Process for producing cyclodextrins

Abstract

A novel process for producing cyclodextrin characterized by subjecting starch to the activity of cyclodextrin glycosyl-transferase produced by cultivation of Bacillus sp. No. 38-2, Bacillus sp. No. 135, Bacillus sp. No. 169, Bacillus sp. No. 13 or Bacillus sp. No. 17-1, at a pH of between 6.0 and 10.5, thereby to hydrolyse the starch.

Description

BACKGROUND OF THE INVENTION:

It is well known that dextrin is produced from starch by hydrolysis with amylase and it has usually a chain structure.

Regarding cyclodextrin, only Schardinger dextrin which has been produced by Bacillus macerans has been known in the "Enzyme Hand Book" edited in 1970 by Shiro Akabori and published from Asakura Publishing Co. Though the Schardinger dextrin is produced by a cyclodextrin glycosyltransferase, said cyclodextrin glycosyltransferase has various defects including an extremely narrow optimum pH range (merely from 6.8 to 7.0), low thermal stability (the optimum temperature is 40.degree.C) and low productivity of the cyclodextrin, and therefore said amylase can not be utilized for commercial production of the cyclodextrin.

SUMMARY OF THE INVENTION

The present invention relates to a novel process for producing cyclodextrin.

An object of the invention is to provide a novel process for producing various kinds of cyclodextrins having the different structures as shown in FIG. 1.

Another object of the invention is to provide an efficient process for producing cyclodextrins, which can be carried out easily under conditions of broad pH range and wide temperature range.

Further object of the present invention is to provide an efficient process which can produce cyclodextrins with high yield.

The above and other objects can be accomplished by a process according to the present invention, which comprises subjecting starch to a cyclodextrin glycosyl-transferase obtained by cultivation of one of the Bacillus named below, at a pH of betweem 6.0 and 10.5.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 shows the structures of various kinds of cyclodextrins which can be obtained according to the present invention;

FIG. 2 is a graph showing relative activities at different pH values of the amylase obtained from Bacillus sp. No. 38-2, in which the relative activity has been calculated on the assumption that activity at pH 9.0 is 100;

FIG. 3 is a similar graph showing relative activities of amylases obtained from Bacillus sp. No. 135 and No. 169;

FIG. 4 is a graph showing relative activities and relative yield of cyclodextrin at different pH values of the amylase obtained from Bacillus sp. No. 13; and

FIG. 5 is a graph showing relative activities and relative yield of cyclodextrin at different pH values of the amylase obtained from Bacillus sp. No. 17-1.

DETAILED DESCRIPTION OF THE INVENTION

As described above the invention of the present application is characterized by employing a specific cyclodextrin glycosyl transferase, namely one obtained by cultivation of a specific amylase-producing microorganism which can grow only in an alkaline medium.

The microrganism which can produce the cyclodextrin glycosyl-trsnsferase described above includes Bacillus species No. 38-2, Bacillus species No. 135, Bacillus species No. 169, Bacillus species No. 13 and Bacillus species No. 17-1, all of them having been found by the inventors of the present invention.

Bacillus sp. No.38-2, No. 135 and No. 169 have been isolated from the soil collected in Hirosawa district of Wako-shi, Saitama Prefecture, Japan.

Bacillus sp. No. 13 and No. 17-1 have been isolated from the soil collected in Karuizawa-shi, Nagano Prefecture, Japan.

Characteristics and properties of those microorganisms will be disclosed below. However we have no intention to restrict the invention to those microorganisms, because any natural or artificial mutant, variant and other species can be used as far as the strain can produce the cyclodextrin producing alkaline amylase.

We have examined the properties and characteristics of the above described species according to the methods described in "Aerobic Spore-forming Bacteria" by Nathan R. Smith, R. E. Gordon and F. E. Clark (United States Department of Agriculture, November 1952) and "Bergey's Manual of Determinative Bacteriology" (1957). Results of examination are shown as follows:

1. Bacillus sp. No. 38-2

a. Growth on Various Media

Table 1 ______________________________________ Growth at Medium pH 7 pH 10 ______________________________________ 1. Bouillon Growth scant Very poor growth 2. Bouillon-agar Growth scant Very poor growth 3. Glucose-bouillon Poor growth Turbid, good growth 4. Glucose-bouillon- Poor growth Good growth agar 5. Gelatin medium -- Growth, liquefied 6. Aqueous peptone -- Growth 7. Potato medium Growth scant Good growth ______________________________________

b. Microscopic Morphology:

Size of the microorganism is 0.5 - 0.6.mu. .times. 2.0 - 3.0.mu.; The spore which is formed near the end of the cell is oval and has a size of 0.9 - 1.0.mu. .times. 1.2 - 1.5.mu.; The sporangium is definitely swollen; The bacteria have pertrichous flagella and are motile and form motile collonies; Gram positive and non acid-fast.

It grows very well on an alkaline medium comprising soluble starch, yeast extract, peptone, K.sub.2 HPO.sub.4 and MgSO.sub.4.sup.. 7H.sub.2 O and containing 1% Na.sub.2 CO.sub.3 whereas growth on any neutral medium is poor.

c. Physiological Properties:

1. Optimum Growth Condition:

pH : around 10

Temperature: 37.degree. - 40.degree.C

Aerobic

2. Conditions under which the microorganism grows:

pH : 7.5 - 11

Temperature : up to 45.degree.C

3. voges-Proskauer reaction : Positive

4. Nitrate : Reduced

5. Catalase reaction : Positive

6. Gelatine, Casein : Liquefied

7. Hydrolysis of Starch : Positive

8. Utilization of Citrate : Utilized but poor

9. Utilization of Ammonium Salt: Utilized

10. Growth in 7% Sodium Chloride Solution : Poor

11. Growth on Glucose-nitrate Medium : Growth

12. Growth under Anaerobic Condition : Growth

13. Growth on Glucose-asparagine Medium : Growth

14. Production of Indole : Negative

d. Utilization of Carbon Source:

Glucose, fructose, xylose, sucrose, maltose, lactose and arabinose are utilized, but galactose, trehalose and inulin are not utilized. Production of acid is observed.

