Method for the preparation of branched cyclodextrins

The invention provides an efficient enzymatic method for the preparation of branched cyclodextrins such as glucosyl and maltosyl cyclodextrins. The inventive method comprises the enzymatic reaction of a branch-splitting enzyme and .beta.-amylase simultaneously with a mixture of a cyclodextrin and starch. Alternatively, a branched cyclodextrin is obtained from a mixture of a cyclodextrin and maltose in an enzymatic reaction with pullulanase, optionally, with admixture of an alcohol such as ethyl and propyl alcohols or a glycol such as ethyleneglycol and propyleneglycol to the reaction mixture. A glucosyl cyclodextrin can be obtained by the steps of first subjecting a mixture of a cyclodextrin and maltose to an enzymatic reaction with pullulanase and then subjecting the reaction product to a second enzymatic reaction in the presence of an enzyme mixture composed of takaamylase and glucoamylase and yeast.

BACKGROUND OF THE INVENTION 
The present invention relates to a method for the preparation of branched 
cyclodextrins. 
Cyclodextrin is an oligosaccharide formed of at least six glucose units 
bonded together through .alpha.-1,4-linkages and principal known 
cyclodextrin compounds include .alpha.-, .beta.- and .gamma.-cyclodextrins 
formed of 6, 7 and 8 glucose units, respectively. 
Molecules of cyclodextrins have a cavity inherently due to the chemical 
structure thereof and the cavity exhibits hydrophobicity so that various 
kinds of oil substances can be incorporated into the cavity and retained 
therein. By virtue of this unique property, cyclodextrins have found very 
wide applications and are highlighted in a variety of industries for the 
manufacture of, for example, medicines, cosmetics and toiletries, 
perfumes, foodstuffs and the like. 
Cyclodextrins generally have low solubility and the values are only about 
14, 2 and 23 for the .alpha.-, .beta.- and .gamma.-cyclodextrins. 
.beta.-Cyclodextrin has a particularly low solubility and this is an 
undesirable and disadvantageous property when practical applications of 
cyclodextrins are intended. 
The inventors have recently conducted extensive investigations on branched 
cyclodextrins to elucidate the properties thereof [see, for example, 
Kobayashi, et al. Starch Science, volume 30, pages 231-239 (1983)]. 
Reportedly, for example, the solubility of the branched cyclodextrin is 10 
times larger than that of the corresponding cyclodextrin. 
Accordingly, a method has been developed for the preparation of branched 
cyclodextrins from starch and various types of branched cyclodextrins are 
being produced by this method. The principle of this method is to roll in 
the branched parts of the starch molecules to effect the cyclization 
reaction so that this method is advantageous in that a variety of the 
branched cyclodextrins are obtained by the method. This method, however, 
is disadvantageous when a single kind of the branched cyclodextrin is 
desired. 
An attempt has already been made [M. Abdullah and D. French, Nature, volume 
210, No. 5052, page 200 (1966)] for the preparation of various kinds of 
branched cyclodextrins utilizing the reverse action of pullulanase on a 
mixture of a cyclodextrin and an oligosaccharide. Their works, however, 
are limited, insofar as in the report, to a mere observation of the 
reverse action of pullulanase in the paper chromatography and details have 
not yet been disclosed. 
SUMMARY OF THE INVENTION 
An object of the present invention is therefore to provide a novel and 
efficient method for the preparation of branched cyclodextrins free from 
the above described problems and disadvantages in the prior art methods. 
Another object of the present invention is to provide a method for the 
preparation of branched cyclodextrins utilizing the reverse action of 
pullulanase and the like branch-splitting enzymes. 
According to the disclosure given below, the method of the present 
invention can be practiced in several different ways. Firstly, branched 
cyclodextrins are produced by the synergistic enzymatic effect of a 
branch-splitting enzyme and .beta.-amylase with a mixture of a 
cyclodextrin and starch. Secondly, branched cyclodextrins are produced by 
the enzymatic effect of pullulanase with a mixture of a cyclodextrin and 
maltose. Thirdly, branched cyclodextrins are produced by the enzymatic 
effect of pullulanase with a mixture of a cyclodextrin and maltose admixed 
with an alcohol selected from the group consisting of ethyl, n-propyl and 
isopropyl alcohols or a glycol compound selected from the group consisting 
of ethyleneglycol and propyleneglycol. Fourthly, the branched 
cyclodextrins produced by the enzymatic effect of pullulanase with a 
mixture of a cyclodextrin and maltose are subjected to column 
chromatographic separation into the individual branched cyclodextrins. 
