WO1989001043A1

WO1989001043A1 - Process and enzyme for preparing cyclodextrins, especially alpha-cyclodextrin

Info

Publication number: WO1989001043A1
Application number: PCT/US1988/002565
Authority: WO
Inventors: Hiroyuki Aoki; Ernest Kar-Cheung Yu
Original assignee: Genetics Institute, Inc.
Priority date: 1987-07-28
Filing date: 1988-07-28
Publication date: 1989-02-09

Abstract

A soil bacterium has been isolated which secretes a cyclodextrin glycosyltransferase enzyme useful in converting gelatinized strain predominantly to alpha-cyclodextrin. A novel process of producing and isolating cyclodextrin, in particular, alpha-cyclodextrin is also disclosed.

Description

PROCESS AND ENZYME FOR PREPARING CYCLODEXTRINS, ESPECIALLY ALPHA-CYCLODEXTRIN

FIELD OF THE INVENTION

This invention relates to the production of cyclodextrins.

BACKGROUND OF THE INVENTION

Cyclodextrins are cyclic oligosaccharides, common species of which are composed of 6, 7 or 8 glucose residues bound through an α-1,4 linkage. They are called α-, β- or γ-cyclodextrins depending on the number of glucose residues; 6, 7 or 8, respectively.

Because its torus configuration provides a hydrophobic cavity, cyclodextrins form inclusion compounds with a wide variety of "guest" compounds and have been used in separation processes, extraction processes, as drug delivery enhancing agents in the medical field, as compound stabilizing agents in the medical field, as compound stabilizing agents in the food industry and in a variety of other applications.

While alternative processes for cyclodextrin production are available and described in the art, the conventional process involves bioconversion of gelatinized starch, by enzyme action. Enzymes useful for this purpose, terms cyclodextrin glycosyltransferases or CGTase fpr brevity, are produced by many different bacteria. Known CGTase-producing bacteria include Bacillus macerans, B. stearothermophilus, B. megaterium, B. circulans, B. ohbensis and other taxonomically distinct Bacillus spp., Klebsiella pneumoniae M5 and species of Hicrococcus such as varians M-849 (ATCC^®31606) and luteus B-645 (ATCC^®31607). While the CGTase produced by these bacteria all function to convert gelantinized starch to cyclodextrin, they differ in terms of reactivity and stability, indicating a difference also in their primary amino acid structure. Some, for example, produce one particular cyclodextrin species i.e. eitheroα, ß or γ in greater amounts than other species of cyclodextrin, a bias which can be controlled in some instances by altering process conditions.

There has been much improvement recently in large scale processing to produce β cyclodextrin, to the extent that the much lower market price for β cyclodextrin is threatening the commercial feasibility of using α-cycl odextrin for its inclusion forming properties. Nevertheless, α-cyclodextrin and β-cyclodextrin can selectivity complex different materials and, because they cannot be used interchangably in these instances, the demand for α-cyclodextrin is being maintained. There remains a need, however, for a more efficient process for producing α-cyclodextrin in order to reduce its market price and increase its availability.

REFERENCE TO THE PRIOR ART

Most processes used to produce α-cyclodextrin utilize as starting material the mixture of cyclodextrins produced by enzymic conversion of gelatinized starch. The majority of enzymes i.e. CGTases, known for this purpose, however, produce a cyclodextrin mixture containing relatively large amounts of β cyclodextrin (about 70% or more by weight ot total cyclodextrin) and smaller amounts of α-cyclodextrin (about 30% or less total weight). Concentration of α-cyclodextrin is therefore necessary, specifically to separate it from β-cyclodextrin and any residual γ-cyclodextrin before purification of it can be completed.

In the presence of β-cyclodextrin, α-cyclodextrin can be concentrated using selective precipitation to provide a concentrate rich in α-cyclodextrin (about 89% α-cyclodextrin, 11% β-cyclodextrin). This concentration takes advantage of the lower solubility of β-cyclodextrin by cooling the reaction products to encourage β-cyclodextrin crystallization and then separating the α-cyclodextrin-rich liquor from the precipitate.

