WO1988008031A1

WO1988008031A1 - Process for preparing cyclodextrins

Info

Publication number: WO1988008031A1
Application number: PCT/US1988/001137
Authority: WO
Inventors: Hiroyuki Aoki; Ernest Kar-Cheung Yu; Masanaru Misawa
Original assignee: Genetics Institute LLC
Current assignee: Genetics Institute LLC
Priority date: 1987-04-08
Filing date: 1988-04-08
Publication date: 1988-10-20
Anticipated expiration: 1989-10-08

Abstract

A soil bacterium, Bacillus licheniformis strain IT25, has been isolated which secretes a cyclodextrin glycosyltransferase enzyme useful in converting starch to cyclodextrin.

Description

PROCESS FOR PREPARING CYCLODEXTRINS

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 anα-1,4 linkage. They are called α-, β- or ɣ-clyclodextrins 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 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 byconversion of gelatinized starch by enzyme action. Enzymes useful for this purpose, termed cyclodextrin glycosyltransferases or CGTase for brevity, are produced by many different bacteria. Known CGTase-producing bacteria include Bacillus macerans, B. stearotherm philus, B. megaterium, B. circulans, B. ohbensis and other taxonomically distinct Bacillus spp., Klebsiella pneumoniae M5 and species of Micrococcus such as varians M-849 (ATCC 31606) and luteus B-645 (ATCC 31607). While the CGTase produced by these bacteria all function to convert gelatinized 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 in greater amounts than other species of cyclodextrin, a bias which can be controlled in some instances by altering process conditions. Efforts to identify bacterial sources of CGTase capable of producing greater amounts of cyclodextrin from substrate are on-going. Efforts are focussed particularly on identifying enzymes which are versatile in terms of substrate action, and stability over wider ranges in pH, temperature and other processing conditions.

Accordingly, it is one object of the present invention to provide a novel bacterial source of CGTase.

It is another object of the present invention to provide a novel CGTase useful in producing cyclodextrin.

It is a further object of the present invention to provide a novel process for producing cyclodextrin, particularly a process which produces one species of cyclodextrin in a predominant amount. SUMMARY OF THE PRESENT INVENTION

It has now been found that bacteria of the species Bacillus licheniformis produce a CGTase having properties which compare favourably with known CGTases. Accordingly, one aspect of the present invention comprises a process for producing cyclodextrin which comprises reacting starch or degraded starch with a cyclodextrin glycosyltransferase produced by a microorganism of the species Bacillus licheniformis.

A strain of B. licheniformis which has been isolated from soil samples taken in Ontario, Canada and is designated herein as IT25, is a particularly valuable source of CGTase and therefore represents, together with the CGTase produced thereby, preferred embodiments of the present invention.

CGTase produced by B. licheniformis IT25 exhibits activity over a wide range of pH and temperature. Notable attributes of this enzyme include stability at high temperature eg. above 65°C, even in the absence of stabilizing agents such as calcium ions, stability at pH from about pH 6 to pH 10.0 and conversion of starch preferentially to β-cyclodextrin. It should be noted as well that no starch pre-hydrolysis is required in the present process of producing cyclodextrin. The CGTase of the present invention actually works better when the starch substrate has not been pre-hydrolyzed with acid or an amylase eg. α-amylase, steps which are required when commercial CGTase preparations are used in cyclodextrin production. The CGTase of the present invention therefore provides a viable alternative to enzymes known in the art of cyclodextrin production.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The preferred strain of Bacillus licheniformis, IT25 was isolated by screening soil samples obtained in Ontario, Canada for microbial growth on a combination of starch and α-cyclodextrin at 37°C. Axenic cultures of B. licheniformis IT25 were deposited with the American Type Culture Collection in Rockville, Maryland on April 6, 1987, under accession number 53603. Samples of the bacterium will be made available while this application is pending only to those entitled access to it by law. After issue of a patent therefor, samples of the bacterium will be available without restriction to all those requesting it from ATCC.

This bacterium can be cultured in a mineral salts medium containing starch 2%, yeast extract 0.5%, peptone 0.5%, K₂HPO₄0.12 and MgSO₄0.02%, at 37°C. Taxonomic data and other analyses reveal the following characteristics of strain IT25;

A. Morphological characteristics Form Rods

Size 0.6 - 0.8 × 1.5 - 3 microns

Motility Motile Gram stain Positive Sporangia swol len Negative Spores 0.7 - 0.9 × 1.1 - 1.4 microns

B. Physiological characteristics Temperature for growth Up to 50°C Catalase Positive Utilization of citrate Positive 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 IT25 exhibits growth at temperatures as low as 28°C. On the basis of these observations and the taxonomic data presented above, it has been concluded that strain IT25 of the present invention most closely resembles the species Bacillus licheniformis.

