CGTase Activity Research Articles

ＣＧＴａｓｅのアミロースへの作用

The action of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) on amylose was reinvesti-gated by determining the product specificity of the cyclization reaction and the substrate specificity of the coupling and hydrolytic reactions of CGTases from alkalophilic Bacillus sp. A2-5a (A2-5a CGTase), Bacillus macerans CGTase (Bmac CGTase) and Bacillus stearothermophilus CGTase (Bste CGTase). The product specificity of the cyclization reaction of CGTase was determined by quantifying each cyclic α-1, 4-glucans with degree of polymerization (DP) from 6 to 31 (CD6 to CD31; CDn, cyclic α-1, 4-glucan with DP of n) produced at the initial reaction stage with HPAEC. All three CGTases produced CD6 to CD31 simultaneously. However, A2-5a CGTase produced more CD7, while Bmac CGTase produced more CD6 than other cyclic α-1, 4-glucans. The speci-ficity to produce CD7 or CD6 was very strict since the cyclic α-1, 4-glucan having a difference of only one glucose unit were not preferably produced by either enzyme. 4n the other hand, Bste CGTase produced more CD6 and CD7 than other cyclic α-1, 4-glucans, respectively. The substrate specificity of the coupling and hydrolytic reactions were determined by using CD6, CD7, CD8 and larger cyclic α-1, 4-glucans (mixture of CD22 to CD50). For the coupling and hydrolytic activities of all three CGTases, CD7 and CD8 were extremely poor substrate, while those activities on larger cyclic α-1, 4-glucans were higher than CD6, CD7 and CD8. The substrate specificity in the cou-pling and hydrolytic reaction could be attributed to the conversion of larger cyclic α-1, 4-glucans into smaller ones. The hydrolytic activity of CGTases on larger cyclic α-1, 4-glucans were 1/1.6 to 1/5 of the coupling activity, which indicated that the hydrolytic activity of CGTase was not so small as negligible. The hydrolytic activities of Bste CGTase were the highest among the three CGTases, while those of Bmac CGTase were the lowest, which would cause the difference in the conversion rate from larger cyclic a-1, 4-glucans into smaller ones. Based on these results, we discussed the action of CGTase on amylose.

Preparation of High-Purity Cyclodextrin Glucanotransferase from Bacillussp. 1070

Two schedules have been developed for chromatographic purification of cyclodextrin glucanotransferase (CGTase) from a culture of Bacillussp. 1070. The purification on butyl-Toyopearl and on Cu(II)-iminodiacetic (IDA) agarose resulted in a 9.5-fold purification of the enzyme. The second schedule for purification (chromatography on butyl-Toyopearl and on DEAE-Sephacel) resulted in a 13.5-fold increase in the specific activity of CGTase. By electrophoresis under denaturing conditions, the enzyme purity was shown to be no less than 90%. According to preliminary data, CGTase consists of two isoenzymes with pI 5.1 and 5.3.

Characterization of cyclodextrin glycosyltransferase from Bacillus firmus strain no. 37.

The enzyme cyclodextrin glycosyltransferase (CGTase), EC 2.4.1.19, which produces cyclodextrins (CDs) from starch, was obtained from Bacillusfirmus strain no. 37 isolated from Brazilian soil and characterized in the soluble form using as substrate 100 g/L of maltodextrin in 0.05 M Tris-HCl buffer, 5 mM CaCl2, and appropriate buffers. Enzymatic activity and its activation energy were determined as a function of temperature and pH. The activation energy for the production of beta- and gamma-CD was 7.5 and 9.9 kcal/mol, respectively. The energy of deactivation was 39 kcal/mol. The enzyme showed little thermal deactivation in the temperature range of 35-60 degrees C, and Arrhenius-type equations were obtained for calculating the activity, deactivation, and half-life as a function of temperature. The molecular weight of the enzyme was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, giving 77.6 kDa. Results for CGTase activity as a function of temperature gave maximal activity for the production of beta-CD at 65 degrees C, pH 6.0, and 71.5 mmol of beta-CD/(min x mg of protein), whereas for gamma-CD it was 9.1 mmol of gamma-CD/(min mg of protein) at 70 degrees C and pH 8.0. For long contact times, the best use of the enzymatic activity occurs at 60 degrees C or at a lower temperature, and the reaction pH may be selected to increase the yield of a desired CD.