Fifthly, glucosyl cyclodextrins are produced by the combined effect of 
yeast and an enzyme mixture composed of takaamylase and glucoamylase with 
the reaction product obtained by the enzymatic effect of pullulanase on a 
mixture of a cyclodextrin and maltose. The cyclodextrin used in the above 
described various ways for practicing the inventive method is not limited 
to a cyclodextrin of a particular type among the known cyclodextrins and 
any of them or a mixture thereof can be used.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
In the first of the above described embodiments of the inventive method, 
one of the starting materials is starch which is not particularly 
limitative in respect of the source plant and starch obtained from any 
starch-producing plant can be used including potatoes, sweet potatoes, 
corns, glutinous corns, barleys, wheats, cassavas and the like. Starches 
are composed, for example, of amylose and amylopectin as the compositional 
fractions. Further, as the decomposition products of starches are named, 
for example, roasted dextrins such as white dextrin, yellow dextrin, 
British gum and the like; processed starches such as low-viscosity 
starches modified by treatment with an enzyme or acid or high-speed 
mechanical agitation; derivatives of starch such as starch ethers and 
starch esters including, typically, starch phosphate, starch acetate and 
the like; physically-treated starches such as those subjected to an 
irradiation treatment with ionizing radiations or neutron beams, 
high-frequency treatment or wet-heat treatment; .alpha.-starches and the 
like. These starches and derivatives thereof can be used either singly or 
as a mixture of two kinds or more. 
The mixing ratio of the starch and the cyclodextrin is not particularly 
limitative but usually 100 parts by weight of the cyclodextrin are mixed 
with from 50 to 100 parts by weight of the starch material. 
The branch-splitting enzyme is preferably pullulanase although isoamylase 
can also be used. The isoamylase is used advantageously in the preparation 
of branched cyclodextrins having a branch formed of a compound composed of 
three or more of the glucose units, e.g. maltotriose, bonded thereto such 
as a maltotriosyl group. In addition to the conventional pullulanase, the 
pullulanase used here may be a heat-resistant or acid-resistant 
pullulanase. The use of a heat-resistant enzyme is advantageous because 
the solubility of the substrate material can be increased by the increaase 
of the temperature so that the reverse reaction is accelerated. 
The origin of the .beta.-amylase used here is also not particularly 
limitative including various kinds of plants and microorganisms. Not only 
highly purified products but also crude products of these enzymes can be 
used. A continuous process can be designed by use of an enzymatic 
bioreactor with an immobilized enzyme. 
In the first embodiment of the inventive method, the starting material is a 
mixture of a cyclodextrin and starch so that the combined use of a 
branch-splitting enzyme and .beta.-amylase is essential in order to 
hydrolyze the starch into maltose units. In the second to fifth 
embodiments of the inventive method, on the other hand, the starch in the 
first embodiment is replaced with maltose so that the .beta.-amylase is no 
longer required. Incidentally, the pullulanase also pertains to the 
splitting of the branches in the starch. 
In the third embodiment of the inventive method, an alcoholic compound or a 
glycolic compound is added to the reaction mixture and the addition of 
these compounds is effective to further improve the efficiency of the 
reverse reaction by the branch-splitting enzyme so that the yield of the 
branched cyclodextrins can be increased. The amount of addition of the 
alcoholic or glycolic compound in this case should be such that the 
concentration of the added compound in the reaction mixture is in the 
range from 10 to 40% by weight or, preferably, from 25 to 30%. When the 
amount of addition is too small, the desired effect mentioned above cannot 
be fully exhibited while an excessively large amount of the compound gives 
no additional improvement corresponding thereto sometimes with rather 
decreased yield of the branched cyclodextrins in comparison with the yield 
in the case of the absence of these compounds. An additional advantage 
obtained by the addition of an alcoholic or glycolic compound to the 
reaction mixture is that the enzymatic reaction can be performed with an 
increased concentration of each of the cyclodextrin and maltose as the 
substrates up to about 10 to 15% by weight. 
In the fourth embodiment of the invention, the reaction product obtained in 
the above described second embodiment is subjected to a column 
chromatography and the maltosyl cyclodextrin as a branched cyclodextrin is 
isolated therefrom. 