2. Bacillus sp. No. 135:

a. Growth on Various Media:

Table 2 ______________________________________ Growth at Medium pH 7 pH 10 ______________________________________ 1. Buillon Poor growth Poor growth 2. Buillon-agar Poor growth Fairly good growth 3. Glucose-Buillon Very poor growth Very good growth 4. Glucose-Buillon- Growth Very good growth agar 5. Gelatine medium -- Growth 6. Aqueous peptone -- Growth 7. Potato medium Growth Good growth ______________________________________

b. Microscopic Morphology:

Size of the microorganism is 0.6 - 0.8.mu. .times. 2.5 .times. 4.mu.; The sporangium is slightly swollen and the spore is oval having size of 1.0 - 1.2.mu. .times. 1.5 - 1.8.mu.. The microorganism has pertrichous flagella and motile. This Bacillus grows very well on an alkaline medium comprising soluble starch, yeast extract, peptone, K.sub.2 HPO.sub.4, MgSO.sub.4.sup.. 7H.sub.2 O and containing 1% of Na.sub.2 CO.sub.3, and appears white. The characteristic of the species is that it grows very well on alkaline medium, though grows a little on neutral medium.

c. Physiological Properties:

1. Optimum Growth Condition:

pH : around 10

Temperature : 37 - 40.degree.C

Aerobic

2. Conditions under which the bacteria can grow:

pH : 7 - 11

Temperature: up to 42.degree.C

Aerobic

3. Gram Stainability : Positive

4. Voges-Proskauer reaction : Positive

5. Nitrate : Reduced

6. Catalase : Positive

7. Gelatine and Casein : Liquefied

8. Hydrolysis of Starch : Positive

9. Utilization of Citrate : Not utilized

10. Utilization of Ammonium Salt : Utilized

11. Growth in 7% NaCl Solution : No detected

12. Growth on Glucose-Nirtate Medium : Growth scant

13. Growth under Anaerobic Condition : Detected

14. Production of Gas in Nitrate Medium under Anaerobic Condition : No produced

15. Growth on Glucose-Asparagine Medium : Growth

d. Utilization of Carbon Source:

Glucose, mannose, salicin, cellobiose, lactose, sucrose, arabinose, mannitol and xylose are utilized, but production of acid can not be observed, because a lot of carbonate is used.

3. Bacillus sp. No. 169:

a. Growth on Various Media:

Table 3 ______________________________________ Growth at Medium pH 7 pH 10 ______________________________________ 1. Bouillon Growth scant Poor growth 2. Bouillon-agar Growth scant Poor growth 3. Glucose-Bouillon Growth scant Turbid, good growth 4. Glucose-Bouillon- Growth scant Good growth agar 5. Gelatine medium -- Good growth liquefied 6. Peptone water -- Growth 7. Potato medium Poor growth Good growth ______________________________________

b. Microscopic Morphology:

Size of the microorganism is 0.5 - 0.6.mu. .times. 2 - 3.mu.; The sporangium is slightly swollen and the spore is oval having a size of 1.0 - 1.2.mu. .times. 1.3 - 1.7.mu.. The microorganism has pertrichous flagella and is motile. It grows very well on an alkaline medium comprising soluble starch, yeast extract, peptone, K.sub.2 HPO.sub.4 and MgSO.sub.4.sup.. 7H.sub.2 O and containing 1% Na.sub.2 CO.sub.3 but it hardly grows on any neutral medium.

c. Physiological Properties:

1. Optimum growth condition:

pH : 8 - 10

Temperature : 37.degree. - 40.degree.C

Aerobic.

2. Conditions under which the bacteria can grow:

pH : 7.5 - 11

Temperature : up to 45.degree.C

Aerobic.

3. Gram stainability : Positive, Changeable

4. Voges-Proskauer reaction : Positive

5. Nitrate : Reduced

6. Catalase reaction : positive

7. Hydrolysis of gelatin and casein : Positive

8. Hydrolysis of starch : Positive

9. Utilization of citrate : Not utilized

10. Growth in 7% NaCl solution : Not growth

11. Growth on glucose-nitrate medium : Scant

12. Growth under anaerobic condition: Growth

13. Production of gas in nitrate medium under anaerobic condition : Not produced

14. Growth on glucose-asparagine medium : Not growth

15. Production of indol : Negative

d. Utilization of Carbon Source:

Glucose, mannose, cellobiose, lactose, sucrose, mannitol and salicin are utilized very well, but arabinose and xylose are not utilized.

Production of acid can not be determined since the medium contains a lot of carbonate.

4. Bacillus sp. No. 13:

a. Growth on Various Media:

Table 4 ______________________________________ Growth at Medium pH 7.0 pH 10.0* ______________________________________ 1. Bouillon liquor Growth scant Growth, turbid; and sediment; membranous. 2. Bouillon-agar plane Growth scant Circular, flat or raised; entire; smooth, brilliant surface; translucent, milky white. 3. Bouillon-agar slant Growth scant Spreading with dull edges and brilliant center; milky white translucent; no pigment. 4. Bouillon-gelatin thrust Growth scant; not liquefaction of gelatin Liquefied in layer. ______________________________________ *1% Na.sub.2 CO.sub.3 is added to the medium in order to adjust pH value to 10.

b. Microscopic Morphology:

Size of the cell is 0.5 - 0.7.mu. .times. 2.0 - 4.0.mu.; oval spore is formed at the end of the cell; size of the spore is 1.3 - 1.4.mu. .times. 1.5 - 1.6.mu.; the sporangium is definitely swollen; the microorganism has pertrichous flagella and is motile. The gram stainability thereof is positive and the acid-fast test is negative.

Note: Above morphological observation has been made on a medium comprising 10 grams sodium carbonate, 5 grams peptone, 5 grams yeast extract, 20 grams starch, 1 gram K.sub.2 HPO.sub.4, 0.2 gram MgSO.sub.4.sup.. 7H.sub.2 O, 15 grams agar and 1 liter water.

c. Physiological Properties:

The following results have been observed on a media which has been described in "Aerobic Spore-forming Bacteria" by N. R. Smith, et al., modified by addingg 1% Na.sub.2 CO.sub.3 respectively.