For analysis, CGTase produced by this strain was recovered initially in culture broth filtrate, purified using standard biochemical techniques and ultimately crystallized. From gel-filtration chromatography the molecular weight of the CGTase is estimated as 140,000. Subsequent evaluation revealed that the enzyme is dimeric in structure, the sub-unit molecular weight being an estimated 72,000 by SDS-PAGE. Isoelectric focussing revealed an isoelectric point of 4.3 for the enzyme. Amino acid composition analysis revealed the presence of cysteine residues which conceivably contribute to its thermostability. The N-terminal sequence of IT25 CGTase has been shown to be different from the corresponding sequence of both B . macerans and B. stearothermophilus CGTase.

Comparison of this data with data available for known CGTases indicates that IT25 CGTase is a distinctly different enzyme which functions in a related manner by converting starch to cyclodextrin. The activity of IT25 CGTase, under a variety of conditions, is revealed in the examples which follow this discussion.

The process by which cyclodextrins are produced using this enzyme is similar to known processes. For example, substrates for the reaction may include any starches, preferably gelatinized, suitable for use in known processes of this type such as potato starch and corn starch at concentrations within known ranges eg. 2% to 50% (w/v). However, it is recommended that one avoid starch pre-hydrolysis in the process. The enzyme described herein is able to convert pre-hydrolysed starch eg. acid treated or enzyme treated starch, but this pre-hydrolysis proves to be unnecessary when IT25 CGTase is used. In fact, cyclodextrin production is enhanced when pre-hydrolysis is avoided, rendering the cost and time of pre-hydrolysis unnecessary in the preferred process of the present invention.

For reaction with the gelatinized but otherwise untreated starch, the IT25 CGTase may be presented in any suitable form. For example, because the enzyme is a secretory product of the bacterium, whole cells may be used as culture broth or a crude filtrate thereof. For more precise control of reagent proportions, concentrated enzyme preparation comprising enzyme and a carrier such as buffer or culture broth or an immobilized enzyme preparation may be used. It is unnecessary to include in the preparation or in the reaction medium any supplements which serve to stabilize the enzyme. While addition of a calcium ion source may be included, as required in some known processes, there is no need to do so in the present process. The IT25 CGTase is equally stable in the presence or absence of calcium. If desired, however, stabilizing amounts of ions such as calcium, manganese, cobalt, zinc, copper or magnesium may be added. The relative amounts of enzyme and substrate to be used in the reaction may also vary in accordance with established limits. Our trials have indicated that an enzyme:substrate weight ratio of 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 should be designed to accommodate the enzyme to achieve maximum efficiency. Temperature may range from about 10°C to higher than 70°C but temperatures higher than 70°C may cause some enzyme instability. More preferably, the reaction is conducted at a temperature at which enzyme activity and stability are suitable i.e. from about 50°C to about 70°C especially between 60 and 70°C. In terms of pH variation, the reaction may be carried out between about pH 4 and pH 11 but the enzyme stability dictates a preferred pH range of from 5 - 9 eg. pH 6 - pH 8.

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 β-cyclodextrin with minor amounts of α-cyclodextrin also being formed.

Embodiments of the invention are described hereinafter by way of example only and with reference to the accompanying drawings in which;

Figure 1 illustrates results of isoelectric focussing of the enzyme; Figures 2A and 2B illustrate graphically the activity of the enzyme in terms of pH and temperature, respectively; and

Figures 3A and 3B illustrate graphically the stability of the enzyme over ranges of pH and temperature, respectively.

Example 1 - Isolation and Harvest of Strain IT25

CGTase producing strains were screened by 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 starch and α-cyclodextrin (α-CD) plates (pH 5-10) and incubated at 37°C for 24 hours. Any colony that showed clearance of starch and α-CD was picked up and transferred into the 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. IT25 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 (NH4)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 × 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 × 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 liter of the same buffer, and then eluted with 0.1 M phosphate, pH 7.5. CGTase active fractions were combined and concentrated with an Amicon concentrator to a final volume of 30 ml.

Solid ammonium sulphate was added to 30% saturation and continued until the solution became faintly turbid. After standing for a few days, crystals appeared in a plate form.

The activities, measured by standard assay (see Nakamura and Horikoshi Agr. Biol. Chem.40(4) (1976) 753-757) and yield of the enzyme at various stages in the crystallization process are summarized below in Table 1: Table 1

Purification Summary of IT25 CGTase

Total Activity Specific Activity Yield

Step (Units x 10³) (Units/mg) (%)

Culture broth 26.3 3.65 100 Starch adsorption 25.8 16.4 98 (NH4)₂SO₄ ppt. 22.4 135 85 DEAE-Zetaprep 21.0 319 80 Crystallization 19.8 349 75

The enzyme was purified about 95-fold from the extract with a recovery of about 75% of the original activity. A chromatogram of the last step of the purification run gave only one protein peak. Purified CGTase was judged to be homogeneous by SDS-PAGE and isoelectric focussing.

