Α-cyclodextrin Production Research Articles

Enhancing the α-Cyclodextrin Specificity of Cyclodextrin Glycosyltransferase from Paenibacillus macerans by Mutagenesis Masking Subsite -7.

Cyclodextrin glycosyltransferases (CGTases) (EC 2.4.1.19) catalyze the conversion of starch or starch derivates into mixtures of α-, β-, and γ-cyclodextrins. Because time-consuming and expensive purification procedures hinder the widespread application of single-ingredient cyclodextrins, enzymes with enhanced specificity are needed. In this study, we tested the hypothesis that the α-cyclodextrin selectivity of Paenibacillus macerans α-CGTase could be augmented by masking subsite -7 of the active site, blocking the formation of larger cyclodextrins, particularly β-cyclodextrin. Five single mutants and three double mutants designed to remove hydrogen-bonding interactions between the enzyme and substrate at subsite -7 were constructed and characterized in detail. Although the rates of α-cyclodextrin formation varied only modestly, the rate of β-cyclodextrin formation decreased dramatically in these mutants. The increase in α-cyclodextrin selectivity was directly proportional to the increase in the ratio of their kcat values for α- and β-cyclodextrin formation. The R146A/D147P and R146P/D147A double mutants exhibited ratios of α-cyclodextrin to total cyclodextrin production of 75.1% and 76.1%, approximately one-fifth greater than that of the wild-type enzyme (63.2%), without loss of thermostability. Thus, these double mutants may be more suitable for the industrial production of α-cyclodextrin than the wild-type enzyme. The production of β-cyclodextrin by these mutants was almost identical to their production of γ-cyclodextrin, which was unaffected by the mutations in subsite -7, suggesting that subsite -7 was effectively blocked by these mutations. Further increases in α-cyclodextrin selectivity will require identification of the mechanism or mechanisms by which these small quantities of larger cyclodextrins are formed.

Mutations enhance β-cyclodextrin specificity of cyclodextrin glycosyltransferase from Bacillus circulans

The effects of amino acid residue at position 31 in the neighborhood of calcium binding site I (CaI) on product specificity of cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) were investigated by replacing Ala31 in the CGTase from Bacillus circulans STB01 with arginine, proline, threonine, serine and glycine. The results showed that the mutations A31R, A31P, and A31T resulted in the increases in β-cyclodextrin-forming activity and β-cyclodextrin production, indicating that these mutations enhanced β-cyclodextrin specificity of the CGTase. Especially the mutant A31R displayed approximately 26% increase in β-cyclodextrin production with a concomitant 41% decrease in α-cyclodextrin production when compared to the wild-type CGTase. Thus, it was much more suitable for the industrial production of β-cyclodextrin than the wild-type enzyme. The enhanced β-cyclodextrin specificity of the mutants might be a result of stabilizing CaI, which also suggested that CaI might play an important role in cyclodextrin product specificity of CGTase.

Crystallization and preliminary X-ray diffraction studies of Tyr167His mutant α-cyclodextrin glucanotransferase from Bacillus macerans.

Improving the specificity of α-cyclodextrin glucanotransferase is a significant issue in the field of α-cyclodextrin production. In this study, a constructed Y167H mutant α-cyclodextrin glucanotransferase with enhanced α-cyclodextrin specificity was successfully expressed and purified. Single crystals were grown using PEG 4000 as a precipitating agent by the hanging-drop vapour-diffusion method at 293 K. The crystals exhibited two kinds of morphology in different crystallization conditions. The crystals diffracted to at least 2.2 Å resolution (space group P2₁2₁2₁), with unit-cell parameters a=65.69, b=78.70, c=137.00 Å. Assuming the asymmetric cell to be occupied by a monomer of 75 kDa, the unit cell contains 43.77% solvent with a crystal volume per protein mass, VM, of 2.19 Å3 Da(-1).

Mutation of Tyrosine167Histidine at Remote Substrate Binding Subsite −6 in α-Cyclodextrin Glycosyltransferase Enhancing α-Cyclodextrin Specificity by Directed Evolution

α-Cyclodextrin glycosyltransferase (α-CGTase) can convert starch into α-cyclodextrin with various proportions of β-cyclodextrin and/or γ-cyclodextrin in the products. To improve the α-cyclodextrin-forming specificity, directed evolution on the wild-type α-CGTase was performed by constructing mutant library with error-prone PCR method. The positive mutant strains were selected in combination of starch plate screening with HPLC detection of the products. An α-CGTase from the mutant strain (assigned No. 95) was found to be able to increase the α:β ratio in product mixture from 3.4 to 7.8 in comparison with the wild-type α-CGTase. Sequence alignment indicated that two mutations occurred in the No. 95 mutant α-CGTase, which were Y167H and A536V. Reverse mutation revealed that Y167H was responsible for this change. A series of 167 site-substituted mutants could improve the α:β ratio to different extents as indicated by saturated mutagenesis, with Y167H as the best substitution. In conclusion, Y167 was confirmed to be one of the main subsites in the -6 domain of α-CGTase that is responsible for the α:β ratio in the product mixture. Y167H is most preferable among all types of mutant enzymes tested at this site. The reconstructed Y167H (i.e., No. 95) α-CGTase showed better potential for α-cyclodextrin production on industrial scale.

