Process for producing cyclodextrins

A process for producing cyclodextrins which comprises using cultured medium of the strains belonging to genus Micrococcus, filtrate thereof or enzyme preparation obtained therefrom, especially, the process in which the ratio of .alpha.-cyclodextrin to .beta.-cyclodextrin can be desirably varied by regulating the condition of the reaction mixture, is disclosed.

BACKGROUND OF THE INVENTION 
(1) Field of the Invention 
The present invention relates to a process for producing cyclodextrins and, 
more particulary, to a process which comprises cultivating microorganisms 
belonging to genus Micrococcus capable of producing cyclodextrin 
glycosyltransferase (E.C. 2.4.1.19), treating starch or degraded starch 
with the enzyme obtained by said cultivation or a composition containing 
such enzme to provide cyclodextrins and, if desired, recovering 
cyclodextrins thus produced. 
The cyclodextrins according to the present invention are crystalline 
dextrin, which are also called "Schardinger dextrin". 
They are cyclic oligosaccharide composed of 6, 7 or 8 glucose residues 
which are bound by .alpha.-1,4-bonds. They are called .alpha.-, .beta.- or 
.gamma.-cyclodextrin depending on the number of carbon residue, 6, 7 or 8, 
respectively. 
The cyclodextrin has been known as monomolecular host molecule which has 
torus in the molecule capable of including various kinds of organic 
compounds (guest compounds). This specific inclusion has been widely used 
in fields of medicine, agricultural chemicals, foods, cosmetics, perfumes 
and the like. 
(2) Description of the Prior Art 
The process for preparation of the cyclodextrins have already described by 
Tilden and Hudson (J. Am. Chem. Soc. 64, 1432 (1942) ) and D. French (J. 
Am. Chem. Soc. 71, 353 (1949) ), in which the cyclodextrins have been 
produced by treating starch with the enzyme from Bacillus macerans, the 
so-called "macerans amylase". 
Recently Bacillus stearothermophilus (Proc. Symp. Amylases (Osaka) 18, 43 
(1973) ) (Japan Kokai SHO 50-63189), Bacillus megaterium (Agr. Biol. Chem. 
38, 387 (1974) ) (Japan Kokai SHO 48-40996), Bacillus circulans (Proc. 
Symp. Amylases (Osaka) 18, 21, (1973) ), Bacillus ohbensis sp. (Japan 
Patent Publication SHO 52-31949) and Bacillus sp. No. 38-2 (ATCC 21783) 
(Die Starke, 27, 410 (1975) ) have been known as a microorganism which 
produces a similar enzyme. All the microorganisms belong to genus 
Bacillus. As such strain belonging to genus other than Bacillus, only 
Klebsiella pneumoniae M5 (Arch. Microbiol. 111, 271 (1977) ) has been 
reported. But there has been no report on production of such enzyme by any 
microorganism except for the microorganisms belonging to genus Bacillus 
and Klebsiella pneumoniae M5. 
Characteristics of the Process of the Invention 
It has been discovered by the present inventors that two strains newly 
isolated from soil and belonging to genus Micrococcus produce a similar 
enzyme to known one produced by the microorganisms belonging to genus 
Bacillus and Klebsiella. 
It has been further discovered that the enzyme produced by the micrococci 
is highly thermostable in wide range of pH and has high producibility of 
the cyclodextrins. 
It has been furthermore found that industrial production of the 
cyclodextrins can be advantageously accomplished by use of the enzyme. 
Furthermore, the present invention provides a process for producing 
cyclodextrins, as one of the embodiments, in which .alpha.-cyclodextrin 
and .beta.-cyclodextrin can be produced in desired ratio. Namely it has 
been reported that, in conventional processes, for example, cyclodextrin 
glycosyltransferases from Bacillus macerans, Bacillus stearothermophilus 
and Klebsiella pneumoniae M5 mainly provide .alpha.-cyclodextrin and those 
from Bacillus megaterium, Bacillus circulans, Bacillus ohbensis sp. and 
Bacillus sp. No. 38-2 give .beta.-cyclodextrin as their main product. 
On the other hand, although cyclodextrin glycosyltransferase according to 
the present invention usually produces .beta.-cyclodextrin, appropriate 
regulation of pH and substrate and enzyme concentrations in the enzyme 
reaction mixture enables to raise a ratio of .alpha.-cyclodextrin to 
.beta.-cyclodextrin in the reaction product. 
OBJECTS OF THE INVENTION 
Accordingly, it is an object of this invention to provide a new process for 
producing cyclodextrins from starch or degraded starch by using cultured 
medium of newly isolated strains belonging to genus Micrococcus, filtrate 
thereof or enzyme preparation obtained therefrom. 
Another object of this invention is to provide an enzyme source of 
cyclodextrin glycosyltransferase which is high thermostable in wide range 
of pH and has highly producibility of the cyclodextrins. 
Still another object of this invention is to provide a new process in which 
the ratio of .alpha.-cyclodextrin to .beta.-cyclodextrin can be desirably 
regulated. 
SUMMARY OF THE INVENTION 
Therefore, the present invention provides a process for producing the 
cyclodextrins which comprises cultivating a microorganism belonging to 
genus Micrococcus capable of producing cyclodextrin glycosyltransferase in 
a nutrient medium and treating starch or degraded starch with a cultured 
medium obtained by said cultivation, medium filtrate, concentrate thereof 
or an enzyme preparation obtained from various steps of purification, to 
give cyclodextrins. 
Furthermore, the present invention also provides a process for desirably 
regulating a ratio of .alpha.- and .beta.-cyclodextrins in the enzyme 
reaction product.

