WO1988008031A1 - Procede de preparation de cyclodextrines - Google Patents

Procede de preparation de cyclodextrines Download PDF

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Publication number
WO1988008031A1
WO1988008031A1 PCT/US1988/001137 US8801137W WO8808031A1 WO 1988008031 A1 WO1988008031 A1 WO 1988008031A1 US 8801137 W US8801137 W US 8801137W WO 8808031 A1 WO8808031 A1 WO 8808031A1
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WO
WIPO (PCT)
Prior art keywords
cgtase
enzyme
starch
cyclodextrin
produced
Prior art date
Application number
PCT/US1988/001137
Other languages
English (en)
Inventor
Hiroyuki Aoki
Ernest Kar-Cheung Yu
Masanaru Misawa
Original Assignee
Genetics Institute, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genetics Institute, Inc. filed Critical Genetics Institute, Inc.
Publication of WO1988008031A1 publication Critical patent/WO1988008031A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/1074—Cyclomaltodextrin glucanotransferase (2.4.1.19)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins

Definitions

  • This invention relates to the production of cyclodextrins.
  • Cyclodextrins are cyclic oligosaccharides, common species of which are composed of 6, 7 or 8 glucose residues bound through an ⁇ -1,4 linkage. They are called ⁇ -, ⁇ - or ⁇ -clyclodextrins depending on the number of glucose residues; 6, 7 or 8, respectively.
  • 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.
  • CGTase cyclodextrin glycosyltransferases
  • 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).
  • 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.
  • 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.
  • 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 ⁇ -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.
  • the CGTase of the present invention therefore provides a viable alternative to enzymes known in the art of cyclodextrin production.
  • 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 ⁇ -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%, K 2 HPO 4 0.12 and MgSO 4 0.02%, at 37°C. Taxonomic data and other analyses reveal the following characteristics of strain IT25;
  • 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.
  • 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.
  • 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).
  • starch pre-hydrolysis 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.
  • 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.
  • the IT25 CGTase may be presented in any suitable form.
  • the enzyme is a secretory product of the bacterium, whole cells may be used as culture broth or a crude filtrate thereof.
  • 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.
  • 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.
  • 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 ⁇ -cyclodextrin with minor amounts of ⁇ -cyclodextrin also being formed.
  • 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
  • FIGS 3A and 3B illustrate graphically the stability of the enzyme over ranges of pH and temperature, respectively.
  • 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 ⁇ -cyclodextrin ( ⁇ -CD) plates (pH 5-10) and incubated at 37°C for 24 hours. Any colony that showed clearance of starch and ⁇ -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.
  • ⁇ -CD ⁇ -cyclodextrin
  • 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.
  • 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.
  • 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 .
  • these val ues are compared wtih those for the CGTase of B . macerans:
  • N-terminal sequences of IT25 CGTase and B. macerans CGTase are set out below: N-Terminal Sequences of CGTases
  • 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 ⁇ l of starch (0.75 mg/mL from Sigma Chemical Co. Ltd., Missouri, U.S.A.) mixed with appropriate buffer and reacted with 10 ⁇ l of diluted enzyme solution for 60 minutes at 50°C.
  • the reaction was stopped by added 50 ⁇ l HCl (0.5N) and activity measured at 620 nm after adding 50 ⁇ l 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.
  • FIG 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
  • IT25 CGTase is compared with a commercial B. macerans CGTase preparation available from Amano Pharmaceutical Co. Ltd., Nagoya, Japan, at equal activity.
  • 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.
  • 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.
  • 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.
  • 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.
  • CGTase 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.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
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  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
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Abstract

On a isolé une bactérie se trouvant dans le sol, Bacillus licheniformis de souche IT25, secrétant une enzyme glycosyltransférase de cyclodextrine utile pour convertir l'amidon en cyclodextrine.
PCT/US1988/001137 1987-04-08 1988-04-08 Procede de preparation de cyclodextrines WO1988008031A1 (fr)