Safety evaluation of an α-cyclodextrin glycosyltranferase preparation

Alpha-cyclodextrin glucosyltransferase (α-CGTase, EC 2.4.1.19) is an amylolytic enzyme used for the production of α-cyclodextrin (α-CD), a novel, soluble dietary fiber, from food-grade starch. The safety of an α-CGTase preparation obtained by batch fermentation from a recombinant strain of Escherichia coli K12 harboring the α-CGTase gene from Klebsiella oxytoca strain M5a1 was examined. In a 13-week subchronic toxicity study in rats, the administration by gavage of the α-CGTase preparation at levels of up to 20 ml/kg bw/day, corresponding to a total organic solids dosage of 260 mg/kg bw/day, did not cause any systemic toxic effect. Some signs of irritation were observed in the respiratory tract which occurred, however, in one sex only and/or were not dose-related. Accordingly, these changes were considered to be an unspecific consequence of the reflux and aspiration of the dosing solution. There was no evidence of a genotoxic activity in Ames tests and a chromosome aberration test in cultured human lymphocytes. It is concluded that the examined α-CGTase preparation is safe when used for the production of α-CD.

Process modeling and simulation can guide process development: case study α-cyclodextrin

Modeling and simulation of biotechnological processes enables the quantification of possible process variations. In this way it provides a tool for guiding research towards the most promising directions. The method is shown on the basis of the case study of α-cyclodextrin production. The production process was modeled and the model is used to evaluate the potential impact of CGTases with new properties. Sensitivity analyses show that yield and selectivity are the crucial parameters. Reaction time has only limited impact and substrate concentration has to be above 20%. The use of thermostable enzymes provides some energy savings. Furthermore, the effect of new CGTases leading to a modified downstream processing was assessed.

Purification and properties of a novel raw starch degrading-cyclodextrin glycosyltransferase from Klebsiella pneumoniae AS- 22

A novel raw starch degrading α-cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19), produced by Klebsiella pneumoniae AS-22, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The specific cyclization activity of the pure enzyme preparation was 523 U/mg of protein. No hydrolysis activity was detected when soluble starch was used as the substrate. The molecular weight of the pure protein was estimated to be 75 kDa with SDS-PAGE and gel filtration. The isoelectric point of the pure enzyme was 7.3. The enzyme was most active in the pH range 5.5–9.0 whereas it was most stable in the pH range 6–9. The CGTase was most active in the temperature range 35–50°C. This CGTase is inherently temperature labile and rapidly loses activity above 30°C. However, presence of soluble starch and calcium chloride improved the temperature stability of the enzyme up to 40°C. In presence of 30% (v/v) glycerol, this enzyme was almost 100% stable at 30°C for a month. The K m and k cat values for the pure enzyme were 1.35 mg ml −1 and 249 μM mg −1 min −1, respectively, with soluble starch as the substrate. The enzyme predominantly produced α-cyclodextrin without addition of any complexing agents. The conditions employed for maximum α-cyclodextrin production were 100 g l −1 gelatinized soluble starch or 125 g l −1 raw wheat starch at an enzyme concentration of 10 U g −1 of starch. The α:β:γ-cyclodextrins were produced in the ratios of 81:12:7 and 89:9:2 from gelatinized soluble starch and raw wheat starch, respectively.

Rational design of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 to increase α-cyclodextrin production

Cyclodextrin glycosyltransferases (CGTase) (EC 2.4.1.19) are extracellular bacterial enzymes that generate cyclodextrins from starch. All known CGTases produce mixtures of α, β, and γ-cyclodextrins. A maltononaose inhibitor bound to the active site of the CGTase from Bacillus circulans strain 251 revealed sugar binding subsites, distant from the catalytic residues, which have been proposed to be involved in the cyclodextrin size specificity of these enzymes. To probe the importance of these distant substrate binding subsites for the α, β, and γ-cyclodextrin product ratios of the various CGTases, we have constructed three single and one double mutant, Y89G, Y89D, S146P and Y89D/S146P, using site-directed mutagenesis. The mutations affected the cyclization, coupling; disproportionation and hydrolyzing reactions of the enzyme. The double mutant Y89D/S146P showed a twofold increase in the production of α-cyclodextrin from starch. This mutant protein was crystallized and its X-ray structure, in a complex with a maltohexaose inhibitor, was determined at 2.4 Å resolution. The bound maltohexaose molecule displayed a binding different from the maltononaose inhibitor, allowing rationalization of the observed change in product specificity. Hydrogen bonds (S146) and hydrophobic contacts (Y89) appear to contribute strongly to the size of cyclodextrin products formed and thus to CGTase product specificity. Changes in sugar binding subsites −3 and −7 thus result in mutant proteins with changed cyclodextrin production specificity.