U.S. 3,541,077 issued Nov. 17, 1970 to CPC International Inc. describes an α-cyclodextrin purification process which uses a concentrated α-cyclodextrin liquor, recovered after β-cyclodextrin precipitation as described above, as starting material. The α-cyclodextrin-rich liquor is treated with an α-cyclodextrin complexant to precipitate the complex which is then washed and further treated to release the complexant, enabling recovery of pure e.g. 95%, α-cyclodextrin.

U.S. 3,640,847 issued Feb. 8, 1972 to the same assignee describes in greater detail the nature of the α-cyclodextrin complexant useful in the α-cyzlodextrin purification process. Saturated or unsaturated aliphatic radicals of at least eight carbons in length bonded terminally to an electronegative atom are indicated as useful complexants.

The two patents described above are exemplary of approaches described in the art to produced-cyclodextrin economically. Much of the available literature focusses on methods for improving the recovery of α-cyclodextrin from enzymic reaction products. By contrast, the present invention is concerned primarily with providing means and a method for improving yields of α-cyclodextrin resulting from the initial enzymic reaction.

Accordingly, it is an object of the present invention to provide a bacterial source of enzyme, i.e. cyclodextrin glycosyltransferase enzyme, which converts amylose preferentially to α-cyclodextrin rather than β-cyclodextrtn.

It is a further object of the present invention to provide enzyme which is useful in converting amylose to a mixture of cyclodextrins in which α-cyclodextrin predominates.

A further object of the present invention is to provide processes for producing and for recovering α-cyclodextrin.

SUMMARY OF THE INVENTION

The present inventors have succeeded in identifying an enzyme which converts gelatinized starch preferentially and indeed predominantly to α-cyclodextrin. The enzyme, herein termed a cyclodextrin glycosyltransferase to reflect its function, is capable of converting gelatinized starch to a cyclodextrin mixture, the α-cyclodextrin component of which comprises at least 70% of the total weight of the cyclodextrin mixture. Yields of α-cyclodextrin are, in fact, typically within the range of about 80% by weight to as high as 95% by weight of the total weight of cyclodextrins produced when the enzyme is used under the specific processing conditions exemplified herein.

Thus according to one aspect of the present invention, there is provided an enzyme which is capable of converting gelatinized starch to a mixture of cyclodextrins the α-cyclodextrin component of which comprises at least 70% of the total weight of the mixture. In accordance with preferred embodiments of this aspeet of the present invention, the enzyme is in a form suitable for presentation to a reactor containing suitable substrate. Alternatively, the microbial source of the enzyme is cultured in the presence of the substrate under conditions which favour enzyme production thereby.

The enzyme of the present invention is produced naturally by a soil microorganism of the genus Bacillus which has been isolated by the present inventors. The particular Bacillus strain preferred as enzyme source is named herein by reference to a'π internal code i.e. Bacillus strain AL35, cultures of which have been deposited with the American Type Culture Collection on April 6, 1987 under accession number 53604. It is to be understood that the present invention encompasses, in addition to the deposited material, those microorganisms which exhibit the enzyme producing characteristics of the deposited microorganism. Microorganisms exhibiting the enzyme producing characteristics of the deposited microorganism are those which are able to produce an enzyme capable of converting gelatinized starch to a mixture of cyclodextrin, the α-cyclodextrin component of which represents at least 70% by weight of the total weight of the cyclodextrin mixture. Microorganisms possessing these characteristics will include clones of the ATCC^®deposited material. Such clones will also share the taxonomic characteristic of Bacillus strain AL35. Microorganisms possessing this characteristic will also include sub-clones of-the deposited material having taxonomic characteristics which may or may not be shared completely by the deposited strain. It is, for example, well within the expected skill of those familiar with the art to sub-clone parental bacterial strains to enhance its growth and other characteristics using such techniques as continuous recycling under selective conditions, chemical mutagenesis, gamma irradiation etc. Provided that such sub-clones retain the ability to express the enzyme of the present invention as characterized above, such sub-clones will be useful sources of the enzyme of the present invention.

Thus, according to another aspect of the present invention, there is provided a microorganism capable of producing an enzyme which is able to convert gelatinized starch to a cyclodextrin mixture the α-cyclodextrin component of which comprises at least 70% by weight of the total weight of the cyclodextrin mixture. According to a preferred embodiment of the invention, the microorganism capable of producing the enzyme has the taxonomic characteristics of Bacillus strain AL35. Most preferably, the microorganism is Bacillus strain AL35 ATCC ^® 53604 per se.