Example 3 - Enzyme Analysis

Isoelectric focussing was conducted in a column (110 ml) containing 0.5% "Pharmalyte" carrier ampholyte with a pH range from 3.0 to 10 at 4°C and 800 volts for 36 hours. The results appear in Figure 1, which reveals a pI of 4.3 for the purified enzyme. Molecul ar weight estimation by gel fil tration was performed wi th a col umn of Sephacryl S-200 (2.4 × 200 cm) equil ibrated with 0.05 M phosphate buffer, pH 7.0, reveal ing an estimated molecul ar weight of 140,000 on Sephacryl S-200 and about 145,000 on Sephacryl S-300, but only 72,000 on SDS-PAGE. Thus CGTase of IT25 appears to be a dimer consisting of two identical sub-units .

Table 2 bel ow shows the amino acid composition of the IT25 CGTase. The val ues are expressed as the number of residues per molecule on the assumption of 140,000. The enzyme was found to consi st of approximately 680 amino acid residues . For contrast, these val ues are compared wtih those for the CGTase of B . macerans:

Table 2 Amino Acid Composition of CGTases

Mol %

Amino Acids IT#25 B. macerans¹

Ala 11.2 9.1

Arg 1.9 2.7

Asp(n) 18.8 14.2

Cys 0.2 0.0

Glu(n) 6.1 6.4

Gly 12.3 11.2 Table 2 Cont' d.

Mol %

Amino Acids IT#25 B. macerans¹

His 1.3 1.6

He 1.8 5.1

Leu 5.1 5.7

Lys 3.3 4.1

Met 1.2 1.8

Phe 4.5 4.8

Pro 4.7 3.6

Ser 5.9 6.9

Thr 13.2 9.5

Trp 1.1 1.7

Tyr 3.6 4.4

Val 4.0 7.2

1_T. Takeno et al., J. Bact. 166 118 (1986)

For additional comparison, N-terminal sequences of IT25 CGTase and B. macerans CGTase are set out below: N-Terminal Sequences of CGTases

1 B. sp IT25 Ser-Gly-Asp-Thr-Tyr-Val-Thr-Asn-Lys-Gln B. macerans^a Ser-Pro-Asp-Thr-Ser-Val-Asp-Asn-Lys-Val

11 -Asn-Phe-Ser-Thr-Asp-Val-Ile-Tyr-Gln-IIe -Asn-Phe-Ser-Thr-Asp-Val-Ile-Tyr-Gln-Val

21

-Phe-Thr-Asp-Ala-Phe-Leu-Asp-Gly-Asn- -Val-Thr-Asp-Arg-Phe-Ala-Asp-Gly-Asp-Arg aT. Takeno et al., J. Bact. 166, 1189 (1986)

Example 4 - Enzyme Activity Analysis

Purified IT25 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 μl of starch (0.75 mg/mL from Sigma Chemical Co. Ltd., Missouri, U.S.A.) mixed with appropriate buffer and reacted with 10 μl of diluted enzyme solution for 60 minutes at 50°C. The reaction was stopped by added 50 μl HCl (0.5N) and activity measured at 620 nm after adding 50 μl 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. IT25 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 65-70°C is optimum for IT25 CGTase.

Figure 3A illustrates the results of pH stability analysis conducted on buffer adjusted enzyme solutions held at 40°C for 2 hours, as revealed by starch degrading activity. IT25 CGTase is stable over a pH range of 6.0 to 9.5. 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. IT25 CGTase did not lose its activity even at 65°C. Addition of calcium chloride in 1.0 mM to the CGTase produced by IT25 caused a 5°C rise in the limit of heat stability.

Example 5 - Cyclodextrin Production Using IT25 CGTase For purposes of comparison, IT25 CGTase is compared with a commercial B. macerans CGTase preparation available from Amano Pharmaceutical Co. Ltd., Nagoya, Japan, at equal activity.

In general, starch was gelatinized by heating for 15 minutes at 121°C and then cooling to 60°C. Enzyme was then added and incubated with shaking at 60ºC for 20-40 hours. The reaction products were then assayed after glucoamylase digestion for the presence of glucose. Total cyclodextrin produced was calculated as the difference in the glucose levels in control and CGTase-treated solutions.