Enzymatic production of α-cyclodextrin with the cyclomaltodextrin glucanotransferase of Klebsiella oxytoca 19-1

A microorganism capable of producing extracellular cyclomaltodextrin glucanotransferase (E.C.2.4.1.19; CGTase) was isolated and identified as Klebsiella oxytoca. Properties of the CGTase and the optimal conditions for production of α-cyclodextrin were also studied. The most effective carbon sources for inducing the production of CGTase were amylopectin and soluble starch, whereas monosaccharides, disaccharides, and amylose did not induce CGTase. The optimal conditions for production of α-cyclodextrin were as follows: 10% (w/v) corn starch; ratio of CGTase to corn starch, around 10 units g -1 substrate; pH 6.0; 40°C; 19 h. The most important property of the CGTase was its ability to produce large amounts of almost exclusively α-cyclodextrin from starch in a short reaction time without the aid of solvents or detergents. Under optimal conditions, cyclodextrins were produced up to 14.75 g l -1 in 19 h, and the ratio of cyclodextrins produced was α: β: γ = 96.5:3.5:0.

A novel process of cyclodextrin production by the use of specific adsorbents: Part II. A new reactor system for selective production of α-cyclodextrin with specific adsorbent

We have developed a novel process of α-cyclodextrin (α-CD) production by using a new adsorbent that is characterized by its exceedingly powerful selectivity for α-CD compared with other CDs. α-CD production was carried out in a closed reactor system that was composed of a main reactor, wherein liquefied starch was converted to CDs by cyclodextrin glucosyltransferase (CGTase: EC 2.4.1.19), and a column packed with the adsorbent. While the reaction mixture was circulated in the system, α-CD was selectively adsorbed in the column and its concentration in the mixture of the main reactor was kept at a low level. This low concentration of α-CD stimulated the conversion of starch to CDs and as a result, enhanced its yield based on added starch. When 8.3 % (w/v) of liquefied starch was used in the reactor system, the yield of α-CD was 22.2% and α-CD occupied 58.7 % of the reaction mixture of total CDs synthesized. Meanwhile, in a batch system without the adsorbent, the yield of α-CD and its fraction were 10.8% and 45.0%, respectively. After the conversion reaction, and following the preliminary washing with water through the column. α-CD was easily eluted with hot water, resulting in a high purity of about 95%.

各種ＣＧＴａｓｅおよびサイクロデキストリンの生産

Three kinds of cyclomaltodextrin glucanotransferases (CGTases) produced by Bacillus ohbensis, Bacillus macerans and Bacillus circulans were respectively purified to a single protein and the CGTase from B. ohbensis was successfully crystallized. Comparative studies of three CGTases revealed that the CGTase from B. ohbensis had the smallest molecular weight (35, 000) and low affinity on starch adsorption while in other properties, i.e., optimum pH, pH stability, optimum temperature and etc., the three enzymes showed similar properties.CGTase from B. macerans had advantages on the production of α-cyclodextrin (CD) but the reaction conditions must be more strictly controlled. CGTase from B. ohbensis can be favorably used for the production of β-and γ-CD. Futhermore, only CGTase from B. ohbensis produced negligible amount of α-CD.

シクロデキストリンについて α‐シクロデキストリンの生産とその利用を中心として

It has been said that cyclodextrins will be successfully used in various fields of industries because of its ability to make inclusion compounds on complexes with various kinds of substances, especially unstable materials against oxidation, heat and light. However, cyclodextrins have not been widely used for processing foods and others up to now. The main reason not to be used may be owing to high price of cyclodextrins production. To solve this problem, we have been working on production of cyclodextrin producing enzyme and cyclodextrins and established a component of cultivating medium for producing the Bacillus macerans enzyme. The enzyme in a crude enzyme preparation (broth) was adsorbed on moist heated starch granules to make starch adsorbed enzyme which was most useful for production of cyclodextrins. A new method of α-cyclodextrin production was also established. The principle of the method is based on the action of glucoamylase, taka amylase and yeast; taka amylase hydrolyzes β- and γ-cyclodextrins effectively to maltooligosaccharides which are hydrolyzed to glucose by the action of glucoamylase. And glucose is converted to ethanol by yeast. As the result, after being treated with the above mentioned enzyme system, the reaction mixture of cyclodextrin producing enzyme on starch is composed of ethanol and a-cyclodextrin, which is easily crystallized by concentrating the mixture. Cyclodextrins syrup which contains α-cyclodextrin as a main component was also produced for use in the food industries and several examples of its utilization were reported.