According to another aspect of the present invention, there is provided a process for preparing cyclodextrin which comprises react ng enzyme, capable of converting substrate to a cyclodextrin mixture, theα-cyclodextrin component of which comprises at least 70% of the total weight of the mixture, with a substrate suitable therefore. Substrates useful in the process include processed starch e.g. gelatinized starch, as described in more detail herein.

The cyclodextrin mixture resulting from the process defined above comprises an α-cyclodextrin component as previously stated, which represents at least 70% of the total weight of the mixture. When enzyme extracted from the preferred, deposited source specifically is used in the process, the % by weight of the α-cyclodextrin is in the range from 80% to 95%. At the upper limit of this range e.g. above 90% by weight, it may be unnecessary to purify further the α-cyclodextrin. The mixture per se may be used to complex desired inclusion compounds with α-cyclodextrin in all but the most purity-strict applications. Substantially pure α-cyclodextrin, i.e. greater than 95% pureα-cyclodextrin, may be recovered from the cyclodextrin mixture, if desired. Because of the constitution of the cyclodextrin mixture, the recovery process of the present invention may be modified, providing a simplified process by comparison with known α -cyclodextrin recovery methods (see U.S. 3,541,077, supra). Cyclodextrin mixtures produced using most known enzymes comprise predominant amounts of β -cyclodextrin, and minor amounts of α-cyclodextrin and γ-cyclodextrin. Whereas β-cyclodextrin can, in an α-cyclodextrin recovery process, be separated to a major extent from α-cyclodextrin using the differential solubilities of β- and α-cyclodextrin e.g. by crystallization of β-cyclodextrin, γ-cyclodextrin has substantially the same solubility as α-cyclodextrin and cannot in practise be separated from it on the basis of solubility. Separation of α-cyclodextrin from γ-cyclodextrin is usually performed by treating the mixture with α-amylase which digests both β- and γ-cyclodextrin but to which α-cyclodextrin is substantially inert. While digestion witho α-amylase as taught in the literature is useful in concentrating α-cyclodextrin, it results also in destruction of other cyclodextrin species in the mixture which may otherwise be recoverable.

In accordance with the present invention, an alternative to α-cyclodextrin recovery process is made possible when the enzyme of the present invention is used to prepare cyclodextrin. Significantly, the process using the enzyme of the invention to produce cyclodextrin results in a cyclodextrin mixture comprising no detectable γ-cyclodextrin. Accordingly, the recovery process of the present invention can exclude the step of γ-cyclodextrin separation e.g. by α-amylase digestion. The β-cyclodextrin component can be removed in useful form using any nondestructive technique e.g. crystallization optionally followed by fractionation of the remaining mixture, to recovery substantially pure α-cyclodextrin.

Thus a further aspect of the present invention provides a process for producing substantially pure α-cyclodextrin which comprises the steps of:

1. reacting enzyme capable of converting substrate to a cyclodextrin mixture comprising at least 70% by weight α-cyclodextrin, β-cyclodextrin and substantially no detectable γ-cyclodextrin;

2. separating the α-cyclodextrin component from the α-cyclodextrin component; and

3. recovering the α-cyclodextrin component

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The preferred microbial source of the enzyme of the present invention, Bacillus strain AL35, was isolated from soil samples obtained in Ontario, Canada by screening for microbial growth on starch as a broad spectrum screen and on α-cyclodextrin as a more selective screen,α-cyclodexrin being the only species of cyclodextrin which can be degraded by the cyclodextrin glycosytransferase enzyme. One strain, exhibiting superior growth in the screening process was isolated and designated strain AL35 also referred to herein as Bacillus strain AL35. This bacterium, the preferred source of the enzyme of the present invention, can be cultured in a minimal salts medium preferably containing 2% starch as carbon substrate. A typical medium will comprise starch 2%, yeast extract 0.5% peptone 0.5%, K₂HPO₄ 0.1% and MgSO₄ 0.02% with growth being maintained preferably at 37°C. Other conventional media useful herein will be known to those skilled in the art, particularly in the light of the taxonomic and other characteristics of the bacteria, as listed below:

A. Morphol ogical Characteristics

Form Rods

Size 0.6 - 0.8 x 1.5 - 3 microns

Motil ity motil e

Gram stain Positive

Sporangia swoll en Negative

Spores 0.7 - 0.9 x 1.1 - 1.4 microns B. Physiological Characteristics

Temperature for growth Up to 50°C

Catalase Positive

Utilization of citrate Negative

Nitrate reduction Positive

Hydrolysis of starch Positive

Gelatin stab Positive

Milk agar streak plate Positive

C. Utilization of Sugars

Acid from glucose Acid from arabinose Acid from mannitol

On the basis of this data and in view of the aerobic growth of the organism, it is clear that it belongs to the Bacillus genus. Because it does not distend sporangium distinctly, the strain is considered to be different from other, known CGTase-producing species of Bacillus, including circulans, polymyxa and macerans. It is apparently different as well from B. stearothermophilus, another known CGTase-producing species, since AL35 exhibits growth at temperatures aas low as 28°C. On the basis of these observations and the taxonomic data presented above, it has been concluded that strain AL35 of the present invention most closely resembles the species B. amyloliouefaciens. Cultures of this strain were, as mentioned, deposited with ATCC on April 7, 1987 and have been allotted accession number ATCC^®53604. Samples of the bacterium will be made available while this applictioπ is pending only to those entitled access to it by law. After issue of a patent therefore, samples of the bacterium will be made available without restriction to all those requesting same from ATCC^®.

The term "culture" is used herein to encompass a population of the novel strain substantially free of natural soil contaminants and in the substantial absence of foreign microorganisms having a deleterious effect on the ability of the novel strain to produce the enzyme of the present invention.

Production of the enzyme of the present invention by Bacillus strain AL35 will proceed under standard culturing conditions, for example, using culture broth containing nutrients as described above and with incubation at around 37ºC. The growth medium preferably contains a component which serves to induce cyclodextrin glycosytransferase expression by the cultured cells e.g. around 2 - 4% starch. The enzyme of interest is secreted into the growth medium by the cultured cells and may be recovered therefrom or from the intact cells following lysis, but the former method is much preferred.

To be useful in the process of the present invention, the enzyme may be presented to the substrate in any number of suitable forms. Extra- cellular medium in which cells secreting the enzyme have been cultured maybe used per se. Alternatively, the extracellular medium may be concentrated and/or fractionated to provide a composition of increased enzyme concentration such as by microfil tration or ultrafiltration, ammonium sulfate precipitation, chromatographic separation or a combination thereof. Lyophilized forms, including immobilized forms, of the raw or concentrated extracellular optionally re-constituted in aqueous buffer are suitable for inoculating a reaction medium. It will be appreciated as well that whole cells may be used in the reaction medium provided that reaction conditions are suited to its survival and that the enzyme is circulated into contact with the substrate either continuously or intermittently.

The process by which cyclodextrins are produced using this enzyme of the present invention is similar to known processes. The substrates suitable for the reaction will include polymers of glucose bonded through an α-1,4 linkage such as amylose and hydrolysed forms of amylose. For economic reasons, the preferred substrate is gelatinized starch prepared by aqueous heating of granular starch to expose amylose. Gelatinized starch further treated with acid or α-amylase to provide shorter lengths of amylose i.e. dextrins, is also suitable herein as substrate. Sources of starch for use herein include rice starch, wheat starch and, more preferably potato starch and corn starch.

To produce cyclodextrin according to the process of the present invention, suitable substrate for example an aqueous solution comprising from 1 to 50% by weight e.g. 4 to 20% by weight, of gelatinized potato or corn starch, is brought into contact with the enzyme. An enzyme:substrate weight ratio of about 10^-4: 1 is suitable but clearly this value can range from about 10^-6: 1 to 1:1 i.e. within a range which strikes a balance between efficient enzyme conversion of starch to cyclodextrin and the economic feasibility of the process in general.