1. Nitrate : Reduced

2. Denitrogenation : Negative

3. Methyl red test : No change of color, due to basicity of the medium

4. Voges-Proskauer reaction : Positive

5. Production of indole : Negative

6. Production of hydrogen sulfide: Negative

7. Hydrolysis of starch : Positive

8. Utilization of citric acid : Utilized

9. Utilization of nitrate and ammonium : Slightly utilized

10. Production of pigment : Negative

11. Catalase reaction : Positive

12. pH range for growth : 7.5 - 11

13. Temperature range for growth : up to 42.degree.C; optimum 37.degree. - 40.degree.C

14. behavior to oxygen : Aerobic

15. Growth in 5% NaCl solution : Slightly growth

d. Utilization of Carbon Source:

Lactose, arabinose, xylose, glucose, mannose, inositole, fructose, galactose, maltose, sucrose, trehalose, mannittol, starch, sorbitol and glycerine are utilized, and acids are produced.

Production of gas is not detected.

5. Bacillus sp. No. 17-1:

a. Growth on various media:

Table 5 ______________________________________ Growth at Medium pH 7.0 pH 10.0* ______________________________________ 1. Bouillon liquor Scant Growth, turbid and sedi- ment: membranous. 2. Bouillon-agar Slightly Circular, flat or raised; plane growth smooth, brilliant, sur- face; translucent; milky - white. 3. Bouillon-agar Slightly Spreading; milky white, growth translucent. No pigment in medium. 4. Bouillon-gelatin Scant Liquefied as crater. thrust No lique- fication ______________________________________ *1% Na.sub.2 CO.sub.3 is added to the medium in order to regulate pH valu to 10.0.

b. Microscopic Morphology:

The vegitative cell is a rod having a size of 0.5 - 0.7.mu. .times. 2.0 - 4.0.mu.. Oval spore is formed at subterminal. The size of a spore is 0.8 - 1.0.mu. .times. 1.2 - 1.5.mu.. The sproangium is definitely swollen. The microorganism has pertrichous flagella and is motile; gram positive and non acid-fast.

The above observation has been made on a medium comprising 10 grams sodium carbonate, 5 grams peptone, 5 grams yeast extract, 20 grams starch, 1 gram K.sub.2 HPO.sub.4, 0.2 gram MgSO.sub.4.7H.sub.2 O, 15 grams agar and 1 liter of water.

C. Physiological Properties:

The following results have been observed on media described in "Aerobic Spore-forming Bacteria" by N. R. Smith, et al., and modified by adding 1% Na.sub.2 CO.sub.3 respectively.

1. Nitrate : Reduced

2. Denitrogenation : Negative

3. Methyl Red reaction : No change of colour, due to basicity of the medium.

4. Vogel-Proskauer reaction : Negative

5. Production of indole : Negative

6. Production of hydrogen sulfide : Negative

7. Hydrolysis of starch : Positive

8. Utilization of citric acid : Utilized very well

9. Utilization of nitrate and ammonium salt : Utilized very well

10. Production of pigment : Not produced

11. Catalase reaction : Positive

12. pH range for growth : 7 - 11

13. Temperature range for growth : up to 42.degree.C, optimum: 37.degree.-40.degree.C

14. behavior to oxygen : Aerobic

15. Growth in 7% NaCl solution : Growth well

c. Utilization of Carbon Source:

Lactose, arabinose, xylose, glucose, mannose, inocitole, fructose, galactose, maltose, sucrose, trehalose, mannitol, strach, sorbitol and glycerine are utilized and acids are produced. Production of gas has not detected.

Studying the bacteriological properties disclosed above, Bacillus species No. 38-2, Bacillus species No. 135, Bacillus species No. 169, Bacillus species No. 13 and Bacillus species No. 17-1 shall belong to the Bacillus genus, because those miroorganisms are an aerobic and spore-forming bacteria respectively.

Further we found that for identification of these microorganisms, Bacillus polymixa, Bacillus macerans and Bacillus circulans shall be selected as known species for comparison, because every sproangium thereof is definitely swollen. Even our new microorganisms are similar in some properties to the known species, they are entirely different in characteristic properties, particularly by the fact that the optimum pH value of our microorganisms reside in alkaline side, whereas that of known species in neutral.

The following Table 6 shows various characteristic properties of Bacillus polymixa, Bacillus macerans and Bacillus circulans (known species) as well as Bacillus sp. No. 38-2, Bacillus sp. No. 135, Bacillus sp. No. 169, Bacillus sp. No. 13 and Bacillus sp. No. 17-1 (new species).

Table 6 __________________________________________________________________________ Growth in Utilization Utilization Growth under Reduction Bacillus species 5% NaCl of citric of nitrate anaerobic con- of nitrate acid dition __________________________________________________________________________ Bacillus polymixa - - + + - Bacillus macerans - - + + Bacillus circulans - - + +or - Bacillus sp. 38-2 - .+-. + ++ + Bacillus sp. No.135 - - .+-. - + Bacillus sp. 169 - - .+-. - - Bacillus sp. No.13 + + .+-. + - Bacillus sp. No. 17-1 ++ ++ ++ + + __________________________________________________________________________

From the above table it is noted that not only every species of Bacillus sp. No. 38-2, No. 135, No. 169, No. 13 and No. 17-1 has different characteristics from the known three species, but also they are not identical each other. Judging differences shown in the table, we concluded that every species of Bacillus sp. No. 38-2, Bacillus sp. No. 135, Bacillus sp. No. 169, Bacillus sp. No. 13 and Bacillus sp. No. 17-1 shall be identified as a new species respectively.

The strains identified as said Bacillus sp. No. 38-2, Bacillus sp. No. 135, Bacillus sp. No. 169, Bacillus sp. No. 13 and Bacillus sp. No. 17-1 have been deposited with the American Type Culture Collection (ATCC) at 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. as ATCC access numbers 21783, 21595, 21594, 31006 and 31007, in unrestricted condition permitting the public to have full access to the cultures, as of Mar. 27, 1972, Aug. 19, 1970, Aug. 19, 1970, Feb. 21, 1974 and Feb. 21, 1974, respectively. All restrictions on the availability of the culture deposit to the public will be irrevocably removed upon the granting of a patnet from this application. Further, the above cultures will be maintained by the depositor throughout the effective life of the patent.

The medium to be used for cultivation of the microorganism above described must be a basic medium containing carbonate, though it may be any of solid or liquid medium. Thus a medium comprising essential components for growth of the microorganism, such as carbon source, nitrogen source, inorganic salt and the like and containing added carbonate are used. Starch, soluble starch and the like are used as the carbon source. Yeast extract, peptone, corn-steep liquor and the like are used as the nitrogen source. Thus a medium comprising soluble starch, peptone, yeast extract, K.sub.2 HPO.sub.4, MgSO.sub.4.sup.. 7H.sub.2 O and an added carbonate is used. Any carbonate selected from anhydrous sodium carbonate, potassium carbonate, sodium bicarbonate and the like may be used.