DETAILED EXNATION OF THE INVENTION 
The microorganism according to the present invention belongs to genus 
Micrococcus, which differs from the conventional bacilli known as 
cyclodextrin glycosyltransferase-producing strain. For this purpose, two 
strains which are highly capable of producing such enzyme and named M 849 
and B 645, respectively, have been selected and taxonomically identified. 
These strains are deposited with Fermentation Research Institute, Agency 
of Industrial Science and Technology as the culture number FERM-P No. 4912 
and FERM-P No. 4913, respectively. 
The taxonomical study of the strains was performed according to a paper, 
"Methods for Classifying Staphylococci and Micrococci", presented by Baird 
Parker appeared in "Identification Methods for Microbiologist" I (Part A) 
(Ed. by B. M. Gibbs and F. A. Skinner, 1966) and papers described by the 
same author in J. Gen. Microbiol. 30, 409 (1963) and 38, 363 (1965). 
Reports by J. B. Evans and W. E. Kloos appeared in Appl. Microbiol. 23, 
326 (1972), and M. Kocur and T. Martinec appeared in International Journal 
of Systematic Bacteriology 22, 218 and 228 (1972), and also Bergy's Manual 
of Determinative Bacteriology, 8th Ed. (1974) were also referred. 
(1) Taxonomical characteristics of strain M 849 (FERM-P No. 4912) (ATCC 
31606) 
(a) Morphology 
(i) Vegetative cell: Spheres, 0.9-1.5 .mu.m in diameter. 
(ii) Occurring singly, in pairs or tetrads, sometimes in irregular 
clusters. 
(iii) Non-motile. 
(iv) Spores not formed. 
(v) Gram-positive. 
(b) Cultural characteristics (Cultivated at 30.degree. C. for 2 days unless 
otherwise noted) 
(i) Nutrient agar plate 
Colonies were circular, white, smooth glistening, opaque and convex with a 
regular edge. 
(ii) Nutrient agar slant 
Good growth in filiform. White, opaque and glistening colonies with a 
smooth surface and regular edge. Sometimes wavy edge in old culture. 
Medium unchanged. 
(iii) Nutrient broth 
Turbid with viscous sediment. Pellicle and ring not formed. Odorless. 
Pigment not formed. 
(iv) Nutrient gelatin stab (20.degree. C.) 
Slow and only surface growth at early stage. Very slight liquefaction after 
more than 15 days. Slight ability to liquefy gelatin was shown by other 
test. 
(v) Litmus milk 
Unchanged at early stage but acidic after 5-10 days. No coagulation, 
peptonization and decolorization of litmus were shown. 
(c) Physiological characteristics 
(i) Reduction of nitrates: Positive. 
(ii) Gas production in nitrate broth: Negative. 
(iii) MR test: Slightly positive. 
(iv) Acetoin production: Negative. 
(v) Indole production: Negative. 
(vi) H.sub.2 S production: Negative. 
(vii) Starch hydrolysis (in nutrient medium supplemented with 0.2-1.0% 
soluble starch): Negative at pH 7.0 and Positive at pH 9.5. 
(viii) Citrates utilization: Negative in Christensen's and Simmons' media. 
(ix) Pigment: Not produced. 
(x) Urease: Positive. 
(xi) Oxidase (Kovac's method): Negative. 
(xii) Catalase: Positive in nutrient broth and nutrient agar supplemented 
with 1% glucose. 
(xiii) Growth conditions: Optimum pH for growth was 6.8-8.5 and good growth 
even at pH 9.5. Almost no growth below pH 5.0. Optimum temperature was 
27.degree.-37.degree. C. Growth occurred at 40.degree. C. or 20.degree. C. 
but scant growth at 10.degree. C. No growth at 45.degree. C. 
(xiv) Behavior to oxygen: Aerobic. 
(xv) OF test: Oxidative. Acid was produced oxidatively from glucose and 
mannitol but no gas produced. No gas and acid production under anaerobic 
condition. 
(According to the method recommended by International Subcommittee on 
Staphylococci and Micrococci, Intern. Bull. Bact. Nomencl. Taxon. 15 109, 
1965.) 
(xvi) Acid production from various sugars under aerobic condition: 
______________________________________ 
(1) L-Arabinose - 
(2) D-Xylose - 
(3) D-Glucose + 
(4) D-Mannose + 
(5) D-Fluctose + 
(6) D-Galactose + 
(7) Maltose + 
(8) Sucrose + 
(9) Lactose + 
(10) Trehalose + 
(11) D-Sorbitol - 
(12) D-Mannitol + 
(13) Inositol - 
(14) Glycerine + 
(15) Starch - 
______________________________________ 
+: Production 
-: Not produced 
No gas production from all the carbohydrates. 
(xvii) Degradation of esculin: Negative (Slight degradation after a 
long-term culture). 
(xviii) Degradation of hippuric acid: Positive. 
(xix) Degradation of arginine: Negative. 
(xx) Deamination of phenylalanine: Negative. 
(xxi) Coagulase: Negative. 
(xxii) Hemolysis: Negative. 
(xxiii) Sodium chloride tolerance: Good growth in nutrient medium 
containing 15% and less NaCl. Slow and scant growth in 18% or 20% 
NaCl-containing medium. 
(xxiv) Phosphatase: Negative. 
(xxv) Degradation of Tween 80: Negative. 
(xxvi) Growth on an inorganic nitrogen agar: 
Almost no growth (according to Kloos' method. International J. System. 
Bacteriol. 24, 79, 1974). 
(xxvii) Liquefaction of gelatin: Slight liquefaction (according to Frazier 
gelatin agar method). 
(xxviii) Hydrolysis of casein: Negative (according to solubilization in 
skim milk agar). 
(xxix) DNase: Negative. 
