7780]. As the A230V mutation introduces steric hindrance at subsite +1, this mutation is expected to negatively affect the use of sugar acceptors. Thus, the characteristics of mutant A230V strongly support the existence of the proposed induced-fit mechanism in which sugar acceptor binding activates CGTase in a transglycosylation reaction.     

Intermolecular transglycosylating reaction of cyclodextrin glucanotransferase immobilized on capillary membrane

The intermolecular transglycosylating reaction of cyclodextrin glucanotransferase ([EC 2.4.1.19]; CGTase) immobilized on a capillary membrane was investigated using low molecular weight substrates such as cyclodextrin (CD), maltooligosaccharide (MOS), and a CD-MOS mixture. The immobilized CGTase catalyzed the conversion reaction of α-CD to β-CD and MOS or β-CD to α-CD and MOS within a short residence time. The conversion ratio increased as the amount of immobilized CGTase increased. The addition of glucose, maltose, and sucrose as acceptors in the substrate solution containing CD resulted in the acceleration of CD degradation compared with only CD substrate. Furthermore, the MOS substrate (degree of polymerization =2–6) was disproportionated with a conversion ratio exceeding 70% by the immobilized CGTase. These data demonstrate that immobilized CGTase can catalyze intermolecular transglycosylation between low molecular substrates in a few minutes by regulating the amount of immobilized enzyme and the residence time. This might contribute to our comprehension of CGTase-immobilized bioreactors for CD production as well as to the development of new glycosides through its excellent transglycosylation ability.     

Enzymatic synthesis of piceid glycosides by cyclodextrin glucanotransferase

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

Direct enzymatic glucosylation of naringin in grapefruit juice by alpha-D-glucosidase from the marine mollusc Aplysia fasciata

The enzymatic glucosylations of naringin, performed using alpha-D-glucosidase, identified in the Mediterranean mollusc Aplysia fasciata is reported. The enzyme actively operates on maltose and has an interesting transglycosylation potential using this donor. We also investigated the use of this marine alpha-glucosidase for a food-compatible glucosylation of naringin to produce new enzymatically modified carbohydrate possessing naringin derivatives. The regioselective formations of the beta-gluco-C6 alpha-glucosyl derivative and of the corresponding isomaltosyl diglucoside of naringin were obtained in high yield and efficiency of reaction. Suspensions of naringin can be used up to approximately 90 mg/mL initial acceptor concentration. In different experiments it was demonstrated that the enzyme was still active after 48 h in presence of this high amount of acceptor and that one of the diasteromers of the naringin is preferred by the enzyme from A. fasciata during glucosylation/deglucosylation enzymatic steps. Finally, the feasibility of efficient naringin glucosylation in grapefruit juice was also demonstrated at optimal pH of the enzyme and low maltose concentrations.     

Fusion of Self-Assembling Amphipathic Oligopeptides with Cyclodextrin Glycosyltransferase Improves 2-O-d-Glucopyranosyl-l-Ascorbic Acid Synthesis with Soluble Starch as the Glycosyl Donor

In this study, we fused six self-assembling amphipathic peptides (SAPs) with cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans to catalyze 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G) production with cheap substrates, including maltose, maltodextrin, and soluble starch as glycosyl donors. The results showed that two fusion enzymes, SAP5-CGTase and SAP6-CGTase, increased AA-2G yields to 2.33- and 3.36-fold that of wild-type CGTase when soluble starch was used as a substrate. The cyclization activities of these enzymes decreased, while disproportionation activities increased. Enzymatic characterization of the two fusion enzymes was performed, and kinetics analysis of AA-2G synthesis confirmed the enhanced soluble starch specificity of SAP5-CGTase and SAP6-CGTase compared to that in the wild-type CGTase. As revealed by structure modeling of the fusion and wild-type CGTases, enhanced substrate-binding capacity may result from the increased number of hydrogen bonds present after fusion. This study demonstrates an effective protein fusion approach to improving the substrate specificity of CGTase for AA-2G synthesis. Fusion enzymes, especially SAP6-CGTase, are promising starting points for further development through protein engineering.     

Enzyme-assisted extraction of flavonoids from Ginkgo biloba leaves: improvement effect of flavonol transglycosylation catalyzed by Penicillium decumbens cellulase

We report a novel enzyme-involved approach to improve the extraction of flavonoids from Ginkgo biloba, in which the enzyme is employed not only for cell wall degradation, but also for increasing the solubility of target compounds in the ethanol-water extractant. Penicillium decumbens cellulase, a commercial cell wall-degrading enzyme with high transglycosylation activity, was found to offer far better performance in the extraction than Trichoderma reesei cellulase and Aspergillus niger pectinase under the presence of maltose as the glycosyl donor. TLC, HPLC and MS analysis indicated that P. decumbens cellulase could transglycosylate flavonol aglycones into more polar glucosides, the higher solubility of which led to improved extraction. The influence of glycosyl donor, pH, solvent and temperature on the enzymatic transglycosylation was investigated. For three predominant flavonoids in G. biloba, the transglycosylation showed similar optimal conditions, which were therefore used for the enzyme-assisted extraction. The extraction yield turned to be 28.3mg/g of dw, 31% higher than that under the pre-optimized conditions, and 102% higher than that under the conditions without enzymes. The utilization of enzymatic bifunctionality described here, naming enzymatic modification of target compounds and facilitation of cell wall degradation, provides a novel approach for the extraction of natural compounds from plants.     