We found that it is very important to add a sufficient amount of carbonate to the medium to make the pH value of the medium alkaline.

The amount of the carbonate to be added to the medium shall be preferably in the range of 0.5% to 1.5% by weight. This fact has been determined by the following experiments.

The experiments have been carried out using a standard neutral medium comprising 5 gs. peptone, 5 gs. yeast extract, 20 gs. starch, 1 g k.sub.2 HPO.sub.4, 0.2 g MgSO.sub.4.sup.. 7H.sub.2 O, 15 gs. agar and 1 liter of water and modified media containing various salts and carbonates in amount shown in the Table 7.

Media having pH value of 10 have been prepared by adding sodium hydroxide to the neutral medium.

Each of the media is innoculated with the strain of Bacillus sp. No. 17-1 and cultured with shaking at a temperature of 37.degree.C.

Growth of the microorganism has been observed by taking the culture broth after 18 hours into a cuvette of 1 cm and measuring the absorbance of light at 660 m.mu..

The yield of cyclodextrin has been determined by measuring the activity of the culture broth after 3 days cultivation under the condition described in later.

These results have been shown in the Table 7.

Table 7 ______________________________________ Medium salt pH before Growth Activity U/ml added cultivation ______________________________________ Standard 7.0 0.6 <1.0 (no additive) 10.0 0.8 <1.0 1% NaCl 7.0 0.8 <1.0 10.0 0.8 <1.0 1% KCl 7.0 0.8 <1.0 10.0 1.0 <1.0 NaHCO.sub.3 0.5% 9.0 1.0 15 1.0% 9.2 1.1 18 1.5% 9.3 1.1 21 2.0% 9.5 1.0 20 Na.sub.2 CO.sub.3 0.5% 9.6 1.1 16 1.0% 10.0 1.1 22 1.5% 10.2 1.2 22 2.0% 10.5 1.0 20 K.sub.2 CO.sub.3 0.5% 9.8 1.1 14 1.0% 10.2 1.1 21 1.5% 10.3 1.0 22 2.0% 10.5 1.1 20 ______________________________________

From the results shown in the table, it is noted that the presence of a suitable amount of the carbonate in the medium is indispensable to produce the subject enzyme to be used in the present invention.

The cultivation of the microorganism described above can be carried out by means of conventional aerobic shaking culture or air bubbling culture. It is preferable to culture for 24 - 96 hours at a temperature between 30.degree.C and 37.degree.C.

The resulting enzyme can be isolated from the culture broth by any conventional method. For example, when the cultivation is finished, the microorganism is removed from the culture broth and then after neutralization of the broth with acetic acid or the like or without any neutralization, the broth is treated with an organic solvent such as methanol or ammonium sulphate to precipitate enzyme and then the precipitated enzyme is separated from the liquid and dehydrated, thereby to obtain crude powdery enzyme. The resulted crude enzyme can be used for production of the cyclodextrin of the present invention as it is.

Purified enzyme can be obtained from the above crude enzyme as follows: The crude powdery enzyme is dissolved in water and the resulting solution is dialyzed against water overnight. The solution is passed through a column of diethyl aminoethyl cellulose (DEAE Cellulose) equilibrated with 0.01 M Tris HCl buffer solution of pH 9.0. Thus the enzyme in the solution is completely adsorbed in the cellulose. The adsorbed enzyme is eluted by changing the concentration of NaCl in the buffer solution from 0.01M to 0.5M.

The active fractions are collected and concentrated. Then the concentrated solution is purified by gelfiltration chromatography using Sephadex G-75 and Sephadex G-100 ("Sephadex" is a resistered trade name) and the resulted cake is freez-dried, thereby to obtain a purified enzyme powder.

The activity of the resulting enzyme is determined as follows:

0.05 ml of the enzyme solution with a suitable concentration is mixed with 0.5 ml of 1% soluble starch solution in 0.1M glycine buffer (pH 10.5).

The resulting mixture is subjected to a temperature of 40.degree.C for 2 hours, after reaction has been completed the solution is neutralized with acetic acid and further heated at 100.degree.C for 10 minutes.

The resulting solution is mixed with 50.mu.g of gluco amylase, the solution is subjected to a temperature of 40.degree.C for one hour to decompose the residual starch and the amount of glucose produced in the solution is determined by means of the dinitrosalicylic acid method.

The same process is repeated except using water instead of the enzyme solution.

The difference of the determined amount of glucose shows the amount of produced cyclodextrin.

One unit of the enzyme was defined as that amount of enzyme producing 1 mg of the cyclodextrin under the method described above.

The activity of the enzyme can be assayed by the iodine method as follows:

The enzyme solution (0.01 ml), which has been suitably diluted so as to reduce the absorbance at 700 m.mu. by from 10% to 20%, is mixed with 0.2 ml of 0.2% potato starch aqueous solution and 0.2 ml of 0.1M acetic acid buffer solution having pH value of 4.5, then the resulted mixture is heated at 40.degree.C for 10 minutes. After reaction, the resulted solution is mixed with 0.3 ml of 0.2M hydrochloric acid and then 3 ml of 0.005% iodine sulution. The absorbance at 700 m.mu. of the sample is measured.

The physicochemical properties of the enzymes produced from Bacillus sp. No. 38-2 (ATCC 21783), Bacillus sp. No. 135 (ATCC 21595), Bacillus sp. No. 169 (ATCC 21594), Bacillus sp. No. 13 (ATCC 31006) and Bacillus sp. No. 17-1 (ATCC 31007) will be explained in detail.

I. enzyme produced from Bacillus sp. No. 38-2 (ATCC 21783)

1. Substrate Specificity:

the enzyme produced by cultivation of the above identified microorganism under the specific conditions described above is active to starch, and reduces the starch-iodine reaction. However, reducing power can not be detected. Then the enzyme is determined to be a liquefying amylase.

2. Optimum pH:

The optimum pH of the enzyme has been determined by measuring activity of the enzyme at various pH values by means of the method described above.

Each pH value has been achieved by use of the following buffers respectively.