Further in the fifth embodiment of the invention, the same reaction product 
as above is used for the preparation of a glucosyl cyclodextrin by the aid 
of yeast and an enzyme mixture composed of takaamylase and glucoamylase. 
The reverse reaction in the above described inventive method with the 
branch-splitting enzyme is performed usually with the pH value of the 
reaction mixture at 4.5 to 6.0 and at a temperature in the range from 
30.degree. to 50.degree. C. and the reaction is continued for 24 to 72 
hours. 
The branched cyclodextrin product obtained by the above described inventive 
methods is mainly composed of maltosyl cyclodextrins. The first embodiment 
of the invention is advantageous in the possibility of direct use of 
starch while the advantage of the second embodiment of the invention is 
obtained in the improved efficiency for the preparation of the branched 
cyclodextrins by virtue of the use of maltose as the substrate. Further, 
the third embodiment of the invention gives an advantage that the reaction 
proceeds more efficiently than in the second embodiment so that the yield 
of the branched cyclodextrins is increased. 
When a glucosyl cyclodextrin is desired as the branched cyclodextrin 
product, the reaction product obtained by the above described method is 
subjected to the reaction by the aid of an enzyme mixture composed of 
takaamylase and glucoamylase as combined with yeast. Suitable species of 
the yeast used here are yeasts belonging to the Genus Saccharomyces which 
include Saccharomyces cerevisiae Saccharomyces diastaticus and the like. 
They are effective to remove the glucose and maltose contained in the 
reaction mixture by fermentation without decomposing the cyclodextrins and 
branched cyclodextrins. 
In addition to the above described method, the branched cyclodextrin 
product such as the maltosyl cyclodextrin and the like can be isolated 
from the reaction mixture by merely standing the reaction mixture at a 
relatively low temperature of 2.degree. to 10.degree. C. for 20 to 100 
hours or, preferably, for 24 to 72 hours. As an alternative method, the 
reaction mixture is admixed with a precipitant such as trichloroethylene, 
tetrachloroethane, bromobenzene and the like and shaken for 10 to 20 hours 
at a temperature of 5.degree. to 10.degree. C. followed by a solid-liquid 
separation procedure such as centrifugal separation to give the desired 
branched cyclodextrins. These methods of separation are applicable to the 
isolation of the branched cyclodextrins other than the maltosyl or 
glucosyl cyclodextrins from a mixture thereof with other saccharides. 
Further, these methods can of course be combined with other methods of 
separation such as a method using an activated carbon adsorbent or ion 
exchange resin, a method with Sephadex and the like utilizing the 
difference in the molecular weights, a method using a membrane and the 
like. 
The form of the branched cyclodextrin product obtained by the inventive 
method can of course be a pure material after further purification but the 
reaction mixture after the above described back synthesis reaction as such 
can be the final product depending upon the intended application. These 
branched cyclodextrins are useful in a wide variety of applications such 
as solubilization of medicines, cosmetics, perfumes, foods and the like. 
In the following, the method of the present invention is described in more 
detail by way of Testing and Preparatory Examples. 
TESTING EXAMPLE 1 
Reaction mixtures were prepared each by admixing .gamma.-cyclodextrin with 
one of the six oligosaccharides ranging from G.sub.1, i.e. glucose, to 
G.sub.6, i.e. maltohexaose, in a concentration of 20% by weight to give an 
overall saccharide concentration of 40% by weight and the mixture was 
admixed with a commercially available crude enzyme product of pullulanase 
in an amount of 200 I.U. per g of the overall amount of the substrates to 
be kept at 40.degree. C. for 48 hours with the pH adjusted to 5.5. The 
yield of the thus formed branched .gamma.-cyclodextrin is shown in Table 1 
for each of the oligosaccharides in a weight percentage based on the 
overall amount of the starting substrates. 
TABLE 1 
______________________________________ 
G.sub.1 
G.sub.2 G.sub.3 
G.sub.4 G.sub.5 
G.sub.6 
______________________________________ 
0 12.5 8.8 6.2 4.1 3.8 
______________________________________ 
The analysis of the branched cyclodextrins was performed by means of the 
high-performance liquid chromatography (HPLC) and paper chromatography. 