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.

The role of arginine 47 in the cyclization and coupling reactions of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 implications for product inhibition and product specificity.

Cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19) is used for the industrial production of cyclodextrins. Its application, however, is hampered by the limited cyclodextrin product specificity and the strong inhibitory effect of cyclodextrins on CGTase activity. Recent structural studies have identified Arg47 in the Bacillus circulans strain 251 CGTase as an active-site residue interacting with cyclodextrins, but not with linear oligosaccharides. Arg47 thus may specifically affect CGTase reactions with cyclic substrates or products. Here we show that mutations in Arg47 (to Leu or Gln) indeed have a negative effect on the cyclization and coupling activities; Arg47 specifically stabilizes the oligosaccharide chain in the transition state for these reactions. As a result, the mutant proteins display a shift in product specificity towards formation of larger cyclodextrins. As expected, both mutants also showed lower affinities for cyclodextrins in the coupling reaction, and a reduced competitive (product) inhibition of the disproportionation reaction by cyclodextrins. Both mutants also provide valuable information about the processes taking place during cyclodextrin production assays. Mutant Arg47-->Leu displayed an increased hydrolyzing activity, causing accumulation of increasing amounts of short oligosaccharides in the reaction mixture, which resulted in lower final amounts of cyclodextrins produced from starch. Interestingly, mutant Arg47-->Gln displayed an increased ratio of cyclization/coupling and a decreased hydrolyzing activity. Due to the decreased coupling activity, which especially affects the production of larger cyclodextrins, this CGTase variant produced the various cyclodextrins in a stable ratio in time. This feature is very promising for the industrial application of CGTase enzymes with improved product specificity.

Application of factorial designs for optimization of cyclodextrin glycosyltransferase production fromKlebsiella pneumoniae pneumoniae AS-22

Production of cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae pneumoniae AS-22 was optimized in shake flasks using a statistical experimental design approach. Effect of various components in the basal medium, like carbon, nitrogen, phosphorus, and mineral sources as well as initial pH and temperature, were tested on enzyme production. The optimum concentrations of the selected media components were determined using statistical experimental designs. Two level fractional factorial designs in five variables, namely, dextrin, peptone, yeast extract, ammonium dihydrogen orthophosphate, and magnesium sulphate concentrations were constructed. The optimum medium composition thus found consisted of 49.3 g/L dextrin, 20.6 g/L peptone, 18.3 g/L yeast extract, 6.7 g/L ammonium dihydrogen orthophosphate, and 0.5 g/L magnesium sulphate. The maximum CGTase activity obtained was 21.4 U/mL in 28 h of incubation. The cell growth and CGTase production profiles were studied with the optimized medium in shake flasks and in 1-L fermenters. It was observed that the enzyme production was growth associated both in shake flask and in fermenter, although it was slower in shake flask. The maximum CGTase activity obtained in the fermenter was 32.5 U/mL in 16 h. The optimized medium resulted in about 9-fold increase in the enzyme activity as compared to that obtained in the basal medium in shake flask as well as in fermenter.

Further Characterization of Dormant Reaction of Cyclodextrin Glucanotransferase with Dextran and Subsequently Produced Dextran.

Structurally modified dextran was produced by the action of cyclodextrin glucanotransferase (CGTase) on clinical dextran. The dormant activity of CGTase was further confirmed by using the enzyme preparation from a different source. Although an increase in reducing sugar in the reaction mixture was small, a clear difference in the molecular weight distribution of CGTase-treated dextran was observed, as in previous experiments. Therefore the susceptibility of CGTases on dextran was verified with the other origin of CGTase. CGTase-treated dextran showed a higher interaction with Con A and fluorescent reagent, 6-p-toluidinylnaphthalene-2-sulfonate. The susceptibility of CGTasetreated dextran to isoamylase and glucoamylase varied significantly according to the reaction temperatures for the CGTase action. NMR-HMQC and methylation analyses both indicated additional occurrence of α-1, 4-branch points in the dextran molecule, which opposed the knowledge of sole branch point of -1, 3-linkages in the dextran. CGTase action seemed to increase the content of α-1, 4-branch points in the CGTase- treated dextran by introducing the cleaved fragments as the new branch into the back bones of α-1, 6-linkages. These results suggested that CGTasetreated dextran had increasing amounts of α-1, 4-branch points, which caused the structural changes demonstrated by the above analysis.