______________________________________ pH Buffer solution 4 - 5 Acetate 5 - 8.5 Tris-maleate 9 - 11 Sodium hydroxide 11 - 12 Sodium carbonate and Sodium hydroxide ______________________________________

The sample of the enzyme has been previously desalted with Sephadex G-25.

The results obtained are shown in FIG. 2 in which the activity at each pH value is shown as relative activity calculated on the assumption that the activity at pH 9.0 is 100. As clearly shown in the drawing, the optimum pH values of the enzyme are found at pH 4.5 pH 7 and pH 9, thus the optimum pH range is very broad.

3. Stable pH:

The enzyme solution (0.01 ml) desalted with Sephadex is mixed with 0.1 ml of various kinds of buffer solutions containing 1.mu. mole of CaCl.sub.2 as a stabilizer.

The resulting mixture is heated at 60.degree.C for 30 minutes and then 0.2 ml of a buffer solution of pH 9.0 and 0.2 ml of a substrate are added to the mixture, thus the residual activity has been determined. The results are shown in the following Table 8.

Table 8 ______________________________________ pH Buffer Solution Residual Activity(%) ______________________________________ 4 Acetic acid 0 5 " 12 6 Tris maleate 100 7 " 100 8 " 100 10 Na.sub.2 CO.sub.3, NaHCO.sub.3 70 11 Na.sub.2 CO.sub.3 10 ______________________________________

It is clearly indicated that the enzyme is quite stable in the range of pH 6 to 8.

4. Thermal Stability:

The thermal stability of the enzyme has been studies as follows:

The same enzyme solution in Tris-maleate buffer of pH 8 as shown in the above paragraph (3) is prepared (no Ca.sup.+.sup.+ ). Said solution is hold at different temperature for different time of period and the residual activity has been determined.

The following results were obtained.

Table 9 ______________________________________ Temperature Time Residual Activity minutes % ______________________________________ 50 15 100 50 30 100 55 15 80 60 15 10 ______________________________________

5. Inhibition by Temperature:

The same enzyme solution of pH 8 as shown in (3) is prepared and 5 m M of Ca.sup.+.sup.+ is added to the solution.

The solution is heated for 30 minutes varying the temperature as shown in the following table. The residual activities are determined and results obtained are shown in the following Table.

Table 10 ______________________________________ Temperature Residual Activity, % pH 4 pH 9 ______________________________________ 50 100 100 55 100 100 60 100 100 65 100 100 70 61 65 75 10 10 80 0 0 ______________________________________

From the above examination, we found that any increase of activation by addition of Ca.sup.+.sup.+ could not be observed, whereas the thermal stability had been improved.

6. Purification of the Enzyme:

CaCl.sub.2 (5M) is added to the culture broth of Bacillus sp. No. 38-2 (ATCC 21783) to adjust the pH value to 10 and produced precipitates are removed by centrifuge. 1/2 acetone is added to the culture broth to form precipitates.

The resulting precipitates are collected and dissolved in water. After dialysis overnight, the solution is concentrated with polyethylene-glycol.

After gel-filtration chromatography with Sephadex G-100, active fractions are collected.

The enzyme has been adsorbed on DEAE cellulose column equilibrated with 10 m M of Tris-HCl buffer solution containing 1 m M CaCl.sub.2 at pH 8.5 and then the adsorbed enzyme is eluted varying the concentration of CaCl.sub.2 from 5 m M to 50 m M. Usually about 40 m M is used.

The active fractions are collected and purified by gel-filtration using Sephadex G-75, to obtain the final product.

Two curves showing the relation between the enzyme activity and the pH value which have been measured before and after purification are essentially the same.

7. Homogeneity of the Enzyme:

The homogeneity of this enzyme has been proved by the following observations:

i. Ultra centrifugal analysis gave a single peak of sedimentation constant at approximately 4.

ii. A single peak of the activity was observed by gel-filtration chromatography.

iii. Disc electrophoretic analysis at pH 8.3 shows monodisperse.

iv. The ratio of the enzyme activity at pH values 4 and 9 does not change, even in the partially denatured enzyme by heating.

The comparison of the physicochemical properties of enzyme disclosed above and that of the known saccharifying and liquefying amylases produced from Bacillus subtilis (Ref.: "Advances in Applied Microbiology," 7, p. 293, 1965) is shown in the following Table 11.

Table 11 __________________________________________________________________________ Thermal Stable Optimum Stabilization Hydrolysis Enzyme Type stability pH range pH by Ca.sup.+.sup.+ of starch __________________________________________________________________________ Amylase Liquefying 65-90.degree.C 4.8-10.8 5.4-6.0 + 35% from Saccharify- B. subtilis ing 55-70.degree.C 4.0-7.8 4.8-5.2 - 70% Amylase 4.5 from Liquefying 60-70.degree.C 5 - 10 7.0 + 15% B.sp.No.38-2 (in the pre- 9.0 sence of Ca.sup.+.sup.+) __________________________________________________________________________ Note: .sup.+ : stable - : unstable

Ii. bacillus sp. No. 135 (ATCC 21595) and Bacillus sp. No. 169 (ATCC 21594):

1. Substrate Specificity:

The enzymes produced by cultivation of the above identified two microorganisms under the specific conditions described above are active to starch and reduce the starch-iodine reaction. However the increase of reducing activity is little.

By paper chromatography of the products, a small amount of a series of oligosaccharides such as maltose, maltotriose, maltotetraose etc. were observed. From the facts these enzymes have been determined to be liquefying amylases.

2. Optimum pH:

The optimum pH of the enzymes produced from Bacillus sp. No. 135 and No. 169 have been determined by measuring activities at various pH values as described above.

The enzyme used has been previously desalted with Sephadex G-25. The other conditions have been disclosed above.

Each pH value used has been achieved by use of the following buffers respectively.

______________________________________ pH Buffer ______________________________________ 4 and 5 Acetate 6, 7 and 8 Phosphate 9 and 10 Borate 10, 11 and 12 Glycine-NaOH-NaCl ______________________________________

The results are shown in FIG. 3, in which the activity at each pH value is shown as relative activity calculated on the assumption that the activity at pH 10.5 is 100.

As clearly shown in the drawing, both optimum pH values of the enzymes from Bacillus sp. No. 135 and No. 169 are found at 10.5.