The conditions for the HPLC were as follows: instrument Tri Rotor by 
Nippon Bunko Co.; elution with 60 and 65% acetonitrile; flow rate 2 
ml/minute; detection by RI; attenuation 8X; column combination of a 
precolumn of 4.6 mm diameter and 5 cm length and a main column of 4.6 mm 
diameter and 25 cm length; and stationary phase in the columns Fine 
sil-NH.sub.2 of 10 .mu.m particle diameter. The retention time in minutes 
for each of the saccharide compounds was as tabulated below, in which the 
notations of G.sub.1 -.alpha.-CD, G.sub.2 -.alpha.-CD, etc. have the 
meanings of glucosyl .alpha.-cyclodextrin, maltosyl .alpha.-cyclodextrin, 
etc., respectively. CD is an abbreviation for cyclodextrin. 
TABLE 2 
______________________________________ 
G.sub.1 4.5 G.sub.2 -.alpha.-CD 
16.2 
G.sub.2 5.2 G.sub.1 -.beta.-CD 
17.1 
.alpha.-CD 8.1 G.sub.2 -.beta.-CD 
21.4 
.beta.-CD 11.0 G.sub.1 -.gamma.-CD 
24.9 
G.sub.1 -.alpha.-CD 
12.8 G.sub.2 -.gamma.-CD 
28.6 
.gamma.-CD 14.3 
______________________________________ 
The analysis of the branched cyclodextrins having a branch of G.sub.2 or 
larger by the HPLC and paper chromatography was preceded by the column 
chromatographic purification using a 2.6.times.100 cm column filled with 
Toyopearl HW-40 Super Fine. Further, a similar analysis was undertaken by 
use of pullulanase with the substrate concentration adjusted to 1% or 
smaller. The product saccharide by the reverse reaction with pullulanase 
is a branched cyclodextrin having a single branch per molecule. 
TESTING EXAMPLE 2 
The same experimental procedure as in Testing Example 1 was repeated with a 
mixture of .gamma.-cyclodextrin and maltose in equal amounts except that 
the total sugar concentration was varied in a wide range. The yield of the 
maltosyl .gamma.-cyclodextrin in % for each total sugar concentration is 
shown in Table 3 below, in which the saccharide concentration in % is 
given for each of the substrate saccharides. For example, the 
concentration of 1% given in the table means that the reaction mixture 
contained 1% by weight of .gamma.-cyclodextrin and 1% by weight of maltose 
to give an total sugar concentration of 2% by weight. 
TABLE 3 
______________________________________ 
Saccharide 
concentration, 
% 1 2 5 10 15 20 40 
______________________________________ 
Yield, % 0.2 0.8 2.6 8.2 11.6 12.5 26.3 
______________________________________ 
PREATORY EXAMPLE 1 
Reaction mixtures were prepared by mixing 100 mg of .alpha.-, .beta.- or 
.gamma.-cyclodextrin and 0.5 ml of each of a 20% solution of liquefied 
potato starch colored blue with iodine with further admixture of 80 mg of 
a crude enzyme product of pullulanase having an activity of 2 I.U./mg and 
0.5 ml of the supernatant liquid obtained by the centrifugal separation of 
a solution of 10 mg of a crude enzyme product of soybean .beta.-amylase 
having an activity of 20 I.U./mg in 1 ml of a 0.1M acetate buffer solution 
at a pH of 5.5 and the enzymatic reaction was performed with each of the 
thus prepared reaction mixture at 40.degree. C. for 48 hours with 
stirring. 
The results were that the yields of the branched cyclodextrins were 5.2%, 
1.3% and 6.8% for .alpha.-, .beta.- and .gamma.-cyclodextrins as the 
substrate, respectively. It was noted that dissolution of the 
.beta.-cyclodextrin was incomplete under the above described conditions 
and a considerable portion thereof remained in the reaction mixture in a 
crystalline form. 
PREATORY EXAMPLE 2 
Reaction mixtures were prepared by mixing 200 mg of .alpha.- or 
.beta.-cyclodextrin and 200 mg of each of maltose with further admixture 
of 0.5 ml of the supernatant liquid obtained by the centrifugal separation 
of a solution of 80 mg of a crude enzyme product of pullulanase having an 
activity of 2 I.U./mg in 1 ml of a 0.1M acetate buffer solution having a 
pH of 5.5 together with 0.5 ml of pure water and the enzymatic reaction 
was performed with each of the thus prepared reaction mixtures at 
40.degree. C. for 48 hours with stirring to give a reaction product 
containing the maltosyl .alpha.-cyclodextrin (G.sub.2 -.alpha.-CD) or 
maltosyl .beta.-cyclodextrin (G.sub.2 -.beta.-CD) and the unreacted 
saccharides as the substrates. 