Reaction conditions are suitably designed to accomodate the enzyme to achieve maximum efficiency. As is revealed in the accompanying examples, enzyme obtained from the preferred bacterial source, Bacillus strain AL35, retains activity between about 10ºC to 70ºC and such reaction temperatures are therefore suitable. Reaction at the higher temperature range e.g. 60ºC to 70°C may cause some enzyme instability which can be compensated by adding to the enzyme preparation or to the reaction medium, enzyme- stabilizing amounts of a cationic species such as potassium, calcium, magnesium and cobalt ions and including manganese ions as a preferred enzyme stabilizing cation. The selected enzyme-stabilizing cation may be introduced in salt form such as its chloride or sulphate salt. To avoid the need for stabilizing cations in the reaction, lower reaction temperatures in the range at which both stability and activity of the enzyme is ideal i.e. in the temperature range from 55 to 65°C, may be used. In terms of pH variation, the reaction may be carried out between about pH4 and pH11 but enzyme stability dictates a preferred pH range of from 6-10 e.g 8-10.

Preferred reaction times will, of course, depend on the selected processing conditions described above. Usually the reaction can be terminated 20-48 hours after initiation. The major cyclodextrin product formed during the reaction is d-cyclodextrin with minor amounts of β-cyclodextrin also being formed.

It is desired within the scope of this invention that the α-cyclodextrin, which comprises approximately 80-95% by weight of the cyclodextrin produced using AL35, be recovered from the other cyclodextrin components by novel, relatively simple, recovery means.

REFERENCE TO THE DRAWINGS

The α-cyclodextrin recovery aspect of the present invention, is described herein below together with other exemplified embodiments of the invention, with reference to the accompanying drawings in which:

Figure 1 is a schematic representation of anα-cyclodextrin recovery process;

Figures 2A, B and 3A, B il l ustrate enzyme activity and stabi l ity profil es. Reference is first made to Figure 1 and an α-cyclodextrin recovery process according to the invention. At zone 10, starch is gelatinized by heating for 15 minutes at 121°C and then cooled to 60°C. CGTase enzyme prepared according to this invention is added to the gelatinized starch at mixing zone 12, resulting in a mixture of cyclodextrins comprising about 80-95% α-cyclodextrin the balance being substantially only β-cyclodextrin. This latter mixture, at zone 14, is subjected to the action of glycoamylase, which breaks down unreacted or by-product linear dextrins to glucose. There is no need to add α-amylase in the purification step as in similar known process described herein above to digest the β and γ-cyclodextrins, as they are not present in substantial quantities. Glucose (20) is then separated from the cyclodextrin component (18) at separation zone 16 by an appropriate filtration means eg. fractionation (ultrafiltration). The cyclodextrin component comprises substantially entirelyc α-cyclodextrin with minor amounts of β-cyclodextrin of extremely low solubility. This latter characteristic of α-cyclodextrin (26) facilitates its removal at concentration zone 22 from theod-cyclodextrin component (24). Alternatively, β-cyclodextrin may be initially precipitated from the reaction products by crystallization prior to glucoamylase reaction at zone (14).

Example 1 - Isolation and Harvest of Strain AL35

CGTase-producing strains were screened using the replicator method. Soil samples collected from various locations in Ontario were pre-soaked in 2% starch broth for 48 hours at 50°C. They were then streaked on two separate plates, one containing starch, the other containing α-cyclodextrin, at pH 5-10 and incubated at 37°C for 24 hours. A colony exhibiting superior growth as measured by clearance of starch and α-CD was picked up and transferred into 4% starch broth for growth. After 48 hours of aerobic growth at 37°C, cells were centrifuged and the supernatant was collected for enzyme activity tests.

Example 2 - Purification of IT25 CGTase

All experiments were carried out at 0-5°C. A culture broth of Bacillus sp. AL35 was centrifuged to remove cells and (NH4)₂SO₄ was added to the supernatant to 15% saturation. The solution was passed through a starch column. The adsorbed enzyme was eluted from the starch column with water then (MH4)₂SO₄ was added to the eluate and precipitates formed between saturations of 30 and 55% were recovered. The supernatant, about 0.5 liters, was fractionated by addition of solid ammonium sulfate to 30% saturation and the mixture was allowed to stand in a refrigerator. After centrifugation at 6,000 x g for 30 minutes, solid ammonium sulfate was further added to the supernatant to 55% saturation and the mixture was allowed to stand one hour. The resulting precipitate was collected by centrifugation at 7,000 x g for 30 minutes and dissolved in 10 ml of .015 M phosphate buffer, pH 7.5. The enzyme solution was dialyzed twice against 5 liters of the same buffer. The insoluble material formed during dialysis was removed by centrifugation. This dialyzed enzyme solution (30 to 50% ammonium sulfate fraction, Table 1) was applied to a column of DEAE-Zetaprep equilibrated with 0.05 M potassium phosphate buffer, pH 7.5. The column was washed first with 1 litre of the same buffer, and then eluted with 0.1 M phosphate, pH 7.5. CGTase active fractions were combined and concentrated with an Ami con concentrator to a final volume of 30 ml.