3. Stable pH:

The pH range in which the activity of the enzymes can be maintained in stable has been studied.

The enzyme (0.01 ml) which has been desalted with Sephadex is mixed with 0.1 ml of various kinds of buffer solutions containing 1.0.mu. mole of CaCl.sub.2 as a stabilizer.

The resulted mixture is heated at 55.degree.C for 15 minutes and then 0.2 ml of a buffer solution having pH 10.5 and 0.2 ml of a substrate are added, thus the residual activity has been observed respectively.

The results of the test are shown in the following Table 12.

Table 12 ______________________________________ Residual pH Buffer Activity, % ______________________________________ 4 Acetic acid 0 6 0 7 Tris maleate 25 8 51 10 Na.sub.2 CO.sub.3 -NaHCO.sub.3 47 11 Na.sub.2 CO.sub.3.NaOH 10 ______________________________________

4. Thermal Stability (Inactivation):

Each enzyme produced from Bacillus sp. No. 135 and No. 169 loses about 50 to 70% activity by heating at a temperature of 60.degree.C for 10 minutes at pH 10.0, whereas at 50.degree.C for 15 minutes no activity is lost.

5. Inhibition, Activation and Stabilization:

The same enzyme solution of pH 10 as disclosed in (3) is prepared and a different amount of Ca.sup.+.sup.+ is added to the solution. The solution is heated to 55.degree.C for 15 minutes. The residual activation is determined and shown in the following Table 13.

Table 13 ______________________________________ Amount of Ca.sup.+.sup.+ Residual Activity, % ______________________________________ 0 0 0.15 .mu. mole 0 0.25 .mu. mole 10 0.5 .mu. mole 25 1.0 .mu. mole 54 1.5 .mu. mole 40 ______________________________________

From the above examination, we found that no increase of activation by addition of Ca.sup.+.sup.+ could be observed, whereas the thermal stability had been improved.

Judging the physicochemical properties of the enzymes from Bacillus sp. No. 135 and 169, these enzymes have characteristic by the fact that they have the optimum pH 10.5.

Thus we confirmed that these enzymes are novel amylases.

Iii. bacillus sp. No. 13:

1. Substrate Specificity:

The enzyme produced by cultivation of the above identified microorganism under the specific conditions described above is active to starch and reduces the starch-iodine reaction. However the enzymme does not increase reducing power at pH 10.5 and it produces cyclodextrin. Thus the enzyme has been determined to be a cyclodextrin-glycosyl-transferase and also to be a liquefying amylase. The amylase reduces starch-iodine reaction and increases the reducing power at pH 4.5.

2. Optimum pH:

The optimum pH of the enzyme has been determined by measuring activities and yield of cyclodextrin at various pH values by means of the above described method.

Each pH value has been achieved by use of the following buffers respectively.

______________________________________ pH Buffer ______________________________________ 4 - 5 Acetate 5 - 8.5 Tris maleate 9 - 11 Glycine-NaOH 11 - 12 NaHCO.sub.3 -NaOH ______________________________________

The sample of the anzyme has been previously desalted with Sephadex G-25.

The results obtained are shown in FIG. 4, in which the curve a shows yield of cyclodextrin at variaous pH values and the curve b shows relative activities of the amylase at various pH values.

From the curve a, it is noted that optimum pH value for production of cyclodextrin lies between 10 and 10.5 and from the curve b it is noted that the optimum pH value for production of amylase lies at 4.5.

3. Stable pH:

The pH range in which the activity of the enzyme can be maintained in stable has been studied.

The enzyme solution (0.01 ml) desalted with Sephadex is mixed with 0.1 ml of a buffer solution listed in the following table and the mixture is heated at 50.degree.C for 10 minutes. Then 0.2 ml of buffer solution of pH 10.0 and 0.2 ml of substrate are added to the heated solution. Thus the residual activities at various pH values are observed. The results obtained are shown in the following Table.

Table 14 ______________________________________ pH Buffer Residual Activity, % ______________________________________ 4 Acetate 0 5 10 6 60 7 Tris maleate 65 8 80 10 Na.sub.2 CO.sub.3.NaHCO.sub.3 60 11 Na.sub.2 CO.sub.3 10 ______________________________________

4. Thermal Stability (Conditions for Inactivation):

The thermal stability of the enzyme has been studied.

The same enzyme solution of pH 7 as shown in the above paragraph (3) is prepared. The inactivation of the solution has been observed by keeping the solution at various temperature for 10 minutes respectively.

The results obtained are shown in the following Table 15.

Table 15 ______________________________________ Temperature Time Residual Activity .degree.C minutes % ______________________________________ 45 10 100 50 10 80 55 10 40 60 10 5 ______________________________________

5. Inhibition, Activation and Stabilization:

In same enzyme solution of pH 7 as disclosed in (3) is prepared.

Various amount of Ca.sup.+.sup.+ is added to the solution and the increase of the activity has been studied. Any increase has not been observed.

Then the thermal stability of same solution has been studied by heating the solution comtaining 5 mM Ca.sup.+.sup.+ at different temperature for 20 minutes and measuring the residual activity of the solution. The results obtained are shown in the following Table 16.

Table 16 ______________________________________ Residual Temperature, .degree.C Activity, % ______________________________________ 50 100 55 80 60 20 65 5 70 0 75 0 80 0 ______________________________________

6. Purification of the Enzyme:

CaCl.sub.2 (5M) is added to a culture broth of the microorganism to form precipitates and which are removed from the liquid by centrifuge. A half volume of acetone is added to the filtrate to form precipitates.

The resulting precipitates are collected and dissolved in water. After dialysis overnight the solution is concentrated with polyethylene glycol.

After gel-filtration chromatography with Sephadex G-100, active factions are collected. The enzyme is adsorbed on DEAE cellulose column equilibrated with 10 m M of tris-HCl buffer solution of pH 9.0 containing 10 m M CaCl.sub.2 and then the adsorbed enzyme is eluted by varying the concentration of NaCl from zero to 0.5 M. Usually the enzyme can be eluted at the concentration of about 0.1 M.

Active fractions are collected, the collected solution is gel-filtrated with Sephadex G-75 to obtain the final product.

Two curves showing the relation between the enzyme activity and the pH value which have been measured before and after purification are substantially same.