It is evident from the above-mentioned results that the strain M 849 
capable of producing the enzyme according to the present invention is 
assigned to a bacteria belonging to genus Micrococcus. There are described 
sixteen species of bacteria as micrococci in Bergey's Mannual of 
Determinative Bacteriology, 7th Edition (1957). Genus Sarcina is also 
described as an independent genus. The latest 8th edition of the same 
mannual contains only three species in genus Micrococcus, that is, 
Micrococcus luteus, M. varians and M. roseus, where genus Sarcina is 
eliminated and many species classified to genus Micrococcus before are 
reclassified to the three species based on further studies. Micrococcus 
varians may be selected as the most resembled species by comparing the 
taxonomical characteristics of strain M 849 with those of the said three 
described micrococcal species. According to the described characteristics 
of M. varians, colonies on a nutrient agar or other common media are 
yellow. This is one of the most important characteristics to distinguish 
M. varians from M. roseus. However, in the above-referred Baird Parker's 
paper, the subgroup 5 of genus Micrococcus which he has proposed, contains 
a number of strains producing white colonies, and is considered as 
belonging to M. varians. Strain M 849 showed negative activity in esculin 
degradation but very weak degradation activity was sometimes shown over a 
long period culturing. 
Consequently, the strain according to the present invention differs in 
these characteristics from the description on the neotype strain of M. 
varians presented by M. Kocur and T. Martinec. But strain M 849 may be 
determined to be Micrococcus varians because the morphological, cultural 
and physiological characteristics coincided with those of M. varians while 
there were differences in white colonies and esculin degradation and is 
deposited as Micrococcus varians M 849 with Fermentation Research 
Institute, Agency of Industrial Science and Technology under the culture 
number FERM-P No. 4912 and also deposited as ATCC 31606 with the American 
Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, 
U.S.A. 
(2) Taxonomical characteristics of strain B 645 (FERM-P No. 4913) (ATCC 
31607) 
(a) Morphology 
(i) Vegetative cells:Sphere, 1.0-1.8.mu. m in diameter. 
(ii) Occuring in packets of cells. 
(iii) Non-motile. 
(iv) Spores not formed. 
(v) Gram-positive. 
(b) Cultural characteristics (Cultivated at 30.degree. C. unless otherwise 
noted.) 
(i) Nutrient agar plate 
Small or dott-like, circular, dark yellow to orange-colored, smooth, 
glistering and opaque convex with regular edge. 
(ii) Nutrient agar slant 
Slow, moderate to poor growth. Orange-yellow to orange-colored and slightly 
raised filiform with a smooth and soft surface. Medium unchanged. 
(iii) Nutrient broth 
Slow-growing, turbid with small sediment. Pellicle and ring not formed. 
Odorless, Pigment not produced. 
(iv) Nutrient gelatin stab (20.degree. C.) 
Poor growth. Liquefaction because of non-solidification by cooling after 5 
day incubation at 30.degree. C. 
(v) Litmus milk 
Unchanged at early stage. Coagulation after 10 days to gradual 
peptonization without color change of litmus. 
(c) Physiological characteristics 
(i) Reduction of nitrates:Negative. 
(ii) Gas production in nitrate broth:Negative. 
(iii) MR test:Negative. 
(iv) Acetoin production:Negative. 
(v) Indole production-Negative. 
(vi) H.sub.2 S production:Negative. 
(vii) Starch hydrolysis (by the same procedure as in strain M 849):Negative 
at pH 7.0 and positive at pH 9.5. 
(viii) Citrates utilization:Negative in Simmons' media and positive in 
Christensen's medium. 
(ix) Pigment:Water-soluble pigment not produced in a medium while colonies 
were yellow to orange-colored. 
(x) Urease:Negative. 
(xi) Oxidase (Kovac's method):Negative. 
(xii) Catalase:Positive (even in 1% glucose-supplemented medium). 
(xiii) Growth conditions:Optimum pH for growth was 6.0-8.5. Good growth 
even at pH 9.5 and 10.5. Almost no growth below pH 5.0. Optimum 
temperature was 25.degree.-35.degree. C. No growth above 40.degree. C. or 
below 17.degree. C. 
(xiv) Behavior to oxygen:Aerobic. 
(xv) OF test (According to the same method as in strain M 849):Aerobic 
growth in case of glucose and mannitol as a carbon source but no growth 
under anaerobic condition, which was confirmed by Evans and Kloos' method 
above mentioned. Acid and gas not produced under aerobic and anaerobic 
conditions. 
(xvi) Acid production from various sugars under aerobic condition: 
______________________________________ 
(1) L-Arabinose - 
(2) D-Xylose - 
(3) D-Glucose - 
(4) D-Mannose - 
(5) D-Fructose - 
(6) D-Galactose - 
(7) Maltose - 
(8) Sucrose - 
(9) Lactose - 
(10) Trehalose - 
(11) D-Sorbitol - 
(12) D-Mannitol - 
(13) Inositol - 
(14) Glycerine - 
(15) Starch - 
______________________________________ 
- : Not produced 
No gas production from all the carbohydrates. 
(xvii) Degradation of esculin:Negative. 
(xviii) Degradation of hippuric acid:Negative. 
(xix) Degradation of arginine:Negative. 
(xx) Deamination of phenylalanine:Negative. 
(xxi) Coagulase:Negative. 
(xxii) Hemolysis:Negative. 
(xxiii) Sodium chloride tolerance:Good growth in nutrient medium containing 
5% and less NaCl. No growth in 8% and more NaCl-containing medium. 
(xxiv) Phosphatase-Negative. 
(xxv) Degradation of Tween 80:Negative. 
(xxvi) Growth on an inorganic nitrogen agar:Almost no growth (according to 
the same method as that in strain M 849). 
(xxvii) Liquefaction of gelatin:Positive (according to Frazier gelatin agar 
method). 
