Engineering of cyclodextrin glucanotransferase on the cell surface of Saccharomyces cerevisiae for improved cyclodextrin production

The cyclodextrin glucanotransferase (CGTase) gene (cgt) from Bacillus circulans 251 was cloned into plasmid pYD1, which allowed regulated expression, secretion, and detection. The expression of CGTase with a-agglutinin at the N-terminal end on the extracellular surface of Saccharomyces cerevisiae was confirmed by immunofluorescence microscopy. This surface-anchored CGTase gave the yeast the ability to directly utilize starch as a sole carbon source and the ability to produce the anticipated products, cyclodextrins, as well as glucose and maltose. The resulting glucose and maltose, which are efficient acceptors in the CGTase coupling reaction, could be consumed by yeast fermentation and thus facilitated cyclodextrin production. On the other hand, ethanol produced by the yeast may form a complex with cyclodextrin and shift the equilibrium in favor of cyclodextrin production. The yeast with immobilized CGTase produced 24.07 mg/ml cyclodextrins when it was incubated in yeast medium supplemented with 4% starch.     

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.     

Cyclodextrin glycosyltransferase: a key enzyme in the assimilation of starch by the halophilic archaeon Haloferax mediterranei

A cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) was successfully isolated and characterized from the halophilic archaeon Haloferax mediterranei. The enzyme is a monomer with a molecular mass of 77 kDa and optimum activity at 55°C, pH 7.5 and 1.5 M NaCl. The enzyme displayed many activities related to the degradation and transformation of starch. Cyclization was found to be the predominant activity, yielding a mixture of cyclodextrins, mainly α-CD, followed by hydrolysis and to a lesser extent coupling and disproportionation activities. Gene encoding H. mediterranei CGTase was cloned and heterologously overexpressed. Sequence analysis revealed an open reading frame of 2142 bp that encodes a protein of 713 amino acids. The amino acid sequence displayed high homology with those belonging to the α-amylase family. The CGTase is secreted to the extracellular medium by the Tat pathway. Upstream of the CGTase gene, four maltose ABC transporter genes have been sequenced (malE, malF, malG, malK). The expression of the CGTase gene yielded a fully active CGTase with similar kinetic behavior to the wild-type enzyme. The H. mediterranei CGTase is the first halophilic archaeal CGTase characterized, sequenced and expressed.     

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.     

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.     

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。     

Characterisation of cyclodextrin glucanotransferase fromBacillus circulans ATCC 21783 in terms of cyclodextrin production

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) fromBacillus circulans ATCC 21783 was purified by ultrafiltration and a consecutive starch adsorption. Total enzyme yield of 75.5% and purification factor of 13.7 were achieved. CGTase was most active at 65°C, possessed two clearly revealed pH-optima at 6.0 and 8.6 and retained from 75 to 100% of its initial activity in a wide range of pH, between 5.0 and 11.0. The cyclising activity was enhanced by 1 mM CaCl₂ or 4 mM CoCl₂. The enzyme was thermostable up to 70°C, and 64% of the original activity remained at 70°C after 30 min heat treatment. Up to 41% conversion into cyclodextrins was obtained from 40 g l^?1 starch without using any additives. This CGTase produced two types of cyclodextrins, beta and gamma, in a ratio 73:27 after 4 h reaction time at 65°C. This feature of the enzyme could be of interest for industrial cyclodextrin production.     

Effect of organic solvents on enzymatic production of cyclodextrins from unliquefied corn starch in an attrition bioreactor

Cyclodextrins were produced from unliquefied corn starch in the presence of water-miscible organic solvents using cyclodextrin glycosyltransferase (CGTase) in an attrition bioreactor. The production yield was singnificatly increased by isopropanol and tertiary butanol, and maximum enhancement was observed to be about 40% by 5% tertiary butnol. Increase in the production of cyclodextrins by organic solvents seems to be due to the fact that organic solvents decreased the product inhibition of CGTase by forming an inclusion complex with cyclodextrins.     

Enzymatic production of cyclodextrins from unliquefied corn starch in an attrition bioreactor

A novel enzymatic process for the production of cyclodextrins from unliquefied starch was developed. Cyclodextrins were produced in an attrition bioreactor in which simultaneous hydrolysis of starch and synthesis of cyclodextrins by cyclodextrin glycosyltransferase (CGTase) occur. The CGTase was obtained from isolated Bacillus sp. BE101, and maximum activity of the enzyme was observed at pH 6.0 and a temperature of 45 degrees C. The effect of milling media size and material on the performance of the attrition bioreactor was investigated, and operational parameters such as agitation speed, volume of milling media, ratio of enzyme to starch, and starch concentration were optimized. The production yield of cyclodextrins from unliquefied corn starch of 15% reached 35% at 24 h under optimized conditions. Energy consumption for the production of cyclodextrins in the attrition bioreactor system was estimated to be about 25% of that required for the liquefaction of starch in the conventional process.     