7. Range of Working Temperature:

The activity of the enzyme has been measured at various temperatures and we found that the optimum working temperature of the enzyme is in the range of from 45.degree.C to 50.degree.C.

8. Molecular Weight of the Enzyme:

The molecular weight of the enzyme is determined by means of Gel-filtration method and found being about 60,000.

9. Isoelectric Point:

The isoelectric point of the enzyme has been examined by means of the electrophoresis using filter paper and found that it is about 4.5.

10. Elementary Analysis:

C: 47.8%, h: 7.2%, s: 0.6%,

n: 15.4%, ash: 0.9%.

Iv. bacillus sp. No. 17-1 (ATCC 31007):

1. Substrate Specificity:

The enzyme produced by cultivation of the above identified microorganism under the specific conditions described above is active to starch and reduces the starch-iodine reaction. However no increase of reducing power can be observed. Almost all of the final products of the enzyme is found to be cyclodextrin.

2. Optimum pH:

The optimum pH of the enzyme has been determined by measuring activity of the enzyme at various pH values by means of the above described method.

Each pH value listed has been achieved by use of the following buffers respectively:

pH Buffer ______________________________________ 4, 5 Acetate 6, 7, 8 Phosphate 9, 10 Borate 10, 11, 12 Glycine--NaOH--NaCl ______________________________________

The sample of the enzyme has been previously desalted with Sephadex G-25.

The results obtained are shown in FIG. 5, in which the curve a shows yield of cyclodextrin at various pH values and the curve b shows relative activities of the amylase at various pH values.

From the curve a, it is noted that the optimum pH value for production of cyclodextrin lies between 10 and 10.5 and from the curve b it is noted that the optimum pH value for production of amylase lies at 4.5.

3. Stable pH:

The pH range in which the activity of the enzyme can be maintained in stable has been studied.

The enzyme solution (0.01 ml) desalted with Sephadex is mixed with 0.1 ml of a buffer solution listed in the following table and the mixture is heated at 55.degree.C for 10 minutes. Then 0.2 ml a buffer solution of pH 10.5 and 0.2 ml of substrate are added to the heated solution. Thus the residual activities at various pH values are observed. The results obtained are shown in the following Table 17.

Table 17 ______________________________________ pH Buffer Residual Activity, % ______________________________________ 4 Acetic acid 0 6 Tris maleate 40 7 " 70 8 " 95 10 Na.sub.2 CO.sub.3 --NaHCO.sub.3 80 11 Na.sub.2 CO.sub.3 --NaOH 20 ______________________________________

4. Thermal Stability (Conditions for Inactivation):

About 20 - 30% of the activity of the enzyme has been lost by heating at 55.degree.C for 10 minutes at pH 10 and almost all of activity has been lost by heating at 60.degree.C for 10 minutes.

5. Inhibition, Activation and Stabilization:

We examined the influence of addition of Ca.sup.+.sup.+ on increase of the enzyme activity. No increase of the activity can be found, but the thermal stability has been improved by addition of Ca.sup.+.sup.+.

Thus an enzyme solution having pH 10 is prepared and the solution is heated at 55.degree.C for 20 minutes, and the residual activity has been examined by varying the amount of Ca.sup.+.sup.+ in the solution.

The results are shown in the following Table 18.

Table 18 ______________________________________ Amount of added Residual Activity, % Ca.sup.+.sup.+, .mu.mole ______________________________________ 0 30 0.15 40 0.25 60 0.5 80 1.0 100 1.5 100 ______________________________________

6. Purification:

Purification of the enzyme can be carried out by the same method described in the purification of the enzyme from Bacillus sp. No. 13 (ATCC 31006).

7. Range of Working Temperature:

The enzyme activity has been measured at various temperatures and we found that the optimum working temperature of the enzyme lies in the range of 50.degree.C to 55.degree.C.

8. Molecular Weight:

The molecular weight by gel-filtration method has been found to be about 50,000 to 60,000.

9. Isoelectric Point:

The isoelectric point of the enzyme has been examined by means of the ampholine electro-focusing method and we found that it is at pH 4.5.

10. Elementary Analysis:

C: 48.0%, h: 7.3%, s: 0.65%,

n: 15.7%, ash: 1.01%.

Summing physcochemical properties of amylases produced from Bacillus sp. No. 38-2 (ATCC 21783), Bacillus sp. No. 135 (ATCC 21595), Bacillus sp. No. 169 (ATCC 21594), Bacillus sp. No. 13 (ATCC 31006) and Bacillus sp. No. 17-1 (ATCC 31007), these amylases are characteristic in that they have the optimum pH value for producing cyclodextrin in the range of from 9 to 10.5, particularly from 10 to 10.5. Further we found that these amylase are characteristic in that the ratio of relative activities at pH 4.5 and pH 10 are specific as shown in the Table 19.

Table 19 ______________________________________ Optimum pH of Ratio of amylase activities Species cyclodextrin (relative activity) at glycosyl- transferase pH 4.5 pH 10 ______________________________________ B. sp.No.13 (ATCC 31006) 10 - 10.5 100% 30 - 50% B.sp.No.17-1 (ATCC 31007) 10 - 10.5 100% 40 - 50% B.sp.No.38-2 (ATCC 21783) 9 - 9.5 100% 70 - 80% B.sp.No.135 (ATCC 21595) 10 - 10.5 100% 80% B.sp.No.169 (ATCC 21594) 10 - 10.5 100% 80% ______________________________________

From these characteristics, we have determined that these amylases are novel ones.

The invention of the present application relates to a process for producing cyclodextrin characterized by use of a specific alkaline amylase under a specific pH condition.

The alkaline amylase to be used must be selected from amylases having an optimum pH value within the range of from 7 to 10.5, preferably from 8 to 10.5, more preferably from 10 to 10.5.

The preferable amylase is a cyclodextrin glycosyltransferase having the optimum pH value of from 8 to 10.

The alkaline amylase to be used is preferably selected from amylases produced by cultivation of a microorganism selected from the group consisting of Bacillus sp. No. 38-2 (ATCC 21783), Bacillus sp. No. 135 (ATCC 21595), Bacillus sp. No. 169 (ATCC 21594), Bacillus sp. No. 13 (ATCC 31006) and Bacillus sp. No. 17-1 (ATCC 31007).

The pH condition to be used according to the present invention shall be within the range of pH 6 to 10.5, particularly within the range of pH 9.0 to 10.5.