Optimization of cyclomaltodextrin glucanotransferase production from Bacillus firmus

Production of cyclomaltodextrin glucanotransferase (CGTase) from Bacilus firmus was optimized in shake flasks using a statistical experimental design approach. Two level complete factorial designs in three variables were used for media optimization. The maximum CGTase activity obtained was 7.05 U ml −1 with 21 g l −1 corn starch, 22.6 g l −1 yeast extract, 21.9 g l −1 Pharmamedia, and an initial pH of 9.8. The optimized medium resulted in about a 20-fold increase in the enzyme activity as compared to that obtained in the basal medium.

Development of ELISA and enzyme-linked immunofiltration assay (ELIFA) methods for monitoring cyclodextrin glycosyltransferase (CGTase) production and bacterial growth in Bacillus macerans batch cultures

Immunochemical methods were developed for monitoring cyclodextrin (CD) glycosyltransferase (CGTase) production and growth of an industrial CD-producing Bacillus macerans strain. Extracellular concentrations of CGTase released into a non-transparent culture medium during a 44 h long fermentation were detected by an indirect antigen inhibition enzyme-linked immunosorbent assay (ELISA). The ELISA was sensitive (minimal detection level 6 ng ml−1) and highly reproducible (coefficients of variation ≤1.2 and 5.9%, within-runs and between-runs, respectively) compared to assays of CGTase activity (coefficients of variation ≤4.2 and 7.0%, respectively). The ELISA, in combination with enzyme activity measurements, was useful to detect the decrease in the specific CGTase activities after 36 h of incubation, which was clearly indicative of the proteolytic degradation of CGTase. B. macerans cell numbers were estimated using an enzyme-linked immunofilter assay (ELIFA). The assay took less than 1 h and the coefficients of variation within and between-runs (2.9–6.4%) were considerably less than for viable counting (10.6–15.4%). In the exponential phase of growth, ELIFA results correlated more closely with the cell counting based on total protein than with viable counts. Nevertheless, in the phase of cell lysis, the bacterial cell number was systematically underestimated by ELIFA in comparison to both viable cell number and total protein determinations. Thus cell antigens detected with immunological procedures might be lost during the transition from vegetative cells to spores. On the other hand, the ELIFA procedure was specific for B. macerans cells and was a better indicator of the onset of the different growth phases than the cell numbers calculated from the protein assay.

Cyclodextrins are likely to induce cyclodextrin glycosyltransferase production inBacillus macerans

Cyclodextrin glycosyltransferase (CGTase) activity was monitored inBacillus macerans culture fluids up to 56 h incubation time using glucose (G1), maltose (G2), maltotriose (G3), maltoheptaose (G7), α-,β-,γ-cyclodextrins (CDs) and soluble starch as carbon sources. Highest maximum specific growth rates (µmax) were observed with glucose, γ-CD and soluble starch (µmax values were 0.86, 0.74 and 0.69/h, respectively) while the maximum viable cell numbers were always within the range of 2.3–7.1×1011 CFU/mL independently of the carbon source used. Highest CGTase production was found in the presence of soluble starch and G7 (55.0 and 35.4 nkat/mL, respectively), these saccharides being easily transformed to CDs by CGTase. Moreover, when culture media were supplemented with cyclic malto-oligosaccharides the CGTase activities were about twice higher (19.6–20.6 nkat/mL) than those obtained with the linear G2 and G3 saccharides (8.9 and 11.3 nkat/mL, respectively) which give rise only to negligible quantities of CDs. CDs, which are the major end products of the action of CGTase, are regarded thus as the likely physiological inducers of the enzyme.

