Action of Cyclodextrin Glycosyltransferase from Bacillus megaterium Strain No. 5 on Starch

The amounts of the cyclodextrins G6, G7 and G8 produced by the action of the enzyme from Bacillus megaterium (No. 5 enzyme) and Bacillus macerans amylase (BMA) on starch-¹⁴C (U) were determined by the calculation of radioactivity. Both fractions of No. 5 enzyme produced the cyclodextrin G6, G7 and G8 in the proportion of 1: 2.4: 1. On the other hand, BMA produced the cyclodextrin G6, G7 and G8 in the proportion of 2.7: 1:1. The cyclodextrin G6 and G8 which are smaller parts of the reaction products by both fractions of No. 5 enzyme were found to be produced directly from starch, not from the redecomposition of cyclodextrin G7. The ratio of the cyclodextrin G6, G7 and G8 were almost constant, regardless of the pH range of the reaction system.

By using the maltooligosaccharides terminated at the reducing end by radioactive glucose, the action of both fractions of No. 5 enzyme and BMA on the maltooligosaccharides were compared with each other. The results showed that both fractions of No. 5 enzyme acted on oligosaccharides larger than maltose, producing the radioactive glucose as the major product from each maltooligosaccharide (G2~G8). On the other hand, BMA acted on oligosaccharides larger than maltotriose, producing the radioactive maltose as the major product.     

Two-step enzymatic synthesis of maltooligosaccharide esters

Glucose and maltose esters were synthesised in organic media by employing a lipase (E.C. 3.1.1.3) from Candida antarctica. In a second reaction step, a transglycosylation catalysed by a cyclodextrin glycosyltransferase (E.C. 2.4.1.19) from either Paenibacillus sp. F8 or Bacillus sp. strain no. 169 (DSM 2518) extended the degree of polymerisation (DP) of the carbohydrate moieties of the carbohydrate esters. The donor substrates used were either a cyclodextrin, a maltooligosaccharide or starch. The highest rate of low DP maltooligosaccharide ester formation was obtained when starch was used as glycosyl donor and caproyl maltose as glycosyl acceptor. The structures of two of the products were identified by 1H and 13C NMR and MALDI-TOF MS as capronate monoesters of maltotriose and maltotetraose, with the ester bond at C-6 of the second glucose unit from the reducing end.     

A New Trisaccharide, 2-α-Maltosyl-glucose,Synthesized by the Transglycosylation Reaction of Cyclodextrin Glycosyltransferase

The transglycosylation reaction of the cyclodextrin glycosyltransferase from Bacillus megaterium strain No. 5 was examined in the reaction system containing kojibiose and soluble starch. As the transglycosylation product, a new trisaccharide was chromatographically isolated. It was confirmed that the trisaccharide was 2-α-maltosyl-glucose ([α]d + 162.0°, α-undecaacetate: mp 105~106°C, [α]d + 163.0°), α-d-glucopyranosyl-(1→4)-α-d-glucopyranosyl-(1→2)-α-d-glucose (4²-α-glucosyl-kojibiose).

The transfer action to kojibiose occurred only to the C₄-hydroxyl group of the non-reducing end glucose unit of kojibiose, leading to the formation of 2-α-maltosyl-glucose.     

A novel and efficient method for enzymatic synthesis of high purity maltose using moranoline (1-deoxynojirimycin).

A transglycosylation reaction with moranoline (1-deoxynojirimycin) was done with soluble starch as the glucosyl donor and Bacillus macerans amylase as a cyclodextrin glycosyltransferase [EC 2.4.1.19]. The resultant transglycosylation products with moranoline, obtained by treating the reaction mixture with a strong cation exchange resin, were hydrolyzed by beta-amylase [EC 3.2.1.2] from sweet potatoes. The hydrolysate was treated with a strong cation exchange resin, and high purity maltose was obtained.     

Molecular imprinting of cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans with gamma-cyclodextrin