(xxviii) Hydrolysis of casein:Positive (according to solubilization in skim 
milk agar). 
(xxix) DNase-Negative. 
From the above results, strain B 645 was identified to be one of bacteria 
belonging to genus Micrococcus. The taxonomical characteristics of the 
strain were in good agreement with those of a neotype strain of 
Micrococcus luteus described in Bergey's Mannual of Determinative 
Bacteriology, 8th Edition (1974) and the above-referred M. Kocur, Z. 
Pacova and T. Martinec's paper appeared in Intern. j. Syst. Bact. 22, 218 
(1972). Therefore, strain B 645 according to the present invention was 
determined to be Micrococcus luteus and is deposited as Micrococcus luteus 
B 645 with Fermentation Research Institute, Agency of Industrial Science 
and Technology under the culture number FERM-P No. 4913 and also deposited 
as ATCC 31607 with the American Type Culture Collection, 12301 Parklawn 
Drive, Rockville, Md. 20852, U.S.A. 
CULTIVATION OF THE MICROORGANISM 
Production of the enzyme according to the present invention is accomplished 
by cultivating the microorganisms according to the present invention by 
aerobic shaking culture or submerged culture which is known per se. A 
medium used for this purpose is known one, namely, which contains 4-10% of 
carbohydrate as a carbon source, such as starch from corn, potato or sweet 
potato, soluble starch, dextrin, acid-treated starch, amylose and 
amylopectin, and appropriate amount of nitrogen source such as, soy bean 
meal, dried yeast, milk casein, peptone, meat extracts, corn steep liquer, 
yeast extract, peptides-containing material and amino acids or a mixture 
thereof. Furthermore, a small amount of inorganic salts, for example, 
K.sub.2 HPO.sub.4 and MgSO.sub.4.7H.sub.2 O is added. The enzyme according 
to the present invention can be produced and accumulated by cultivating 
the microorganisms according to the present invention aerobically in said 
medium, pH 7-10.5, at 25.degree.-40.degree. C., preferably 
30.degree.-37.degree. C., for 24-96 hours. 
RECOVERY OF THE ENZYME 
The enzyme according to the present invention may be recovered and purified 
by the known process which has been employed for recovery and purification 
of enzymes from various cultured materials. For example, the following 
methods may be employed independently or in their 
combination:Concentration of a cultured broth filtrate by evaporation 
under reduced pressure or ultrafiltration, salting-out with ammonium 
sulfate, sodium sulfate or sodium chloride, fractional precipitation with 
organic solvent such as methanol, ethanol and acetone, adsorption and 
elution process with an appropriate adsorbant such as starch, DEAE 
cellulose and cross-linked dextran, precipitation with 
protein-precipitation agent, isoelectric precipitation or electric 
dialysis. 
ASSAY OF THE ENZYME ACTIVITY 
Various methods for assay of cyclodextrin glycosyotransferase activity have 
been reported. It has been found that there is a correlationship between 
the two activity values obtained by sugar-transfer method by H. Bender 
(Arch. Microbiol. 11, 271-282 (1977) and modified cyclodextrin production 
method with glucoamylase by M. Matzuzawa et al (Die Starke 27, 410-413 
(1975) ), respectively. Therefore, the sugar-transfer method is mainly 
employed. 
SUGAR-TRANSFER METHOD BY H. BENDER 
A mixture containing 0.50% cycloheptaamylose and 0.025% maltotriose in 0.05 
M phosphate buffer solution (pH 7.0) is prepared. To 1.9 ml of the mixture 
is added 0.1 ml of enzyme solution and the mixture is incubated at 
37.degree. C. for 30 min. The reaction is stopped by heating with boiling 
water for 10 min. A 0.5 ml portion of the reaction mixture is poured into 
0.8 ml of 0.2 M phosphate buffer solution (pH 6.0) containing 40 units of 
.beta.-amylase. The mixture is incubated at 37.degree. C. for 30 min., and 
then reducing activity of the mixture is measured by Somogi-Nelson's 
method. The same procedure is repeated as a control except that water is 
used in place of the enzyme solution. The reducing activity corrected with 
the control value is converted to amount of glucose, which is defined as 
sugar-transfer activity. One unit of the activity is defined as an amount 
of enzyme required for production of 1 mg of glucose calculated from the 
reducing activity at 37.degree. C. for 1 min. 
METHOD USING GLUCAMYLASE 
A mixture of 1.0 ml of 1.0% soluble starch solution in 0.1 M Veronal buffer 
solution, pH 8.0, containing 1 mM CaCl.sub.2 as a substrate, and 0.1 ml of 
an appropriately diluted enzyme solution, is incubated at 50.degree. C. 
for 2 hours. After pH is adjusted to 6.0 and the volume of the reaction 
mixture is filled up to 2.0 ml with water, the mixture is heated at 
100.degree. C. for 10 min. Then, 100.mu. g of glucamylase is added and the 
mixture is incubated at 40.degree. C. for 1 hour to hydrolyze starch and 
oligasaccharide. Glucose thus produced is quantitatively determined by 
Somogi-Nelson's method. The same procedure is carried out using water in 
place of enzyme solution as a control. An amount of cyclodextrins produced 
is calculated from a difference between the above-mentioned two values. 
Purification and physico-chemical properties of the enzyme are shown with 
Micrococcus varians M 849, as there is almost no difference in the 
properties of enzyme between Micrococcus luteus B 645 and M. varians M 
849. 