According to the present invention, any kinds of starch may be used, and a preferred starch is potato-starch.

The process of the present invention can be carried out as follows:

Starch is thoroughly liquefied, preferably by NaOH. After adjusting the pH value of the starch solution to 6.0 - 10.5, preferably to 9.0 - 10.5, the alkaline amylase disclosed above is added to the solution and the solution is maintained at the optimum working temperature for sufficient time to produce cyclodextrin. The optimum working temperature depends on the kind of the alkaline amylase used as described above. The production of cyclodextrin is usually accomplished in twelve hours.

We have confirmed that the cyclodextrin obtained according to the process of the present invention has the same physicochemical properties as that of the known cyclodextrin.

The physical and chemical properties of the cyclodextrin of the present invention are as follows:

1. Elementary Analysis:

C: 44.4%, h: 6.1%, o: 49.5%.

2. molecular Weight:

Crude product : 1200 .+-. 100

.alpha.-dextrin fraction: 970

.beta.-dextrin fraction: 1.140

.gamma.-dextrin fraction: 1.300

3. Melting Point:

200.degree.C (as acetylated)

4. Optical Rotation [.alpha.] D :

.alpha.-dextrin fraction: + 150

.beta.-dextrin fraction: + 160

.gamma.-dextrin fraction: + 170

5. Ultraviolet Absorption Spectrum:

No characteristic

6. Infre-red Absorption Spectrum:

Almost same as that of commercially available .alpha.-dextrin.

7. Color Reaction:

.alpha.-dextrin fraction: Give blue color by iodine

.beta.,.gamma.-dextrin fractions: hardly iodine reaction, give yellowish brown or reddish brown color.

8. Crystallography:

.alpha.-dextrin fraction: Hexagon or blade.

.beta.-dextrin fraction: Parallelogram.

.gamma.-dextrin fraction: Quadrilateral.

9. Acidity:

Neutral.

10. Color:

White.

11. Reducing Power:

No reducing power:

Glucose, maltose and malt-triose are produced by Taka-.alpha.-amylase. But no effect by an enzyme which decomposes poly or oligosuccharides from the chain termini.

As disclosed above, the cyclodextrin produced according to the process of the present invention contains .alpha.,.beta. and .gamma. dextrin fractions.

The enzymes to be used according to the present invention have better thermal stability higher by 15.degree.C to 20.degree.C than that of the known cyclodextrin producing enzyme. Therefore the process of the present invention is extremely useful and effective.

The cyclodextrin produced by the process of the present invention has various uses. Particularly it is useful for manufacture of sweeteners, such as millet jelly, because the cyclodextrin glucosyltransferase has transferase activity.

For instance, a novel and useful sweetener can be produced as follows:

10 gs. dextrin or starch is mixed with 3 gs. sucrose and the mixture is dissolved in water. 100 ml of the enzyme solution containing cyclodextrin-glucosyltransferase is added to the solution and the solution is allowed to stand at a temperature of 37.degree.C overnight.

Unreacted dextrin is removed with trichloroethylene, the solution is concentrated to obtain 10 gs. of a syrup like millet jelly. We found that the product comprises minor sucrose and major compound of sucrose and glucose and has sufficient sweetness for use as a sweetener.

The invention will be explained the following examples, but we do not intend to restrict the invention by them.

EXAMPLE 1

10 gs. of potato starch is mixed in 100 mls of 1N NaOH solution and the resulting mixture is stored in a refrigerator over-night to thoroughly liquefy the starch.

100 mg of the alkaline amylase which has been produced by cultivation of Bacillus sp. No. 38-2 (ATCC 21783) is added to the starch solution which has been adjusted to pH 9.0. After standing the solution at 34.degree.C overnight, any insoluble substance is removed and the product is precipitated by addition of trichloroethylene.

The collected precipitates are thoroughly washed and trichloroethylene is removed by heating.

By recrystallization from aqueous propyl alcohol containing 60% of alcohol, 8 gs. of cyclodextrin is recovered as white crystal.

EXAMPLE 2

The same process explained in Example 1 is repeated except that the alkaline amylase from Bacillus sp. No. 135 (ATCC 21595) is used instead of the alkaline amylase from Bacillus sp. No. 38-2.

5 gs. of cyclodextrin has been obtained as white crystal.

EXAMPLE 3

The process explained in the Example 1 is repeated except that the alkaline amylase from Bacillus sp. 169 (ATCC 21594) is used as the alkaline amylase.

4.5 gs. white crystalline cyclodextrin has been obtained.

EXAMPLE 4

10 gs. of potato starch is mixed in 100 ml of 1 N NaOH solution and the resulted mixture is stored in a refrigelator overnight, to thoroughly liquefy the starch.

100 mg of the crude enzyme powder which has been produced by cultivation of Bacillus sp. No. 13 (ATCC 31006) is added to the solution which has been adjusted to pH 10.0. After standing the solution at 34.degree.C over night, any insoluble substances are removed and the product is precipitated by addition of trichloroethylene.

The collected precipitates are washed thoroughly and trichloroethylene is removed by heating.

By recrystallization from aqueous propylalcohol containing 60% of ethanol, 7.5 gs. of white crystalline cyclodextrin has been obtained.

EXAMPLE 5

The process described in Example 4 is repeated except that substituting the a crude enzyme powder produced by cultivation of Bacillus sp. No. 17-1 (ATCC 31007) is used as the alkaline amylase.

7.7 gs. of white crystalline cyclodextrin has been obtained.

Claims

We claim:

1. A process for producing cyclodextrin which comprises subjecting starch to the activty of a cyclodextrin glycosyltransferase produced by cultivation of a microorganism selected from the group consisting of Bacillus sp. No. 38-2 (ATCC 21783), Bacillus sp. No. 135 (ATCC 21595), Bacillus sp. No. 169 (ATCC 21594), Bacillus sp. No. 13 (ATCC 31006) and Bacillus sp. No. 17-1 (ATCC 31007), at a pH of 6.0 - 10.5 and recovering said cyclodextrin.

2. A process according to claim 1, wherein the reaction is carried out at a pH between 8 and 10.5.

3. A process according to claim 2, wherein the reaction is carried out at a pH between 8 and 10.

4. A process according to claim 1, wherein said cyclodextrin glycosyltransferase has an optimum pH for producing cyclodextrin of between 8 and 10.