Chapter 22: Cyclodextrins.

I. Introduction
Cyclodextrins are produced from starch by the action of an enzyme, cyclodextrin
glycosyltransferase (CDTGase; ED 2.4.1.19). A chemical synthesis has been reported, 1 but it
is too tedious for commercial production of cyclodextrins. Alpha-, beta- and gammacyclodextrins contain 6, 7 and 8 (1 4)-linked -D-glucopyranosyl units, respectively, and
are the predominate cyclodextrins formed by the enzyme. Deltaand nu-cyclodextrins, which
respectively contain 9 and 12 glucosyl units in the ring, have been isolated in small quantities
and characterized, 2,3 and evidence has been reported for cyclodextrins containing up to 16
glucosyl units in the ring. 4 Because of the predominance of the -, - and -cyclodextrins,
they are the cyclodextrins of current commercial interest.
Enzymes for the production of cyclodextrins (CD) are produced by various bacteria
(Table 22.1 ). No fungi or other organisms have been reported to produce CDTGase.
Enzymes obtained from these bacteria differ in the relative amounts of -, - and
-cyclodextrins produced. Typically, a mixture of two or three cyclodextrins is produced. As
the reaction proceeds, a second or third cyclodextrin also begins accumulating. While
-cyclodextrin might be the cyclodextrin initially produced, -cyclodextrin might accumulate
in larger quantities by the time the reaction is complete. In some cases, some of the
-cyclodextrin might be converted to -cyclodextrin. The fi nal ratio of the -, - and
-cyclodextrins depends on the enzyme used, the conditions of the reaction and the duration of
the reaction.
CDTGase

will

catalyze

three

different

enzyme

reactions:

cyclization;

transglycosylation; and hydrolysis 18 20 (see Chapter 7). Figure 22.1 shows a reaction
scheme for the reaction. Starch or a starch hydrolyzate can be used as the substrate. The
starch or starch hydrolyzate forms a complex with the enzyme; cleavage and joining of the
ends results in cyclization to form the cyclodextrin. The reaction is reversible, and a
cyclodextrin can serve as the substrate for the enzyme, resulting in ring opening. If the
reaction is allowed to go to equilibrium, either with starch or any one of the cyclodextrins as
the substrate, a mixture of cyclodextrins and linear maltooligosaccharides result.
In the transglycosylation reaction, a cyclodextrin or starch chain is cleaved and
another starch chain is added to the cleaved chain, resulting in one longer linear chain and
one shorter linear chain of glucopyranosyl units. This allows for the formation of a mixture of
cyclodextrins from a single reaction.

CDTGase displays -amylase activity. In this case, water is the acceptor and starch or
the cyclodextrin is cleaved, resulting in a shorter starch chain or a linear maltooligosaccharide
when a cyclodextrin is the substrate.
By using water-soluble organic solvents, usually referred to as precipitants, the
reaction can be directed to produce only one cyclodextrin. In the presence of toluene, the
enzyme from B. macerans produces only -cyclodextrin and linear starch chains. Betacyclodextrin can be separated from the soluble starch chains and recovered as a precipitated
toluene complex. In the presence of decanol, -cyclodextrin is the only cyclodextrin
produced using the enzyme from B. macerans . 21,22 Precipitants such as large cyclic
compounds similar to musk oil 23 or -naphthol and methyl ethyl ketone (butanone)24 can
be used to produce -cyclodextrin.
II. Production
Two processes are used for the commercial production of cyclodextrins. One is
referred to as the non-solvent process; in it, the enzyme is allowed to react with starch in a
completely aqueous environment. In the other, referred to as the solvent process, an organic
precipitant is added to direct the reaction to produce only one cyclodextrin.
Both processes use starch (corn, potato, rice, wheat, etc.) as the substrate. Corn and
potato starches are most commonly used. In both processes, the starch is hydrolyzed to a
dextrose equivalent (DE) of 3 to 8 (see Chapter 21) prior to use. 25 If liquifaction/hydrolysis
is not suffi cient, retrogradation occurs. This limits the availability of substrate, resulting in
low yields of cyclodextrins, and interferes with later recovery steps. If the starch is overhydrolyzed, the disproportionation reaction dominates and yields of cyclodextrins are low. If
an -amylase is used to hydrolyze the starch, it must be inactivated by acidifi cation, raising
the temperature, otherwise, the yield will be greatly reduced.
For the non-solvent process, 26 28 the pH of the starch hydrolyzate is adjusted to
the optimum pH before adding the enzyme. Starch concentration is usually _ 15%. At higher
concentrations, the yield of cyclodextrins decreases. At lower concentrations, the amount of
cyclodextrins per unit weight of the starch increases, but the yield per unit volume of the
reaction decreases. It is economically benefi cial to utilize more starch than to recover a
smaller amount of cyclodextrin per unit volume.
When the reaction is complete, a mixture of -, - and -cyclodextrins and linear
malto-oligosaccharides are present. The relative amounts of these components are dependent
on the source of CDTGase, which is inactivated, usually by heating, to prevent the loss of