Production of cyclodextrin homologues using aqueous two-phase system

Cyclodextrin homologues (CDs), produced by cyclodextrin glycosyltransferase (CGTase), were simultaneously partitioned in aqueous two-phase system (ATPS). Partition coefficients of CDs were measured in PEG/salt and PEG/dextran systems. Phosphate, citrate, sulfate were tested as salt. ATPS of PEG/salt and PEG/dextran had the partition coefficients of the CDs, larger than unity. However, PEG/dextran system was observed better than PEG/salt as CGTase activity decreased sharply with salt concentration. Enzymatic reaction occurred mainly in PEG-rich bottom phase because of the low partition coefficient of CGTase. The resulting CDs transferred to the PEG-rich top phase, obeying the diffusional partition. In the ATPS of 7% PEG (M.W. 20,000) and 9% dextran (M.W. 40,000), 7 mg/ml of CDs were obtained in top phase at 4.5 hours.

Cyclodextrins Are Not the Major Cyclic α-1,4-Glucans Produced by the Initial Action of Cyclodextrin Glucanotransferase on Amylose

The initial action of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from an alkalophilic Bacillus sp. A2-5a on amylose was investigated. Synthetic amylose was incubated with purified CGTase then terminated in the very early stage of the enzyme reaction. When the reaction mixture was treated with glucoamylase and the resulting glucoamylase-resistant glucans were analyzed with high performance anion exchange chromatography, cyclic alpha-1,4-glucans, with degree of polymerization ranging from 9 to more than 60, in addition to well known alpha-, beta-, and gamma-cyclodextrin (CD), were detected. The time-course analysis revealed that larger cyclic alpha-1, 4-glucans were preferentially produced in the initial stage of the cyclization reaction and were subsequently converted into smaller cyclic alpha-1,4-glucans and into the final major product, beta-CD. CGTase from Bacillus macerans also produced large cyclic alpha-1, 4-glucans except that the final major product was alpha-CD. Based on these results, a new model for the action of CGTase on amylose was proposed, which may contradict the widely held view of the cyclization reaction of CGTase.

Regulation of cyclodextrin glucanotransferase synthesis in Bacillus ohbensis

The production of cyclodextrin glucanotransferase (CGTase) by Bacillus ohbensis is dependent on the presence of starch and inhibited by glucose in the medium. Northern blot analysis revealed that the CGTase gene ( cgt) was transcribed to almost the same level irrespective of the presence or absence of starch, but glucose completely repressed the transcription. Furthermore, a relatively high amount of CGTase protein was detected on Western blotting only in the medium with starch, showing the lack of posttranslational control of the CGTase activity. These findings suggest some starch induction mechanism for the cgt gene, possibly at the posttranscriptional level, besides negative transcriptional regulation by glucose.

Synthesis of Cyclodextrin glycosyl transferase by immobilized cells of Bacillus circulans

The cells of Bacillus circulans (ATCC 21783) immobilized in sodium alginate gel matrix were able to synthesize the extracellular enzyme, Cyclodextrin glycosyl transferase (CGTase, E.C. 2.4.1.19) which is industrially employed for the preparation of cyclodextrins. Optimization for the maximum production of enzyme was carried out by varying the cell density (3.3–53.5 kg/m3) in the gel and the incubation temperature (30°–42°C). The CGTase activity was found to be the highest (45 units/cm3) with maximum cell loading at 37°C. The reusability of immobilized cells was ascertained by repeated batch experiments. The enzyme activity exhibited was in the range of 50 to 55 units/cm3 in each batch. The continuous synthesis of CGTase by immobilized cells has been demonstrated by operating a fluidized bed reactor at a dilution rate 1.1 · 10−4 sec−1 for a period of 15 days. The enzyme activity has decreased to 42.5 units/cm3 from an initial value of 61 units/cm3 during continuous operation.

Cyclodextrin glycosyltransferase may be the only starch-degrading enzyme in Bacillus macerans.