Each of the thus obtained reaction mixtures was divided into two equal 
portions, of which one was kept standing as such for 48 hours at 4.degree. 
C. and the other was admixed with 200 .mu.l of tetrachloroethane or 
bromobenzene for the mixture from the .alpha.-cyclodextrin or from the 
.beta.-cyclodextrin, respectively, and shaken overnight at 10.degree. C. 
followed by centrifugal separation at 5000 r.p.m. for 20 minutes to give a 
supernatant liquid to be subjected to the determination of the maltosyl 
cyclodextrin therein. The results are shown in Table 4 in which the 
content of the maltosyl cyclodextrin in the supernatant liquid is given in 
% by moles based on the overall content of the cyclodextrin in moles 
including the values obtained by the analysis of the reaction mixture as 
such, after the low-temperature standing and after the precipitant 
treatment. 
TABLE 4 
______________________________________ 
Type of sub- 
Reaction After low- After pre- 
strate cyclo- 
mixture temperature 
cipitant 
dextrin as such standing treatment 
______________________________________ 
.alpha. 7.0 24.9 45.3 
.beta. 1.6 38.6 64.2 
______________________________________ 
PREATORY EXAMPLE 3 
A 2.6.times.100 cm chromatographic column filled with Toyopearl HW-40 Super 
Fine was loaded with 1 ml of the reaction mixture obtained in the reaction 
of Preparatory Example 2 with the .alpha.-cyclodextrin as the starting 
substrate and elution was performed with 10% ethyl alcohol as the eluant 
at a flow rate of 22 ml/hour to give fractions each having a volume of 2.2 
ml. The figure of the accompanying drawing illustrates a chromatogram 
obtained in this manner showing three definitely splitted peaks 
corresponding to the respective constituents as indicated. 
PREATORY EXAMPLE 4 
A mixture composed of 1.5 g of .alpha.-cyclodextrin and 1.5 g of maltose 
was admixed with 400 I.U. of a heat-resistant, acid-resistant pullulanase 
dissolved in 750 .mu.l of a buffer solution having a pH of 4.5 to 5.5 and 
further with 250 .mu.l of ethyl alcohol and the enzymatic reaction in this 
reaction mixture was performed at 70.degree. C. for 48 hours to give a 
yield of the maltosyl .alpha.-cyclodextrin as high as 42%. 
PREATORY EXAMPLE 5 
A mixture composed of 300 mg of .beta.-cyclodextrin and 300 mg of maltose 
was admixed with 750 .mu.l of the same enzyme solution as used in the 
preceding example and 250 .mu.l of ethyl alcohol and the enzymatic 
reaction was performed in this reaction mixture at 70.degree. C. for 48 
hours to give a yield of the maltosyl .beta.-cyclodextrin of 21%. Further 
experimentation established that the optimum concentration of ethyl 
alcohol in the reaction mixture was in the range from 25 to 30% and the 
yield of the desired product with a concentration of 45% or larger was 
even lower than the yield without addition of ethyl alcohol. 
PREATORY EXAMPLE 6 
The reaction mixture prepared in the preceding example was subjected to 
thermal deactivation and 1 ml portion of the mixture was, after adjustment 
of the pH to 4 to 5, admixed with water to have a total sugar 
concentration of 20% by weight followed by further addition of 2 mg of 
glucoamylase, 1 mg of takaamylase and 50 mg of wet fungus body of yeast 
(Saccharomyces cerevisiae). After the enzymatic reaction in this mixture 
at 30.degree. C. for 48 hours, the reaction mixture was subjected to 
centrifugal separation and the supernatant liquid was concentrated to give 
glucosyl .beta.-cyclodextrin. The purity of this product was 78% and the 
recovery was 65% based on the maltosyl .beta.-cyclodextrin produced in the 
reaction mixture. 
The glucosyl .beta.-cyclodextrin also could be prepared from the reaction 
mixture obtained in the preceding example by passing the mixture through a 
column of immobilized enzymes including glucoamylase, takaamylase and 
yeast.