The activities, measured by standard assay (see Nakamura and Horikoshi Agr. Biol. Chem. 40(4) (1976) 753-757) are summarized below in Table 1:

Example 3

Purified AL35 CGTase was assayed for its starch degrading activity in an acetate buffer at pH 3.0 to 5.5, MES Buffer at pH 6.0-7.0 and with a Tris-HCl buffer at pH 7.5 to 9.0, for optimum pH. The assay was conducted by mixing 50 ul of starch (0.75 mg/mL from Sigma Chemical Co. Ltd., Missouri, USA) mixed with appropriate buffer and reacted with 10 ul of diluted enzyme solution for 60 minutes at 50ºC. The reaction was stopped by adding 50 ul HCl (0.5N) and the activity measured at 620 nm after adding 50 ul of 0.02% Iodine/0.2% potassium iodide.

Figure 2A shows the profile of starch degrading activity of the CGTase over the pH range tested. AL35 CGTase showed strong activity in a wide pH range.

Figure 2B illustrates the results of activity as a function of temperature, conducted at pH 6.0 but otherwise as described above. A temperature of about 70ºC is optimum for AL35 CGTase.

Figure 3A illustrates the results of pH stability analysis conducted on buffer adjusted enzyme solutions held at 40ºC for 2 hours, as revealed by starch degrading activity. AL35 CGTase is stable over a pH range of

6.0 to 10.

Heat stability is shown in Figure 3B. The purified CGTase was allowed to stand at various temperatures for 15 minutes in 50 mM Tris-HCl buffer (pH 7.0) with or without calcium ion solution and starch degrading activity was measured, as plotted in Figure 3B. AL35 CGTase did not significantly lose its activity even produced by AL35 caused a 5°C rise in the limit of heat stability.

Example 4 - α-CD Production by AL35 CGTase Preparations

Cyclodextrin production was carried out in sodium acetate buffer (0.05 M, pH 6.0) or sodium phosphate buffer (0.05 M, pH 9.0), both containing 10 mM calcium chloride. Corn starch at 4% (w/v) in the respective buffer was heated to 121°C for 15 minutes and then cooled to 60°C. CGTase enzyme preparation was then added to achieve a final enzyme dosage of 10 U/g of starch. Three enzyme preparations (culture filtrates concentrated by ultrafiltration with molecular weight cut-off at 10,000 daltons) were obtained from different fermentation runs of the AL35 strain:

1. Frozen ultraretentate from a 400 L pilot plant fermentation;

2. Ultraretentate from a 10 L fermentor run operated at pH 7.0;

3. Ultraretentate from a 10 L fermentor run operated at pH 9.0.

These crude CGTase preparations without further purification were tested for CD production at either pH 6.0 or 9.0 by incubating at 60°C for 24 hours. The resulting suspensions were sampled and assayed for α-CD and β-CD by dye-binding methods using methyl orange and o-phenolphthalein, respectively.

Results indicate that the enzyme preparations tested under the present process parameters are specific in the production α-CD, which constitutes around 80-95% of the total CD yield. This finding is of commercial significance since it would greatly simplify the down-stream processing involved in recovering the α-CD from the mixture of CD's. For applications where absolute purity of α-CD is not required, the level of contaminating CD's in the present process liquors may already be acceptable without further separation. The results also show that the enzyme preparations of AL35 can effectively operate at pH 9.0, with yields comparable to processes operated at pH 6.0. This is advantageous since the higher pH will lessen the tendency of gelatinized starch to retrograde, thereby minimizing the problem of viscosity (i.e. agitation and mixing) with high levels of starch substrates are to be used during the processing. The proper mixing of the reaction mixture would likely enhance the efficiency in CD production, resulting ultimately in better process economics in α-CD production.

