Same as Subheading 381 step

2. Liquefy by adding CGTase (1.05 mL; 630 U) and stirring magnetically for 20 min at 25°C. pH is ~5.5 (see Note 4). Addition of streptomycin is not essential.

3. Add Tris (5 mL of stock solution), pullulanase (32 U), and cyclotridecanone (2.3 g; 2.5 mmol). Adjust pH to 7.0, and place stoppered flask in a shaker cabinet at 40°C (see Note —).

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4. At the end of 3 d, add a second portion of CGTase (1.05 mL). Readjust pH to 7.0, if necessary, and continue shaking at 40°C for two more days.

5. Same as Subheading 3.8.1., steps 5-9. Yield, 5.2 g (at 31% RH); 10.5% H^. HPLC analysis of anhydrous sample: 0% a; ~2% p; 97.4% y.

6. Recrystallization from water of 4.2 g of impure y-CD isolated in step 5 follows the same procedure as that in Subheading 3.8.1., step 10. Yield, 3.1 g; 8.8% Hp. HPLC analysis of anhydrous, sample: 0% a; 0% p; 100% y; 0% other.


1. The use of cyclododecanone as complexant to enhance y-CD production, unlike the use of cycloalkanones of larger ring size, necessitates applying CGTase in small increments over a prolonged period of time. A single increment, regardless of size, favors the production of P-CD (5).

2. Should reaction mixtures or solutions of CDs be treated with amyloglucosidase to eliminate any noncyclic maltooligosaccharide and maltodextrin components, consideration must be given to the possibly significant degradative action of this enzyme on y-CD. a-CD and P-CD are relatively inert. To illustrate, when 4 mL of 1.25 mM y-CD (~40 mmol of glucose residues) are treated with amyloglucosidase (10 |L of stock solution; 0.2 U of activity) at pH 4.5 and 55°C for

1 h, 2.5% of the y-CD is lost through conversion to D-glucose (1.0 mmol). A 5% loss occurs after

2 h; D-glucose and a smaller amount maltose are products. (A single HPLC operation permits the quantitative determination of y-CD, d-glucose, and maltose). Thus, when use is made of amyloglucosidase to determine the purity of a y-CD sample, an appropriate correction factor must be applied to take into account the loss from degradation.

Amyloglucosidase that has been applied to a reaction mixture can be effectively and completely inactivated at pH 7.0 without significant action on y-CD by very rapidly heating the mixture to 100°C and keeping it at this temperature for 30 min.

3. In the preparation of a-CD, but not that of either P-CD or y-CD, initial starch concentrations of <10% (w/w) should not be used because of an adverse effect that lower starch concentrations can have on yields (unpublished observations of the author).

4. The flask containing the gel and liquefying enzyme must first be agitated manually in order to weaken the gel structure sufficiently to permit rotation of the stir bar.

5. The pH should not be allowed to fall below 6.0, because below this level the enzyme stability might decrease more rapidly than is desirable. Adjustments of pH are made by additions of NaOH or HCl.

6. Approximately the same yield of a-CD can be obtained by using a single 70-mL application of CGTase (420 U) and a 6-d reaction period.

7. Elevated temperatures can cause extensive dissociation and dissolution of CD complexes in aqueous media.

8. The i-butanol-P-CD complex is highly soluble at room temperature and is not effectively isolated by precipitation.

9. A temperature lower than 60°C for the filtration operation is not recommended, because of the relatively low solubility of P-CD at or near 25°C.

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10. Use of Tris buffer eliminates the need for frequent pH checks. However, as a precautionary measure, it is recommended that a pH determination be made every 2 or 3 d during the course of a reaction. Reduction of the temperature is not essential for the purpose of such measurements.

11. Shaking should be in a rotary fashion and at a rate (~200 rpm) fast enough for efficient mixing, but not so fast that the mixture splashes into the neck of the flask. Use of thin Teflon tape to seal glass-stoppered joints can eliminate the possibility of stoppers becoming difficult or impossible to remove because of the cementing ability that certain reaction-mixture components have once they enter the joint.

12. Because of the relatively low thermostability of pullulanase at 60°C, liquefication of starch gel (through debranching) is first effected with pullulanase alone at 25°C prior to adding complexant and CGTase and raising the temperature to 60°C, the desired level for conversion. Conversions of starch into y-CD in the presence of the complexant cyclododecanone are best conducted at or very close to 60°C. Use of lower temperatures can result in much lower yields; higher temperatures necessitate larger amounts of CGTase because of lower thermostability of the enzyme.

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