Cyclodextrin glycosyltransferase catalyzes the formation of a mixture of cyclodextrins from starch by an intramolecular transglycosylation reaction. To manipulate the product specificity of the Paenibacillus sp. A11 and Bacillus macerans cyclodextrin glycosyltransferases towards the preferential formation of gamma-cyclodextrin (CD(8)), crosslinked imprinted proteins of both cyclodextrin glycosyltransferases were prepared by applying enzyme imprinting and immobilization methodologies. The crosslinked imprinted cyclodextrin glycosyltransferases obtained by imprinting with CD(8) showed pH and temperature optima similar to those of the native and immobilized cyclodextrin glycosyltransferases. However, the pH and temperature stability of the immobilized and crosslinked imprinted cyclodextrin glycosyltransferases were higher than those of the native cyclodextrin glycosyltransferases. When the catalytic activities of the native, immobilized and crosslinked imprinted cyclodextrin glycosyltransferases were compared, the efficiency of the crosslinked imprinted enzymes for CD(8) synthesis was increased 10-fold, whereas that for cyclodextrin hydrolysis was decreased. Comparison of the product ratios by high-performance anion exchange chromatography showed that the native cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans produced CD(6) : CD(7) : CD(8) : > or = CD(9) ratios of 15 : 65 : 20 : 0 and 43 : 36 : 21 : 0 after 24 h of reaction at 40 degrees C with starch substrates. In contrast, the crosslinked imprinted cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans produced cyclodextrin in ratios of 15 : 20 : 50 : 15 and 17 : 14 : 49 : 20, respectively. The size of the synthesis products formed by the crosslinked imprinted cyclodextrin glycosyltransferases was shifted towards CD(8) and > or = CD(9), and the overall cyclodextrin yield was increased by 12% compared to the native enzymes. The crosslinked imprinted cyclodextrin glycosyltransferases also showed higher stability in organic solvents, retaining 85% of their initial activity after five cycles of synthesis reactions.     

Production of Schardinger beta-dextrin by soluble and immobilized cyclodextrin glycosyltransferase of an alkalophilic Bacillus sp.

Succinylated cyclodextrin glycosyltransferase (EC 3.2.1.19) of an alkalophilic Bacillus sp. was adsorbed on a vinylpyridine copolymer. The enzyme had about 25% of the activity of soluble enzyme added. No increase of pH or thermal stability of the enzyme was observed by the adsorption, whereas optimum temperature for the enzyme action was shifted from 50 to 55 degrees C. The enzyme converted starch to cyclodextrine without significant loss of activity under the conditions of 4 times reusing of 6 hr conversion by the batch system or 2 weeks continuous reaction by the column system at 55 degrees C and pH 8.0. About 46% of the potato starch solution [15% (w/v)] was converted to cyclodextrins by the enzyme, and 52% was converted by the simultaneous action of the enzyme and alkaline pullulanase of alkalophilic Bacillus sp. (No. 202-1). These values were almost the same as those obtained by the soluble enzyme or enzymes system.     

Synthesis of Malto-Oligosaccharides Via the Acceptor Reaction Catalyzed by Cyclodextrin Glycosyltransferases

The disproportionation activity (intermolecular transglycosylation) of cyclomaltodextrin glycosyltransferases (CGTases) from Thermoanaerobacter sp. and Bacillus circulans strain 251 was studied. Using soluble starch as donor, the CGTase from Thermoanaerobacter sp. showed the highest transglycosylation activity with all the malto-oligosaccharides tested as acceptors. At ratios of starch: D-glucose from 2:1 to 1:2 (w/w), the formation of cyclodextrins was completely inhibited, and a homologous series of malto-oligosaccharides (G_n) was produced. The conversion of starch into acceptor products was in the range of 63-79% in 48 h. The degree of polymerisation of malto-oligosaccharides formed could be modulated by the ratio of starch: D-glucose provided; at a ratio of 1:2 (w/w), the reaction was quite selective for the formation of G2-G3.     

Enzymatic Synthesis of 4-O-α-d-Glucopyranosyl-moranoline

A transglycosylation reaction with moranoline (1-deoxynojirimycin) was carried out with α-cyclodextrin as the glucose donor and Bacillus macerans amylase as cyclodextrin glycosyltransferase [EC 2.4.1.19]. The resultant transglycosylation products were hydrolyzed by glucoamylase [EC 3.2.1.3] from Rhizopus niveus. The hydrolyzate (the transglycosylation product of the lowest molecular weight) was isolated and the structure was found by physico-chemical methods to be 4-O-α-d-glucopyranosyl-moranoline.     

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.     

Production of cyclodextrin from starch by cyclodextrin glycosyltransferase from Bacillus firmus and characterization of purified enzyme

During screening for cyclodextrin-forming microorganisms, an alkalophilic Bacillus sp, which produced high activity of cyclodextrin glycosyltransferase, was isolated and identified as Bacillus firmus. The crude enzyme transformed starch to mainly β-and γ-cyclodextrin. The purified enzyme had an optimum pH of 7.5–8.5 and its optimum temperature was 65°C, which is the highest optimum temperature as compared to other cyclodextrin glycosyltransferases except that produced by Bacillus amyloliquefaciens. Received 06 January 1997/ Accepted in revised form 20 March 1997     

Cyclodextrin production by cyclodextrin glycosyltransferase from Bacillus circulans DF 9R

Cyclodextrins (CD) are cyclic oligosaccharides with multiple applications in the food, pharmaceutical, cosmetic, agricultural and chemical industries. In this work, the conditions used to produce CD with cyclodextrin glycosyltransferase from Bacillus circulans DF 9R were optimized using experimental designs. The developed method allowed the partial purification and concentration of the enzyme from the cultural broth and, subsequently, the CD production, using the same cassava starch as enzyme adsorbent and as substrate. Heat-treatment of raw starch at 70 degrees C for 15 min in the presence of adsorbed cyclodextrin glycosyltransferase allowed the starch liquefaction without enzyme inactivation. The optimum conditions for CD production were: 5% (w/v) cassava starch, 15 U of enzyme per gram of substrate, reaction temperature of 56 degrees C and pH 6.4. After 4h, the proportion of starch converted to CD reached 66% (w/w) and the weight ratio of alpha-CD:beta-CD:gamma-CD was 1.00:0.70:0.16.     