cyclodextrins when -amylase is used to hydrolyze starch to reduce the viscosity. After
hydrolyzing the starch with an -amylase that will not attack the cyclodextrins, the reaction
mixture is deionized with a mixed-bed ion exchange resin and treated with activated charcoal.
Then, the reaction mixture is concentrated to a solids concentration of about 45% using
vacuum evaporation. This concentration is selected so that -cyclodextrin, the least soluble
of the cyclodextrins, will crystallize on cooling, but the other cyclodextrins will remain in
solution. The concentration can vary, depending on the enzyme used and the proportion of
cyclodextrins in the mixture. The concentrated reaction mixture is then cooled to crystallize
-cyclodextrin. If the starch is not suffi ciently hydrolyzed, the viscosity will be too high to
allow recovery of -cyclodextrin crystals. The yield is _ 15%.
Crystals of -cyclodextrin are then recovered by fi ltration or centrifugation and
washed with a small amount of water. They are then dried or dissolved in water to be
recrystallized to make a purer product. The -cyclodextrin produced from this process has a
purity of 98% or greater. If it is recrystallized, the purity can be 99%. The major impurities in
both cases are starch hydrolyzate and - and -cyclodextrins.
The mother liquor contains linear starch hydrolyzate and a mixture of cyclodextrins.
This can be further concentrated to about 70% solids and sold or further treated to recover the
- and -cyclodextrins. 29 To recover the latter, the residual starch hydrolyzate is converted
to D-glucose using glucoamylase/amyloglucosidase. The hydrolyzate is then passed through
an ion exchange resin to separate the glucose from the cyclodextrins. The resulting mixture of
cyclodextrins is then passed through a size-exclusion column to separate the cyclodextrins.
In the solvent process, 30,31 a solvent is used to direct the reaction to produce only
one cyclodextrin. Higher starch concentrations can be used in this process than in the nonsolvent process. (Concentrations of 30% are typically used.) Maximum yields per unit
volume are achieved at 30% solids in the presence of solvents, compared to a maximum yield
at 15% solids using the non-solvent process. The pH of the hydrolyzate is adjusted to the
optimum for the enzyme, and the enzyme and solvent are added to the starch hydrolyzate.
Solvents typically used for -cyclodextrin production include toluene, trichloroethylene and
cyclohexane. As the reaction proceeds, -cyclodextrin precipitates as an insoluble complex.
Yields per unit weight of starch are typically 30 45%, much higher than the yield obtained
by the non-solvent process.
On completion of the reaction, the complex of cyclodextrin and precipitant is
recovered by fi ltration or centrifugation. The precipitate is slurried in water, and the mixture
is heated to distill off the precipitant. At the completion of the distillation an aqueous solution

of cyclodextrin, containing _ 1 ppm of precipitant, results. The solution of -cyclodextrin is