Optimisation of batch culture conditions for cyclodextrin glucanotransferase production from Bacillus circulans DF 9R

Background  

The extracellular enzyme cyclodextrin glucanotransferase (CGTase) synthesizes cyclic malto-oligosaccharides called cyclodextrins (CDs) from starch and related α-1,4-glucans. CGTases are produced by a variety of bacteria, mainly Bacillus species, by submerged culture in complex medium. CGTases differ in the amount and types of CDs produced. In addition, CGTase production is highly dependent on the strain, medium composition and culture conditions. Therefore we undertook this study with a newly isolated strain of Bacillus circulans.     

Cyclodextrin glucanotransferase production by cell biocatalysts of alkaliphilic bacilli

Cells of obligated alkaliphiles Bacillus pseudalcaliphilus 20RF and Bacillus pseudalcaliphilus 8SB isolated from Bulgarian habitats, producers of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19), were immobilized by three different techniques: on two types of polysulphone membranes; entrapped in agar-gel beads containing magnetite and by nano-particles of silanized magnetite covalently bound on the cell surface. The biocatalysts obtained demonstrated the opportunity for a significantly enhanced CGTase production compared to free cells for a long period of time (10 days semicontinuous cultivation) without impact on their mechanical stability. The cell membrane-biocatalysts exhibited the highest enzyme activity after 240 h repeated batch cultivation and retained 1.3–2.3-fold increase of the CGTase yield compared to free cells at the end of the process. Membrane biocatalysts were applied for a direct cyclodextrin (CD) production. The results obtained demonstrated the possibility of starch conversion into cyclodextrins by immobilized cells without using of crude or purified enzyme. The membrane biocatalysts of both obligated alkaliphiles formed mainly β- and γ-CDs after 6 h enzyme reaction at pH 9.0 of the reaction mixture. Under these conditions, the quantity of γ-CDs was a relative high, to 35–37% of the total CD amount.     

Improvement of cyclodextrin glycosyltransferase (CGTase) production by recombinant Escherichia coli pAD26 immobilized on the cotton

The cyclodextrin glycosyltransferase (CGTase) of the recombinants Escherichia coli pAD26 cells immobilized on cotton was optimally produced by statistical methodology. Primarily, carbon and nitrogen sources were selected by one-factor-at-a-time method. Wheat starch, Casamino acid, Edamin and Hy-soy were identified as the best nutrients. These sources were secondly confirmed by Plackett-Burman design (fifteen variables were studied with sixteen experiments), as the most significant components with respect to CGTase production. In the third step, concentration of most significant factors and their interaction were optimized with a Box-Behnken experimental design. Under the optimized conditions (agitation 200 rpm, yeast extract concentration 20 g/L, wheat starch concentration 10 g/L and Hy-soy concentration 2.5 g/L), CGTase yield 145.11 U/mL was 3.6 and 23 folds higher than those obtained by the use of the initial conditions (39.77 U/mL) and free cells (6.37 U/mL), respectively.     

利用高效阴离子色谱快速检测筛选环糊精糖基转移酶产生菌>

利用高效阴离子色谱快速直接地检测微生物发酵液中的环糊精成分,尤其是大环环糊精的组成,进而创造了一种能快速准确地从土壤中筛选产环糊精糖基转移酶菌种的方法。共分离了149个产胞外淀粉水解酶的微生物菌株,利用高效阴离子交换色谱共检测了其中11株菌,其中6株主要产CD6 ,5株主要产CD7,主要产CD8的没有。在直接鉴定产生环糊精糖基转移酶菌株的过程中,也可以定量检测各种环糊精包括大环糊精（CD大于8）的含量。     

Enzymatic production of cyclodextrins from milled corn starch in an ultrafiltration membrane bioreactor

Cyclodextrin glycosyltransferase (CGTase) was found to be severely inhibited by cyclodextrins. In order to increase the conversion yield by reducing product inhibition and reuse the CGTase in the production of cyclodextrins from milled corn starch, an ultrafiltration membrane bioreactor system was employed. In a batch operation with ultrafiltration, the conversion yield was increased 57% compared with that without ultrafiltration. Operating conditions for the continuous production of cyclodextrins in the membrane bioreactor were optimized by taking into consideration the filtration rate and the conversion yield as follows: initial starch concentration, 7% (w/v); starch feeding rate, 240 mg/h; CGTase loading, 350 units/initial gram starch. When cyclodextrins were continuously produced in the membrane bioreactor under optimized conditions, 340 units of CGTase was require to produce 1 g of cyclodextrins for 48 h, while in the case of conventional batch operation, 1 g of cyclodextrins was produced for 24 h by 1410 units of CGTase. (c) 1993 John Wiley & Sons, Inc.     