PURIFICATION OF THE ENZYME 
A cultured medium of Micrococcus varians M 849 is centrifuged to remove 
cells. Acetone is added to the supernatant to precipitate the enzyme. The 
precipitate is dissolved in water. The solution is subjected to fractional 
precipitation by ammonium sulfate salting out. The precipitate obtained 
between 45 and 65% saturation of ammonium sulfate is purified by column 
chromatography with DEAE Sephadex A-50 and then by gel filtration with 
Sephandex G-100. The purified enzyme thus obtained give a single band 
detected with coumassie brilliant blue in disc electrophoresis with 
polyacrylamide gel at pH 8.3. This band also shows the enzyme activity. 
Table 1 shows a typical result of the purification. 
TABLE 1 
______________________________________ 
Specific 
Total activity 
Re- 
Volume Activity activity 
(U/mg of 
covery 
Process (ml) (U/ml) (U) protein) 
(%) 
______________________________________ 
Cultured 
medium 
filtrate 
1800 8.6 1.55 .times. 10.sup.4 
1.8 100 
Acetone 
precipi- 
tate 
dissolved 
in water 
1240 9.5 1.18 .times. 10.sup.4 
2.1 76 
Ammonium 
sulfate 
saturated 
fraction 
30 284.0 0.85 .times. 10.sup.4 
14.0 55 
DEAE- 
Sephadex 
eluate 680 6.8 0.46 .times. 10.sup.4 
25.6 30 
Sephadex 
G-100 
Fraction 
190 18.0 0.34 .times. 10.sup.4 
43.4 22 
______________________________________ 
(1) Enzyme action and substrate specificity 
The enzyme according to the present invention acts on various kinds of 
starch, for example, potato, starch, corn starch and dextrin to make their 
reactivity to iodine negative and produce a large amount of cyclodextrins 
without showing substantial increase of reducing activity. In addition to 
the cyclodextrins, maltotetraose, maltotriose, maltose and glucose are 
produced as the final reaction products. The same final reaction products 
are also obtained by treating oligosaccharides having 
.alpha.-1,4-glycoside bonds with the enzyme. Therefore, the enzyme of the 
present invention can be defined to be cyclodextrin glycosyltransferase 
(E.C. 2.4.1.19). 
However, the cyclodextrin glycosyltransferase according to the present 
invention is capable of producing .alpha.-cyclodextrin as a main reaction 
product by regulating pH, substrate concentration and enzyme concentration 
although the enzyme usually produces .beta.-cyclodextrin dominantly, while 
conventional cyclodextrin glycosyltransferases produce only one type of 
cyclodextrin as a main product. 
(2) Optimum pH and pH stability 
Acetate buffer solution (pH 4.0-6.0), Veronal buffer solution (pH 6-8.5) 
and glycine-sodium hydroxide buffer solution (pH 8.5-12.0) were used for 
studies on the action and stability of the enzyme at 37.degree. C. 
FIGS. 1 and 2 shows the optimum pH for the activity and the yield of 
cyclodextrins of the enzyme, respectively. In FIG. 1, the axis of ordinate 
represents relative activity in % of sugar-transfer activity measured by 
Bender's method and the axis of abscissa represents pH. In FIG. 2, the 
axis of ordinate represents recovery in % of cyclodextrin production from 
starch measured by glucamylase method and the axis of abscissa represents 
pH, where the open circles (curve 1) and closed circles (curve 2) show the 
enzymes from Micrococcus varians M 849 and Micrococcus luteus B 645, 
respectively. The enzyme according to the present invention shows high 
activity in considerably wide range of pH, between 4.5 and 10.5 and the 
optimum pH is between 5 and 8. There is also another activity peak around 
pH 10. 
FIG. 3 shows the pH stability at 37.degree. C. of the enzyme in which 
measurement of residual activity after treatment of the enzyme at various 
pH for 24 hours at 37.degree. C. show almost no inactivation between pH 5 
and pH 9 as shown in FIG. 3. 
(3) Effect of temperature 
FIG. 4 shows the enzyme activity measured at various temperatures and pH 7, 
where the axis of ordinate represents the relative activity (calculated 
the value at 37.degree. C. as 100) and the axis of absissa represents 
temperature in centigrade. As shown in this figure, the activity increases 
with a raise of temperature up to 40.degree.-55.degree. C. The optimum 
temperature is 55.degree.-65.degree. C. 
(4) Inactivation of the enzyme by pH and temperature 
FIG. 5 shows the residual activity measured after treatment of the enzyme 
solution (containing 5 mM Ca) at pH 5.0, 7.0, 9.0 and 11.0 for 15 min, 
where the axis of ordinate represents the residual activity in % and the 
axis of abscissa represents temperature in centigrade and also curves 1, 
2, 3 and 4 represent the results obtained at pH 5.0, 7.0, 9.0 and 11.0, 
respectively. No inactivation is shown between pH 5 and pH 9 below 
50.degree. C. and only 20-30% of the activity is lost at 60.degree. C. 
(5) Inhibition, activation and stabilization 
Tab, 2 shows the residual activity after treatment of the purified enzyme 
with various inhibitors or inorganic salts in 0.05 M phosphate buffer 
solution (pH 7) at 37.degree. C. for 1 hour. The enzyme of the present 
invention is inhibited by metal ions such as copper, iron and mercury and 
ethylendiamine tetraacetic acid. The thermostability of the enzyme is 
enhanced by addition of 10.sup.-2 M-10.sup.-3 M calcium chloride. The 
present enzyme is stable in presence of Ca.sup.++ even at almost 5.degree. 
C. higher than the temperature at which the enzyme is inactivated in 
absence of the metal ion, as shown in FIG. 6, wherein the axis of ordinate 
represents the residual activity in % after treatment at various 
temperatures for 15 min. and the axis of abscissa represents temperature 
in centigrade and also the solid line (1) and interrupted line (2) 
represent the results in absence and in presence of 10.sup.-2 M calcium 
chloride, respectively. 