In the absence of pre-hydrolysis, the following results were obtained:

Table 3

Total CD Produced g/L Substrate

Substrate Conc'n (%, w/v) Amano IT25

Potato Starch 4.0 16.4 18.5

15.0 45.5 61.2

Corn Starch 4.0 13.4 19.3

15.0 55.3 61.2

The effect of pre-hydrolysis on cyclodextrin production was revealed in subsequent experiments. In a first such experiment, starch was pretreated with α-amylase (Termamyl from Novo Industries A/S) using 0.01 units α-amylase per gram of starch and then incubated for 30 minutes at 90°C and then the α-amylase denatured by heating to 121°C for 15 minutes. Then, the treated starch was cooled to 60°C and CGTase added (100 U/g starch). The results are given in Table 4 on the next page: Table 4

Total CD Produced g/L Substrate

Substrate Cone 'n (%, w/v) Amano IT25

Potato Starch 4.0 14.4 14.2

15.0 64.4 44.6

Corn Starch 4.0 13.9 14.5

15.0 38.9 50.8

In a similar experiment, the effect of aci d pre-hydrolysi s was determined. Starch was treated wi th HCl sufficient to l ower the pH to 2.5 and then heated to 121°C for 15 minutes in accordance wi th standard procedures for pre-treatment. The medium was cooled to 60°C and pH adjusted to pH 6.0 with NaOH. The enzyme was then added ( 100 U/g starch) and assayed as described above. The results appear i n Table 5 :

Table 5

Total CD

Produced g/L Substrate

Substrate Cone 'n (%, w/v) Amano IT25

Potato Starch 4.0 21.6 19.6

15.0 62.2 63.2

Corn Starch 4.0 17.0 16.9

15.0 59.6 55.3

From the data appearing in Tables 3, 4 and 5, it is evident that prehydrolysis is not required for efficient cyclodextrin production using IT25 CGTase.

Subsequent specific HPLC analysis of the cyclodextrins produced by action of IT25 CGTase has revealed that β-cyclodextrin is the dominant reaction product with minor amounts ofα-cyclodextrin also being produced. The ratio of β:α cyclodextrin in the reaction products is about 5:1 but this value will vary to some extent depending upon process parameters. By contrast the Amano preparation results usually in β:α ratio of around 2.4:1. Example 6 - Recombinant CGTase

As should be understood by those skilled in this art, the gene encoding the CGTase of this invention should be readily clonable from B. licheniformis strain IT25 using conventional techniques well known in the art and oligonucleotide probes based on the extensive N-terminal peptide sequence depicted in Example 3. The design and sythesis of such probes may be effected with purely conventional procedures, and the probes may used for identification of the gene, again using purely conventional methods. Once obtained, the CGTase gene may be inserted into a routine expression vector, numerous examples of which are well known in the art, for conventional overexpression of the gene in host cells, such as the well known strains of E. coli. The recombinant CGTase enzyme may then be recovered from the culture medium of the genetically engineered host cells, e.g. by the method of Example 2, crystallized if desired, assayed for specific activity and other biological functional characteristics, and used in accordance with this invention, all as previously described.

It should be noted that CGTase, whether obtained from IT25 or by recombinant means may be used in crude form, i.e. recovered from the culture media substantially free from cellular debris, that is in "isolated" form. If desired, however, the natural or recombinant enzyme may be further purified, e.g. by the methods of Example 2, to obtain the CGTase substantially free from other proteins and other components of the conditioned medium.

It is also contemplated that one may make alterations in the protein sequence of recombinant CGTase, e.g. by conducting oligonucleotide-directed or random mutagenesis on the gene encoding the CGTase, without destroying the ability of the enzyme to catalyze the production of cyclodextrin. Such variants of the CGTase described herein are also encompassed by this invention.

Claims

What is claimed is:

1. An isolated cyclodextrin glycosyltransferase (CGTase) enzyme containing within it a polypeptide sequence substantially as follows:

SGDTYVTNKQNFSTDVIYQIFTDAFLDGN.

2. A CGTase enzyme of claim 1 isolated from the culture medium of Bacillus licheniformis strain IT25 (ATCC No. 53603).

3. Bacillus licheniformis strain IT25 (ATCC No. 53603) and clones and subclones thereof.

4. An axenic culture of a microorganism of claim 3.

5 . A CGTase enzyme of claim 1 produced by heterologous expression in a host cell of a gene encoding the CGTase enyzme.

6. An enzyme of claims 1, 2, or 5 in crystalline form.

7 . A process for producing cyclodextrin which comprises contacting starch or pre-hydrolyzed starch with a CGTase of claim 1, 2, 5 or 6 under suitable conditions permitting the production of cyclodextrin.

8. A process of claim 7, wherein the starch is unhydrolyzed, gelatinized starch.

9. A method for producing a CGTase of claim 1 which comprises culturing microorganism of claim 3 under suitable conditions permitting the production and secretion of the CGTase, and isolating the CGTase so produced from the culture medium.