Example 5 - Effect of Metal Ions on the Stability of CGTase from Bacillus AL35

The enzyme from Bacil l us AL35, purified as in Exampl e 2 , was pre-incubated with different metal sal ts in 0.1 M tris-HCl buffer, pH 7.5 at 60ºC for 10 minutes . 10 ul of each enzyme preparation was then added to a substrate mi xture consi sting of 50 ul of 1 0.75 mg/ml sol ution of starch mixed with 50 ul of 100 mM sodium acetate buffer, pH 6.0. The reaction was stopped by the addition of 50 ul of 0.5N HCl . After addition of 50 ul of 0.02% iodine i n 0.2% potassi um iodi de sol ution, activity was determined from the absorbance read at 620 nm. The results are as fol l ows :

As with most known CGTases, the presence of calcium ions improved the thermostability of the CGTase from AL35, however, the stability effect of other divalent cations observed with AL35 CGTase such as magnesium, cobalt, and in particular manganese is not known to have been reported with other CGTases.

Claims

We Claim:

1. A microorganism which produces an enzyme capable of converting gelatinized starch to a cyclodextrin mixture the α-cyclodextrin component of which comprises at least 70% of the total weight of said mixture.

2. A microorganism as defined in claim 1 which has the identifying characteristics of Bacillus strain AL35 ATCC^R 53604.

3. Bacillus strain AL35 ATCC^R 53604.

4. A culture of a microorganism claimed in any one of claims 1, 2 or 3.

5. An enzyme capable of converting gelatinized starch to a cyclodextrin mixture the α-cyclodextrin component of which comprises at least 70% of the total weight of said mixture.

6. An enzyme composition capable of converting gelatinized starch to a cyclodextrin mixture the α-cyclodextrin component of which comprises from 80 to 95% of the total weight of the mixture.

7. An enzyme composition comprising an enzyme as defined in claim 5 or claim 6 in a form suitable for inoculating a reaction medium.

8. The enzyme composition defined in claim 7 in lyophilized form.

9. The enzyme composition defined in claim 7 which comprises a component of the extracellular medium in which a bacterial source of said enzyme has been cultured.

10. The enzyme composition defined in claim 7 which comprises said enzyme and aqueous buffer.

11. An enzyme composition defined in any one of claims 7, 8, 9 or 10 which comprises an enzyme stabilizing amount of a thermally stabilizing cation.

12. The enzyme composition according to claim 11 wherein said cation is manganese cation.

13. An enzyme as defined in claim 5 in substantially pure form.

14. A process for producing enzyme which is capable of converting gelatinized starch to a cyclodextrin mixture the α-cyclodextrin component of which comprises at least 70% of the total weight of said mixture, which comprises culturing bacteria having the identifying characteristics of Bacillus strain AL35 under conditions which promote production of said enzyme by said bacteria.

15. The process according to claim 11 wherein said bacteria are Bacillus strain AL35 ATCC^R 53604.

16. The process according to claim 14 wherein said bacteria are cultured in the presence of an enzyme stimulating amount of starch.

17. Enzyme capable of converting gelatinized starch to a cyclodextrin mixture the α-cyclodextrin component of which represents at least 70% of the total weight of said mixture, whenever prepared by a process defined in any one of claims 13, 14, or 15.

18. A process for producing α-cyclodextrin which comprises reacting substrate with an enzyme capable of converting said substrate to a cyclodextrin mixture the α-cyclodextrin component of which represents at least 70% of the total weight of said mixture.

19. The process according to claim 17 wherein said substrate is gelatinized starch.

20. A process for preparing α-cyclodextrin which comprises reacting an aqueous solution of gelatinized starch with cyclodextrin glycosyltransferase produced by Bacillus strain AL35 ATCC^R 53604, at a temperature in the range from about 50°C to about 70°C and at a pH in the range from 6 to 10.

21. The process according to claim 19 wherein said aqueous solution comprises from 1 to 50% by weight of gelatininzed starch.

22. The process according to claim 20 wherein said aqueous solution comprises from 2 to 20% by weight of a gelatinized starch selected from gelatinized potato starch and gelatinized corn starch.

23. A process for recovering substantially pure α-cyclodextrin which comprises reacting substrate with enzyme to produce a cyclodextrin mixture consisting essentially of at least 70% α-cyclodextrin and β-cyclodextrin, separating β-cyclodextrin and other reaction products there fropi and recovering substantially pure α-cyclodextrin.