TABLE 2 
______________________________________ 
Concentration Residual activity 
Additives (mM) (%) 
______________________________________ 
CaCl.sub.2 1.0 100 
MgCl.sub.2 1.0 100 
ZnCl.sub.2 1.0 100 
MgSO.sub.4 1.0 100 
FeSO.sub.4 0.5 10 
CuSO.sub.4 0.5 6 
CoCl.sub.2 0.5 88 
HgCl.sub.2 0.5 5 
Ethylenediamine- 
tetraacetic acid 
1.0 2 
p-Chloromercuri- 
benzoic acid 1.0 95 
Diisopropylfluoro- 
phosphate 1.0 100 
Monoiodoacetic 
acid 1.0 97 
O-Phenanthroline 
1.0 100 
______________________________________ 
(6) Molecular weight etc. 
(i) The purified enzyme preparation provided a single symmetrical peak in 
ultracentrifugation pattern. Sedimentation constant, S.sub.20, w, was 
5.40-5.60.times.10.sup.-13 which was calculated from the experimental 
results obtained with 0.5, 0.3 and 0.2% protein concentration. Diffusion 
constant, D.sub.(w), was 5.84.times.10.sup.-7 measured with an interfacial 
cell. Therefore, the enzyme of the present invention has a molecular 
weight of 85,000.+-.2,000. 
(ii) The purified enzyme preparation gave a single band in electrofocusing 
with Ampholine and sucrose gradient between pH 3 and pH 10 at 700 V and 3 
mA for 48 hours in a LKB 810 electrofocusing column. The isoelectric point 
has been found to be pH 4.2. 
(iii) Tab. 3 shows amino acid composition of the enzyme. 
TABLE 3 
______________________________________ 
Amount of amino 
acid (g) per Number of amino 
enzyme protein acid residue per 
Amino acid (100g) enzyme molecule 
______________________________________ 
Lysine 2.5 13.7 
Histidine 2.7 13.4 
Arginine 4.7 18.9 
Aspartic 17.0 101.4 
Threonine 7.0 48.4 
Serine 6.7 53.3 
Glutamic 9.4 50.3 
Proline 3.4 23.9 
Glycine 6.2 74.0 
Alanine 3.1 30.4 
Valine 6.2 42.7 
Methionine 0.8 4.3 
Isoleucine 6.9 41.6 
Leucine 5.3 32.5 
Tyrosine 7.3 30.9 
Phenylalanine 
4.8 22.3 
Tryptophan 3.6 13.3 
Half-cystine 
0.7 4.7 
NH.sub.3 1.7 70.7 
Total 100 
______________________________________ 
(7) Crystal structure 
The purified enzyme crystallizes in ammonium sulfate solution. The crystal 
has dodecahedron structure of which specific activity is 45 U/mg of 
protein. 
PRODUCTION OF THE CYCLODEXTRINS 
The substrate, for example, starchs such as potato starch, corn starch or 
soluble starch and degraded starch such as dextrin, is dissolved in water 
with heating. To the solution is added an enzyme preparation obtained from 
various steps of purification, such as a cultured medium of the 
microorganism of the present invention capable of producing the enzyme, 
cultured medium filtrate, concentrate thereof, or purified enzyme, after 
the pH is adjusted to 4.5-10.5, preferably 6-7 for the production of total 
cyclodextrin. After incubation at a temperature of 40.degree.-65.degree. 
C. for an appropriate time, the reaction product is precipitated by one of 
the known methods. The precipitate is collected and washed with water. 
Recrystallization by the known method of the washed precipitate provides 
crystalline cyclodextrins. 
The cyclodextrins thus obtained can be fractionated into .alpha.-, .beta.- 
and .gamma.-cyclodextrin according to a method described by D. French et 
al (J. Am. Chem. Soc. 71, 353 (1949), each of which may be obtained as a 
white crystalline purified preparation. In the physico-chemical properties 
of each preparation, crystal form, melting point, specific optical 
rotation, iodo-starch reaction, reducing sugar content and infrared 
spectrum were confirmed to coincide with those of authentic .alpha.-, 
.beta.- or .gamma.-cyclodextrin. 
Relationship betweein cyclodextrin productions and the condition for 
producing thereof 
EXPERIMENT 1 
A solution of 10 g of soluble starch dissolved in 100 ml of water 
containing 0.05% CaCl.sub.2.2H.sub.2 O was prepared with heating. After 
the pH was adjusted to 6.0, various amounts of enzyme and 5 ml of 
trichloroethylene were added. The mixture was incubated at 50.degree. C. 
or 60.degree. C. for 2 days. Cyclodextrins were recovered as precipitation 
of its trichloroethylene complex. Table 4 shows the results. 
TABLE 4 
______________________________________ 
Amount of 
enzyme used 
Cyclodextrin production (%) 
(U/g of (Recovery from starch 
Enzyme starch) 50.degree. C. 
60.degree. C. 
______________________________________ 
Cultured medium 
1 50 52 
filtrate of 
2 55 60 
strain M 849 
3 58 55 
5 52 45 
10 48 40 
Purified 1 50 51 
preparation 
2 58 62 
3 56 54 
5 50 43 
10 47 38 
______________________________________ 
Cyclodextrins were produced in 50-52% yield by using one unit of enzyme per 
gram of starch and in higher yield such as 60% by using 2 units per gram 
of starch in the case of the enzyme of the present invention and further 
there shows a tendency of rather decrease of the total cyclodextrins yield 
with using more enzyme than this optimal units. 
EXPERIMENT 2 
Soluble starch was dissolved with heating in water containing 0.05% 
CaCl.sub.2.2H.sub.2 O. The solution was diluted to various concentration 
indicated in Table 5. To the solutions, were added various amounts of the 
enzyme of the present invention after the pHs being adjusted to various 
values, as indicated in Table 5. The reaction mixtures were incubated at 
50.degree. C. for 2 days, and then the amounts of .alpha.-, .beta.- and 
.gamma.-cyclodextrins, respectively were determined by thin layer 
chromatography. Table 5 shows the results. 