In vitro synthesis of artificial polysaccharides by glycosidases and glycosynthases

Artificial polysaccharides produced by in vitro enzymatic synthesis are new biomaterials with defined structures that either mimic natural polysaccharides or have unnatural structures and functionalities. This review summarizes recent developments in the in vitro polysaccharide synthesis by endo-glycosidases, grouped in two major strategies: (a) native retaining endo-glycosidases under kinetically controlled conditions (transglycosylation with activated glycosyl donors), and (b) glycosynthases, engineered glycosidases devoid of hydrolase activity but with high transglycosylation activity. Polysaccharides are obtained by enzymatic polymerization of simple glycosyl donors by repetitive condensation. This approach not only provides a powerful methodology to produce polysaccharides with defined structures and morphologies as novel biomaterials, but is also a valuable tool to analyze the mechanisms of polymerization and packing to acquire high-order molecular assemblies.     

Optimisation of growth medium for the production of cyclodextrin glucanotransferase from Bacillus stearothermophilus HR1 using response surface methodology

Cyclodextrin glucanotransferase (CGTase) activity was observed when the bacterium was grown in the medium at various initial pH values, containing carbon, nitrogen, phosphorus and mineral salt sources at 50 °C for 24 h in the shake flasks. The optimisation of this growth medium was carried out using response surface methodology. The design contains a total of 32 experimental trials involving 10 star points and 6 replicates at the centre points. The design was employed by selecting sago starch, peptone from casein, K₂HPO₄, CaCl₂ and initial pH as five independent variables in this study. The optimal calculated values of tested variables for maximal production of CGTase were found to be comprised of: sago starch, 16.02 g/l; peptone from casein, 20 g/l; K₂HPO₄, 1.4 g/l; CaCl₂, 0.2 g/l and initial pH, 7.54 with a predicted CGTase activity of 14.20 U/ml. These predicted optimal parameters were tested in the laboratory and the final CGTase activity obtained was very close to the predicted value at 14.80 U/ml.     

Immobilization of cyclodextrin glucanotransferase on capillary membrane

A bioreactor system with the enzyme immobilized on a capillary membrane is a promising tool for the mass production of valuable substances, because of the good productive efficiency. To investigate the kinetics of immobilized cyclodextrin glucanotransferase ([EC 2.4.1.19]; CGTase) on a capillary membrane in a bioreactor system, the amount of immobilized CGTase and the operating conditions, such as pressure and the reaction temperature, were examined under a constant substrate concentration (1.0%) and a constant flow rate (0.12 m/s). When the CGTase was immobilized at a concentration of 0.04 to 0.62 mg per membrane area (cm²), the decrease in the immobilized amount of CGTase resulted in an increase in the cyclodextrin production rate (g of CD/h·m²; CPR) and the CPR correlated well with the flux of the CGTase-immobilized membrane. Although a higher reaction temperature caused an increase in the CPR within a short operating time of the bioreactor, repeated operation at 60°C led to a reduction in the CPR due to the denaturation of the immobilized CGTase. The percentage of cyclodextrin (CD) to total sugar obtained in the permeate was slightly more than 60% under most operating conditions, but immobilization of the excess amount of CGTase (0.42–0.62 mg/cm²) reduced the CD yield as well as the ratio of α-CD to β-CD, suggesting that it led to a CGTase side-reaction such as intermolecular transglycosylation. These data suggest that the conditions under which the bioreactor with 0.04–0.40 mg/cm² was operated; a reaction temperature of 50°C, a residence time of 1–2 min and adjustable pressure, could be employed to obtain a high CPR using a large scale CGTase-immobilized membrane bioreactor.     

The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms.

Cyclodextrin glycosyltransferase (CGTase) catalyzes three transglycosylation reactions via a double displacement mechanism involving a covalent enzyme-intermediate complex (substituted-enzyme intermediate). Characterization of the three transglycosylation reactions, however, revealed that they differ in their kinetic mechanisms. Disproportionation (cleavage of an alpha-glycosidic bond of a linear malto-oligosaccharide and transfer of one part to an acceptor substrate) proceeds according to a ping-pong mechanism. Cyclization (cleavage of an alpha-glycosidic bond in amylose or starch and subsequent formation of a cyclodextrin) is a single-substrate reaction with an affinity for the high molecular mass substrate used, which was too high to allow elucidation of the kinetic mechanism. Michaelis-Menten kinetics, however, have been observed using shorter amylose chains. Coupling (cleavage of an alpha-glycosidic bond in a cyclodextrin ring and transfer of the resulting linear malto-oligosaccharide to an acceptor substrate) proceeds according to a random ternary complex mechanism. In view of the different kinetic mechanisms observed for the various reactions, which can be related to differences in substrate binding, it should be possible to mutagenize CGTase in such a manner that a single reaction is affected most strongly. Construction of CGTase mutants that synthesize linear oligosaccharides instead of cyclodextrins thus appears feasible. Furthermore, the rate of interconversion of linear and circular conformations of oligosaccharides in the cyclization and coupling reactions was found to determine the reaction rate. In the cyclization reaction this conversion rate, together with initial binding of the high molecular mass substrate, may determine the product specificity of the enzyme. These new insights will allow rational design of CGTase mutant enzymes synthesizing cyclodextrins of specific sizes.