Thermoanaerobacterium thermosulfurigenes cyclodextrin glycosyltransferase.

Cyclodextrin glycosyltransferase (CGTase) uses an alpha-retaining double displacement mechanism to catalyze three distinct transglycosylation reactions. To investigate these reactions as catalyzed by the CGTase from Thermoanaerobacterium thermosulfurigenes the enzyme was overproduced (8 mg.L(-1) culture) using Bacillus subtilis as a host. Detailed analysis revealed that the three reactions proceed via different kinetic mechanisms. The cyclization reaction (cyclodextrin formation from starch) is a one-substrate reaction, whereas the other two transglycosylation reactions are two-substrate reactions, which obey substituted enzyme mechanism kinetics (disproportionation reaction) or ternary complex mechanism kinetics (coupling reaction). Analysis of the effects of acarbose and cyclodextrins on the disproportionation reaction revealed that cyclodextrins are competitive inhibitors, whereas acarbose is a mixed type of inhibitor. Our results show that one molecule of acarbose binds either in the active site of the free enzyme, or at a secondary site of the enzyme-substrate complex. The mixed inhibition thus indicates the existence of a secondary sugar binding site near the active site of T. thermosulfurigenes CGTase.     

Characterization of Bacillus stearothermophilus cyclodextrin glucanotransferase in ascorbic acid 2-O-alpha-glucoside formation

In this study, we characterized cyclodextrin glucanotransferase (CGTase) from Bacillus stearothermophilus in L-ascorbic acid-2-O-alpha-D-glucoside (AA-2G) formation and compared its enzymological properties with those of rat intestinal and rice seed alpha-glucosidases which had the ability to form AA-2G. CGTase formed AA-2G efficiently using alpha-cyclodextrin (alpha-CD) as a substrate and ascorbic acid (AA) as an acceptor. Several AA-2-oligoglucosides were also formed in this reaction mixture, and they could be converted to AA-2G by the additional treatment of glucoamylase. The optimum temperature for AA-2G formation was 70 degrees C and its optimum pH was around 5.0. CGTase also utilized beta- and gamma-CDs, maltooligosaccharides, dextrin, amylose, glycogen and starch as substrates, but not any disaccharides except maltose. CGTase showed the same acceptor specificity as two alpha-glucosidases, whereas its hydrolyzing activity towards AA-2G was very low compared with those of alpha-glucosidases. Cleavage profiles of AA-2-oligoglucosides by CGTase present a possible mechanism for AA-2G formation that CGTase transfers a glucose-hexamer to an acceptor at the first step and then a glucose is stepwisely removed from the non-reducing end of the product through glucoamylase-like action of this enzyme. These results indicate that CGTase is able to synthesize AA-2G more efficiently than rat and rice alpha-glucosidases and utilization of this enzyme makes the mass production of AA-2G possible.     

Succinylation of cyclodextrin glycosyltransferase from Thermoanaerobacter sp. 501 enhances its transferase activity using starch as donor

A simple modification procedure, the succinylation of amino groups, was suitable to increase the transferase (disproportionation) activity of cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacter sp. 501 using different linear oligosaccharides as acceptors. On the contrary, the synthesis of cyclodextrins (CDs), the coupling of CDs with oligosaccharides, and the hydrolysis of starch decreased after chemical modification. The degree of succinylation of amino groups (45%) was accurately determined by MALDI-TOF mass spectrometry. The formation of CDs under industrial conditions was analyzed for native and succinylated CGTases, showing similar selectivity to alpha-, beta-, gamma-CD. The acceptor reaction with D-glucose using soluble starch as glucosyl donor was studied at 60 degrees C and pH 5.5. Malto-oligosaccharides (MOS) production was notably higher using the semisynthetic enzyme at different ratios (w/w) starch:D-glucose. Thus, more than 90% of the initial starch was converted into MOS (G2-G7) in 48 h employing a ratio donor:acceptor 1:2 (w/w).     

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.     