Applications Claiming Priority (2)

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US3595287A 1987-04-08 1987-04-08
US035,952 1987-04-08

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991009962A1 (fr) * 1989-12-22 1991-07-11 Novo Nordisk A/S Procede de conversion par voie enzymatique de l'amidon en cyclodextrines
US5364788A (en) * 1991-07-01 1994-11-15 Ahc Inc. Pure culture of Bacillus subtilis FERM BP-3418
US5501968A (en) * 1987-10-15 1996-03-26 Novo Nordisk A/S Thermostable cyclodextrin glycosyl transferase and processes using it
US9499804B2 (en) 2013-02-05 2016-11-22 Green Biologics Ltd Cyclodextrin glucanotransferase

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4235970A (en) * 1976-08-05 1980-11-25 Cpc International Inc. Protease inactivated α-amylase preparations
JPS60203183A (ja) * 1984-03-29 1985-10-14 Daido Nippon Kk 新規なバチルスｓｐ．ＨＡ３−３−２
US4659667A (en) * 1985-02-26 1987-04-21 Miles Laboratories, Inc. Process to recover crystalline enzymes and crystalline enzymes produced thereby
US4717662A (en) * 1985-01-31 1988-01-05 Miles Laboratories, Inc. Thermal stabilization of alpha-amylase
US4724208A (en) * 1985-11-04 1988-02-09 Miles Laboratories, Inc. Process for the production of solution stable alpha-amylase and liquid alpha-amylase produced thereby

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4235970A (en) * 1976-08-05 1980-11-25 Cpc International Inc. Protease inactivated α-amylase preparations
JPS60203183A (ja) * 1984-03-29 1985-10-14 Daido Nippon Kk 新規なバチルスｓｐ．ＨＡ３−３−２
US4717662A (en) * 1985-01-31 1988-01-05 Miles Laboratories, Inc. Thermal stabilization of alpha-amylase
US4659667A (en) * 1985-02-26 1987-04-21 Miles Laboratories, Inc. Process to recover crystalline enzymes and crystalline enzymes produced thereby
US4724208A (en) * 1985-11-04 1988-02-09 Miles Laboratories, Inc. Process for the production of solution stable alpha-amylase and liquid alpha-amylase produced thereby

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GENE, Volume 47, issued 1986 (Oxford, England), (F. BINDER et al.), "Cyclodextrin-Glycosyltransferase from Klebsiella Pneumoniae M5al: Cloning, Nucleotide Sequence and Expression", see pages 269-277. *
J. BACTERIOL, Volume 166, No. 3, issued June 1986, (Washington D.C.), (T. TAKANO et al.), "Molecular Cloning, DNA Nucleotide Sequencing and Expression in Bacillus Subtilis Cells of the Bacillus Macerans Cyclodextrin Glucanotransferase Gene", see pages 1118-1122, especially fig. 3. *
J. BACTERIOL., Volume 169, No. 9, issued September 1987 (Washington D.C.), (K. KIMURA et al.), "Nucleotide Sequence, Sequence of the -Cyclodextrin Glucanotransferase Gene of Alkalophilic Bacillus Sp. Strain 1011 and Similarity of its Amino Acid Sequence to these of -Amylase", see pages 4399-4402, especially fig. 2. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5501968A (en) * 1987-10-15 1996-03-26 Novo Nordisk A/S Thermostable cyclodextrin glycosyl transferase and processes using it
WO1991009962A1 (fr) * 1989-12-22 1991-07-11 Novo Nordisk A/S Procede de conversion par voie enzymatique de l'amidon en cyclodextrines
US5364788A (en) * 1991-07-01 1994-11-15 Ahc Inc. Pure culture of Bacillus subtilis FERM BP-3418
US9499804B2 (en) 2013-02-05 2016-11-22 Green Biologics Ltd Cyclodextrin glucanotransferase
US9783831B2 (en) 2013-02-05 2017-10-10 Green Biologics Ltd Cyclodextrin glucanotransferase

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