TABLE 5 
______________________________________ 
Concentra- 
Amount of Cyclodextrins production (%) 
tion of enzyme (Recovery from starch) 
starch (U/g of .alpha.-cyclo- 
.beta.-cyclo- 
.gamma.-cyclo- 
(W/V %) starch) pH dextrin 
dextrin 
dextrin 
______________________________________ 
1 10 5 40 19 0 
1 10 6 36 31 0 
1 10 7 19 43 3 
1 10 8 0 55 5 
1 10 10 0 55 10 
1 1 5 6 49 3 
1 5 5 22 40 0 
1 8 5 29 36 0 
1 10 5 40 19 0 
1 15 5 42 15 0 
2 10 5 36 32 0 
3 10 5 20 44 0 
5 10 5 12 45 5 
10 10 5 6 45 9 
1 10 6 36 32 0 
2 10 6 33 30 0 
3 10 6 12 48 0 
5 10 6 5 50 3 
10 10 6 4 46 5 
______________________________________ 
When treating starch with the enzyme of the present invention, a ratio of 
.alpha.-, .beta.- and .gamma.-cyclodextrins in the reaction product 
depends upon the substrate concentration, amount of enzyme used and pH of 
the reaction mixture among various reaction conditions. For example, under 
the conditions that the substrate concentration is less than 2% at pH 
4.5-6.5 with 10 U/g of starch, .alpha.-cyclodextrin production is larger 
than .beta.-cyclodextrin. But, at pH 7-10, dominant reaction product is 
.beta.-cyclodextrin without dependency on substrate concentration and 
enzyme amount used. In both cases .gamma.-cyclodextrin production is very 
small. Namely, it has been found that .alpha.-cyclodextrin is in larger 
amount than .beta.-cyclodextrin under the reaction condition in which the 
pH of the reaction mixture is 4.5-6.5 at a substrate concentration of less 
than 2% (W/V) with enzyme at a concentration of 10 U/g of starch 
substrate. It has also been found that this property of the enzyme is 
common to conventional .beta.-cyclodextrin glycosyltransferase which has 
been known to produce mainly .beta.-cyclodextrin. 
The following examples illustrate the present invention more in detail. 
EXAMPLE 1 
A 60 ml portion of liquid medium consisting of 4.0% soluble starch. 1.0% 
soy bean meal, 0.3% yeast extract, 0.3% disodium glutamate, 0.2% K.sub.2 
HPO.sub.4, 0.05% MgSO.sub.4.7H.sub.2 O and 0.03% CaCl.sub.2.2H.sub.2 O (pH 
8.0) was dispensed to 500 ml-Sakaguchi flasks and sterilized by 
autoclaving. The medium was inoculated with a loop of Micrococcus varians 
M 849, FERM-P No. 4912 slant culture and incubated with shaking at 
32.degree. C. for 3 days. 
One liter of the cultured medium was centrifuged to remove cells. To the 
supernatant was added CaCl.sub.2.2H.sub.2 O at a final concentration of 
0.1%. The mixture was cooled. The enzyme was precipitated by adding 2.5 
volumes of cold acetone. The precipitate was collected by centrifugation 
and dried under reduced pressure to provide 9.5 g of crude enzyme powder. 
A solution of 300 g of soluble starch dissolved in 3 l of water (containing 
6 g of CaCl.sub.2.2H.sub.2 O) with heating was prepared. After the pH of 
the solution was adjusted to 6.0 with HCl, 1 g (770 U) of the crude enzyme 
powder mentioned above was added. The mixture was incubated at 60.degree. 
C. for 48 hours and then cooled with water. To the cooled reaction mixture 
was added 200 ml of toluene with thoroughly stirring and mixing to 
precipitate the reaction product. The precipitate thus formed was 
collected by filtration and washed with water, and then suspended in 1 
liter of water. Toluene was evaporated by heating the suspension. To the 
solution thus obtained was added 4 g of active carbon. The mixture was 
filtered at higher than 60.degree. C. Cooling the filtrate gave 180 g of 
cyclodextrins (60% of yield based on starch) as white crystals. 
EXAMPLE 2 
The same procedure described as in Example 1 was repeated except using 100 
ml (860 U) of a supernatant of the cultured medium collected by 
centrifugation, obtained by cultivation carried out in the same manner as 
in Example 1, in place of 1 g of the crude enzyme preparation. Treatment 
of soluble starch provided 150 g of the cyclodextrins (50% of yield) as 
white crystals. 
EXAMPLE 3 
The same procedure as described in Example 1 was repeated except using 
Micrococcus luteus B 645, FERM-P No. 4913 according to the present 
invention in place of M. varians M 849. Treatment of soluble starch with 
1.0 g (650 U) of the crude enzyme preparation provided 160 g of the 
cyclodextrins (53% of yield) as white crystals. 
EXAMPLE 4 
A solution of 300 g of soluble starch dissolved in 3 l of water (containing 
6 g of CaCl.sub.2.2H.sub.2 O) was prepared with heating. After the pH of 
the solution was adjusted to 6.0 with dilute hydrochloric acid, 1.5 g 
(1155 U) of the crude enzyme preparation obtained in Example 1 was added. 
The mixture was incubated at 50.degree. C. for 72 hours. Then the reaction 
mixture was concentrated under reduced pressure to 1 liter, which was 
cooled to give crystals. The crystals thus produced were collected by 
filtration and dissolved in 0.7 l of water with heating. To the solution 
was added 3 g of active carbon and the mixture was filtered at higher than 
60.degree. C. Cooling the filtrate provided 100 g of cyclodextrins (33% of 
yield) as white crystals. 