treated with activated carbon and then cooled for crystallization. Crystals of -cyclodextrin
are collected by fi ltration or centrifugation and then dried. The purity of the product is at
least 98%, the major impurity being starch hydrolyzate. Since the reaction is directed to
produce only -cyclodextrin, and -cyclodextrins are not present in detectable quantities.
Alpha-cyclodextrin can also be produced using an organic precipitant. 21,22 In the
presence of decanol, the enzyme from B. macerans produces predominantly -cyclodextrin.
In the early stages of the reaction, a mixture of - and cyclodextrins are present, so the
reaction must be allowed to proceed long enough for the -cyclodextrin to disappear. Choice
of enzyme and precipitant is important. Some precipitants, such as cyclohexane, will produce
a mixture of - and -cyclodextrins; by controlling the temperature, relative proportions of
each can be controlled. 32 As the temperature is increased, the relative amount of
-cyclodextrin increases.
Decanol can be removed either by steam distillation 21,22 or by solvent extraction.
For steam distillation, the precipitated complex with decanol is slurried in water, and the
slurry is heated. Alternatively, a slurry can be extracted by mixing it with a solvent of lower
boiling temperature than decanol. Alpha-cyclodextrin forms a complex with the extraction
solvent and precipitates. The precipitate is recovered by centrifugation or fi ltration and
slurried in water, and the extraction solvent is then distilled and recovered for further use.
Alpha-cyclodextrin solution, free of decanol and extraction solvent, is treated with activated
carbon and fi ltered, and the product is crystallized.
Gamma-cyclodextrin can also be formed using a precipitant. Macrocyclic compounds,
such as cyclotridecanone, which do not fi t into the cavity of and -cyclodextrins are
used. 23,33 Such compounds do not dissolve in water or melt at temperatures compatible
with the enzyme and require the presence of a solvent, such as methyl isobutyl ketone, to
dissolve the precipitant and make it available to form a complex with -cyclodextrin. The
precipitated complex of -cyclodextrin is collected by centrifugation or fi ltration. Solvent
extraction is used for purifi cation of -cyclodextrin in the same manner as described for
cyclodextrin.
III. Properties
Cyclodextrins are soluble in water and insoluble in most organic solvents (Table
22.2).34,35 Solubility increases as the temperature increases. Beta-cyclodextrin is the least
soluble cyclodextrin in water. In -cyclodextrin, the C2 and C3 hydroxyl groups of adjacent

 -D-glucopyranosyl units orientate in such a way as to have maximum interaction with each
other, compared to the other cyclodextrins. 36,37 As a result, they are not as much available
to be hydrated compared to the other cyclodextrins. The -cyclodextrin ring is more
strained, resulting in these hydroxyl groups being further apart and more able to interact with
water, providing greater solubility. The -cyclodextrin ring is less strained than that of - or
-cyclodextrins. Water solubility results from the hydroxyl groups interacting much less with
each other and much more with water.
Interference with the hydrogen bonding of the hydroxyl groups increases the water
solubility of the cyclodextrins. In the presence of urea, the solubility of cyclodextrin
increases to 250 g/L. 38 At high pH, ionization of hydroxyl groups increases water solubility;
at pH 12.5 the solubility of -cyclodextrin is 750 g/L. 38,39
Cyclodextrins are insoluble in most organic solvents ( Table 22.3 ). Beta-cyclodextrin
is soluble in some polar aprotic solvents and is more soluble in some of these solvents than in
water. Cyclodextrins are somewhat soluble in mixtures of water and water-miscible organic
solvents. In general, as the concentration of the organic solvent increases, the solubility of the
cyclodextrin decreases.
Cyclodextrins are hydrolyzed by strong acids at a rate dependent on the temperature
and the acid concentration. 40 42 The fi rst step in hydrolysis of the cyclodextrins is ring
opening, a slow step compared to the hydrolysis of starch by acids. Once the ring is opened,
hydrolysis proceeds at the same rate as the hydrolysis of maltooligosaccharides. Depending
on the conditions, the products are a mixture of D-glucose and chains of (1 4)-linked -Dglucopyranosyl units up to a degree of polymerization equal to that of the cyclodextrin being
hydrolyzed. Weak organic acids do not readily hydrolyze cyclodextrins. Cyclodextrins are
stable in the presence of bases.
The differential scanning calorimetry (DSC) thermograms for -, - and
cyclodextrins are identical. Two heat absorption peaks are present: the fi rst occurs at 100 C
as water is evaporated from the crystals; the second, occurring at _ 250 C, is a result of
crystal melting and thermal decomposition.
Hydrolysis of cyclodextrins can be affected by enzymes 43 45 but not by
glucoamylases or -amylases, because both require a non-reducing end group to initiate
hydrolysis. Alpha-amylases are endoenzymes and can open the ring of the cyclodextrin
molecule (see Chapter 7 for a description of various amylases). Fungal amylases are better
catalysts for the hydrolysis of cyclodextrins than bacterial -amylases. The rate of hydrolysis
is dependent upon the cyclodextrin. Alpha-cyclodextrin is hydrolyzed very slowly compared

to -cyclodextrin. Gamma-cyclodextrin is hydrolyzed much more rapidly than

-cyclodextrin. Hydrolysis products consist of a mixture of D-glucose and short maltooligosaccharides.