Cyclodextrin glycosyltransferase (CGTase) was released into the culture fluid by Bacillus macerans predominantly in the late stationary phase of growth and during autolysis in the presence of either glucose or starch as a carbon source. In both cases significant soluble intracellular enzyme activity could be observed in the early stationary phase, and a low non-soluble intracellular CGTase activity could be demonstrated also in the exponential growth phase in the presence of starch. At the end of the exponential phase the non-soluble specific intracellular enzyme activity was found to be constant with a value of 0.63 +/- 0.06 nkat/10(9) viable cells. Since amylase activity could not be detected in any intracellular or extracellular sample taken at any culture time, we conclude that cellbound CGTase is the only starch-digesting enzyme in growing B. macerans and, hence, may be fully responsible for the degradation of starch in the culture fluid.

Production and immobilization of a proteinase-reduced cyclodextrin glycosyltransferase preparation

Cyclodextrin glycosyltransferase (CGTase) was produced by a 3-day cultivation of Bacillus macerans growing in a natural medium containing grated-potatoes. Besides CGTase the culture supernatant contained a mixture of serine proteinases with a predominant subtilisin-like activity. By fractionated precipitation with ammonium sulphate the CGTase (molecular mass approximately 70 kDa) was concentrated and largely separated from the proteinases (molecular mass approximately 28 kDa). Among the various immobilization methods and carrier materials tested the enriched CGTase was covalently bound preferably onto porous glass beads using glutardialdehyde as cross-linker. The discontinuous conversion of soluble starch into beta-cyclodextrin was carried out with native as well as immobilized CGTase over 24 h. The batch re-usability of the fixed enzyme proved to be at least 20 times with a residual CGTase activity of 65%.

Syntheses of glucosides with trimethylolpropane and two related polyol moieties by cyclodextrin glucanotransferase and their esterification by lipase

For the syntheses of glucosyl trimethylolpropane (Glc-TMP), glucosyl trimethylolethane (Glc-TME), and glucosyl pentaerythritol (Glc-PE), Bacillus stearothermophilus cyclodextrin glucanotransferase was much more effective than Bacillus circulans and Bacillus macerans enzymes. The amounts of Glc-TMP synthesized increased with the concentrations of the substrates and the CGTase activity. In the esterification of the glucosides with oleic and lauric acids by a lipase from Candida cylindracea, the esters increased in the following order; Glc-PE < Glc-TME < Glc-TMP. Palmitic acid gave no esters of the three glucosides.

Enhancement of enzymatic production of cyclodextrins by organic solvents

A strain of Bacillus sp. BE101 was isolated from soil and used to obtain the cyclodextrin glycosyltransferase (E.C.3.2.1.19, CGTase). Cyclodextrin glycosyltransferase from Bacillus sp. BE101 synthesized mainly β-cyclodextrin from soluble starch; the composition of α-, β-, and γ-cyclodextrin was 1:7:2. The optimum conditions for CGTase activity were pH 6.0–6.5 and temperature 45°C. An increase in cyclodextrin production by organic solvents such as acetone, propanol, and ethanol was observed. The maximum increase was observed at 10% ethanol, and the enhancement of production yield was about 100%. Studies on the cause of the increase in production yield of cyclodextrins by ethanol were carried out. It seemed that enhancement of the yield by ethanol was due to the increase in hydrolysis of starch by CGTase in the presence of ethanol.

Nucleotide sequence of the raw-starch-digesting amylase gene from [formula omitted] sp. B1018 and its strong homology to the cyclodextrin glucanotransferase genes

The gene coding for the raw-starch-digesting amylase from Bacillus sp. B1018 was cloned into Escherichia coli , and its nucleotide sequence was determined. Starting at an ATG codon, there was an open reading frame composed of 2139 bp (713 amino acids). The NH 2-terminal portion encoded a 27 amino acid-long signal peptide. S1 mapping revealed the presence of a typical promoter region upstream from the transcription initiation site. The deduced amino acid sequence of the extracellular mature enzyme was very similar to those of cyclodextrin glucanotransferases (CGTases), especially that of alkalophilic Bacillus sp. 1011 (87% homology). In fact, the B1018 amylase showed CGTase activity. The COOH-terminal portion of the B1018 amylase shows significant homology with other raw-starch-digesting enzymes.