EXAMPLE 5 
A solution of 300 l g of soluble starch dissolved in 3 l of water 
(containing 6 g of CaCl.sub.2.2H.sub.2 O) was prepared with heating. After 
the pH of the solution was adjusted to 6.0 with dilute hydrochloric acid. 
0.5 g (335 U) of the crude enzyme preparation obtained in Example 1 and 
200 ml of trichloroethylene were added. The mixture was incubated at 
50.degree. C. for 48 hours. Then the reaction product was precipitated by 
cooling with stirring. The precipitate thus obtained was collected and 
washed with water, and then suspended in 1 liter of water. The suspension 
was heated to remove trichloroethylene by evaporation. To the solution 
thus obtained was added 4 g of active carbon and the mixture was filtered 
at higher than 60.degree. C. The filtrate was cooled to which an equal 
volume of isopropanol was added. The mixture was allowed to stand 
overnight to give 190 g of cyclodextrins (63% of yield) as white crystals. 
The ratio of .alpha.-, .beta.- and .gamma.-cyclodextrin of the crystalline 
products was .alpha.:.beta.:.gamma.=5:85:10. 
EXAMPLE 6 
A solution of 100 g of soluble starch dissolved in 10 l of water 
(containing 10 g of CaCl.sub.2.2H.sub.2 O) with heating was prepared. 
After the pH of the solution was adjusted to 5.0 with HCl, 1.5 g (1155 U) 
of the crude enzyme powder obtained in Example 1 was added. The mixture 
was incubated at 50.degree. C. for 48 hours and then cooled with water. To 
the cooled reaction mixture was added 300 ml of toluene with thoroughly 
stirring and mixing. A precipitate was formed. The mixture was filtered to 
collect the precipitate and filtrate separately. The precipitate was 
suspended in 60 ml of water. Toluene was evaporated by heating. To the 
solution thus obtained was added 0.3 g of active carbon. The mixture was 
filtered at higher than 60.degree. C. Cooling the filtrate provided 15 g 
of .beta.-cyclodextrin (15% of yield based on starch) as white crystals. 
The filtrate obtained by adding toluene to the reaction mixture mentioned 
above was concentrated by evaporation under reduced pressure up to 1 liter 
and cooled. To the concentrate was added 30 ml of tetrachloroethane with 
thoroughly stirring and mixing to precipitate the reaction product. The 
precipitate thus produced was collected by filtration, washed with water 
and suspended in 100 ml of water. After tetrachloroethane was removed by 
heating, 0.5 g of active carbon was added to the solution. The mixture was 
filtered at higher than 50.degree. C. The filtrate was cooled. An equal 
volume of isopropanol was added to the cooled filtrate. The mixture was 
allowed to stand overnight to give 25 g of .alpha.-cyclodextrin (25% of 
yield based on starch) as white crystals. 
Two kinds of cyclodextrins obtained separately as described above were 
identified to be .alpha.- and .beta.-cyclodextrins, respectively, based on 
their melting points and by specific optical rotation, iodo-starch 
reaction and thin layer chromatography. 
EXAMPLE 7 
A solution of 300 g of soluble starch dissolved in 3 l of water (containing 
6 g of CaCl.sub.2.2H.sub.2 O) was prepared with heating. After the pH of 
the solution was adjusted to 10 with Na.sub.2 CO.sub.3, 0.5 g (325 U) of 
crude enzyme preparation obtained in Example 3 and 200 ml of toluene were 
added. The reaction mixture was incubated at 55.degree. C. for 48 hours. 
The reaction product was precipitated by cooling the mixture with 
stirring. The precipitate was washed with water and suspended in 1 liter 
of water. The suspension was heated to remove toluene by evaporation. To 
the solution thus obtained was added 4 g of active carbon. The mixture was 
filtered at higher than 60.degree. C. To the filtrate was added an equal 
volume of isopropanol after cooling. The mixture was allowed to stand 
overnight to provide 180 g (60% of yield) of cyclodextrins as white 
crystals. 
The ratio of .alpha.-, .beta.- and .gamma.-cyclodextrins of the crystal was 
analyzed to be .alpha.:.beta.:.gamma.=0:95:5. 
EXAMPLE 8 
The same procedure as described in Example 6 was repeated except using 2.0 
g (1300 U) of the crude enzyme preparation obtained in Example 3 in place 
of 1.5 g of the crude enzyme of Example 6. Treatment of soluble starch 
provided 30 g of .alpha.-cyclodextrin (30% of yield based on starch) and 
12 g of 62-cyclodextrin (12% of yield). 
EXAMPLE 9 
A solution of 300 g of potato starch dissolved in 3 l of water (containing 
6 g of CaCl.sub.2.2H.sub.2 O) was prepared with heating. The pH of the 
solution was adjusted to 6.0 and then 0.06 g of .alpha.-amylase 
(Neospitase K) was added. The mixture was incubated at 80.degree. C. for 
10 min., and then heated at 100.degree. C. for 10 min. After cooling, 1 g 
(770 U) of crude enzyme preparation obtained in Example 1 was added. The 
mixture was incubated at 60.degree. C. for 48 hours and cooled with cold 
water. To the mixture was added 200 ml of toluene with thoroughly stirring 
and mixing to precipitate the reaction product. The precipitate was 
collected by filtration and washed with water, and then suspended in 1 
liter of water. The suspension was heated to remove toluene by 
evaporation. To the solution thus obtained was added 4 g of active carbon. 
The mixture was filtered at higher than 60.degree. C. Cooling of the 
filtrate provided 170 g of cyclodextrins (57% yield based on starch) as 
white crystals.