Unusual starch degradation pathway via cyclodextrins in the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324

The hyperthermophilic archaeon Archaeoglobus fulgidus strain 7324 has been shown to grow on starch and sulfate and thus represents the first sulfate reducer able to degrade polymeric sugars. The enzymes involved in starch degradation to glucose 6-phosphate were studied. In extracts of starch-grown cells the activities of the classical starch degradation enzymes, alpha-amylase and amylopullulanase, could not be detected. Instead, evidence is presented here that A. fulgidus utilizes an unusual pathway of starch degradation involving cyclodextrins as intermediates. The pathway comprises the combined action of an extracellular cyclodextrin glucanotransferase (CGTase) converting starch to cyclodextrins and the intracellular conversion of cyclodextrins to glucose 6-phosphate via cyclodextrinase (CDase), maltodextrin phosphorylase (Mal-P), and phosphoglucomutase (PGM). These enzymes, which are all induced after growth on starch, were characterized. CGTase catalyzed the conversion of starch to mainly beta-cyclodextrin. The gene encoding CGTase was cloned and sequenced and showed highest similarity to a glucanotransferase from Thermococcus litoralis. After transport of the cyclodextrins into the cell by a transport system to be defined, these molecules are linearized via a CDase, catalyzing exclusively the ring opening of the cyclodextrins to the respective maltooligodextrins. These are degraded by a Mal-P to glucose 1-phosphate. Finally, PGM catalyzes the conversion of glucose 1-phosphate to glucose 6-phosphate, which is further degraded to pyruvate via the modified Embden-Meyerhof pathway.     

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.     

Site-saturation mutagenesis of proline 176 in Cyclodextrin Glucosyltransferase from Bacillus sp. Y112 effects product specificity and enzymatic properties

Based on the analysis of amino acid sequence and simulated structure, saturation mutagenesis was performed to explore the role of the site p176 of cyclodextrin glucosytransferase (CGTase) from Bacillus sp. Y112. Compared to the wild-type, mutant P176G showed 10.4 % improvement in conversion from starch to cyclodextrins (CDs), whose β-CD yield increased by 6% and α-CD yield decreased by 8%. Mutants P176L and P176I were increased by 7.9 % and 9.4 % on CDs production, indicating replacement of hydrophobic amino acids significantly improved in cyclization activity. Kinetics studies indicated the substrate affinity of P176G and P176K were increase by 13 % and 14 %, and the catalytic efficiency of P176K was increase by 14 %. In addition, the optimal temperature of mutants transformed from 50℃ to 40℃ and the optimal pH shifted from 10.0 to 8.0. These results indicate that the site P176 plays a critical role in catalytic activity, product specificity and enzymatic properties of CGTase.     

Cyclodextrin-Glucanotransferase von Klebsiella pneumoniae

1. When growing with cyclodextrins, Klebsiella pneumoniae M 5 al produces extracellular cyclodextrin glucanotransferase in amounts comparable to those obtained during the growth with potato starch.
2. Intracellular cyclodextrin glucanotransferase-activity was demonstrated to be present in the homogenates of cells grown with cyclodextrins. In addition, an amylomaltase-like enzyme and the maltodextrin phosphorylase could be pointed out. The cyclodextrins are metabolized to glucose-1-phosphate and glucose by the concerted actions of these three enzymes. paraGlucose-1-phosphate is liberated from cyclohexaamylose by the actions of purified cyclodextrin glucanotransferase and purified maltodextrin phosphorylase. The liberation of the sugar phosphate is increased fivefold by addition of glucose as an acceptor. This sugar, however, retards the formation of glucose-1-phosphate from the cyclic compound by the enzymes of the cell extract: In the presence of glucose the amylomaltase is incapable of synthesizing substrates for the phosphorylase from maltose. This experimental result clearly demonstrates that the amylomaltase is involved in the disproportionation of maltosaccharides arising from the cyclodextrins.
3. A NADP⁺-specific glucose dehydrogenase was demonstrated to be present in the cell extracts. This enzyme, which is activated by ADP, may control the energy-depending pool of free glucose. Glucose originates from the disproportionation of maltosaccharides catalyzed by the glucanotransferases.
4. A glucose-1-phosphate-hydrolysing phosphatase, which is shown to be present in the cell extract, seems to be without physiological significance for the metabolism of the cyclodextrins.
5. Preliminary permeation studies make it probable that the cyclodextrins are transported into the cells as such and degraded only within the cells.
6. A scheme for the metabolism of cyclodextrins in Klebsiella pneumoniae M 5 al is proposed.