Engineering cyclodextrin glycosyltransferase into a starch hydrolase with a high exo-specificity

Cyclodextrin glycosyltransferase (CGTase) enzymes from various bacteria catalyze the formation of cyclodextrins from starch. The Bacillus stearothermophilus maltogenic alpha-amylase (G2-amylase is structurally very similar to CGTases, but converts starch into maltose. Comparison of the three-dimensional structures revealed two large differences in the substrate binding clefts. (i) The loop forming acceptor subsite +3 had a different conformation, providing the G2-amylase with more space at acceptor subsite +3, and (ii) the G2-amylase contained a five-residue amino acid insertion that hampers substrate binding at the donor subsites -3/-4 (Biochemistry, 38 (1999) 8385). In an attempt to change CGTase into an enzyme with the reaction and product specificity of the G2-amylase, which is used in the bakery industry, these differences were introduced into Thermoanerobacterium thermosulfurigenes CGTase. The loop forming acceptor subsite +3 was exchanged, which strongly reduced the cyclization activity, however, the product specificity was hardly altered. The five-residue insertion at the donor subsites drastically decreased the cyclization activity of CGTase to the extent that hydrolysis had become the main activity of enzyme. Moreover, this mutant produces linear products of variable sizes with a preference for maltose and had a strongly increased exo-specificity. Thus, CGTase can be changed into a starch hydrolase with a high exo-specificity by hampering substrate binding at the remote donor substrate binding subsites.     

Effect of different substrates and their preliminary treatment on cyclodextrin production

Summary Various kinds of substrates were tested for cyclodextrin production with cyclodextrin glucanotransferase (CGTase) from Bacillus megaterium. The enzyme formed cyclodextrin from different kinds of starch, dextrins, amylose, and amylopectin. However, the highest degree of conversion was obtained from starch. Corn starch appeared to be the best substrate – the cyclodextrin yield was 50.9%. The effect of molecular mass and preliminary treatment of starch with α-amylase on the CD yield was investigated. It was proved that CGTase preferred native starch with high molecular mass and low dextrose equivalent. The preliminary treatment with α-amylase occurred to be inefficient and unnecessary since it did not lead to an increase in the CD yield. Some of the substrates were treated with pullulanase. The effect of debranching was highest in the case of corn starch: the cyclodextrin yield increased by 10%.     

Inspection of human salivary alpha-amylase action by its transglycosylation action

The course of the action of human salivary alpha-amylase (HSA) on a substrate was examined taking advantage of its transglycosylation action. IG5 phi (IG-G-G-G-G-phi), IG4 phi (IG-G-G-G-phi), and GIG4 phi (G-IG-G-G-G-phi) were used as the substrates and p-nitrophenyl alpha-glucoside (GP, G-P) as the acceptor. HSA hydrolyzes IG5 phi, IG4 phi, and GIG4 phi to IG3 (IG-G-G) and G2 phi (G-G-phi), to IG3 and G phi (G-phi), and to GIG3 (G-IG-G-G) and G phi, respectively. In the presence of GP, a part of the glycon residues, IG3 and GIG3, were transferred to the acceptor to give IG4P (IG-G-G-G-P) and GIG4P (G-IG-G-G-G-P), respectively. Whenever the enzyme attacks the substrate, G phi or G2 phi is liberated in both transglycosylation and hydrolysis. The extent of transglycosylation can be, therefore, estimated from the molar ratio of the transfer product to the liberated aglycon, G phi or G2 phi. HPLC analysis of the reaction mixtures revealed that the value of IG4P/G phi in the digest of IG4 phi was nearly equal to that of GIG4P/G phi in the digest of GIG4 phi and these values were ten times larger than that of IG4P/G2 phi in the digest of IG5 phi. These data suggested that G phi residue would fall away from aglycon binding site more rapidly than G2 phi residue after the cleavage of the alpha-1,4-glycosidic linkage to offer GP more chance to attack to the activated glycon and also indicated that the space of the glycon binding site corresponds to three glucose residues.     

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.     

Purification and Properties of Cyclodextrin Glycosyltransferase of an Alkalophilic Bacillus sp.

Extracellular cyclodextrin glycosyltransferase (α-1,4-glucan 4-glycosyltransferase, cyclizing, EC 3.2.1.19) of an alkalophilic Bacillus sp. (ATCC 21783) was purified about 74-fold and shown to be a single, homogeneous protein by disc polyacryl amide gel electrophoresis and ultracentrifugation. The molecular weight and isoelectric point were 88,000 and pH 5.4. The optimum pH for the enzyme action was 4.5-4.7. The apparent V_max and Km values for α-, β- and γ-cyclodextrin at the constant concentration of sucrose were 133.3, 23.4, 12.3 µmoles glucose/min per mg protein and 5.88, 0.39, 0.25 mm, respectively. The enzyme converted about 73% of starch, 65% of amylopectin, 45% of glycogen and 25% of amylopectin (β-limit dextrin to cyclodextrins.