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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
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/07—Bacillus
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/832—Bacillus

Definitions

• the invention relates primarily to cyclodextrin glycosyltransferases producing cyclodextrin.
• cyclodextrin glycosyl transferase (abbr .: CGTase) E.C.2.4.1.19 -catalyzes the formation of cyclodextrins from starch.
• CGTase cyclodextrin glycosyl transferase
• E.C.2.4.1.19 catalyzes the formation of cyclodextrins from starch.
• CD cyclodextrin glycosyl transferase
• the cyclodextrin glycosyltransferase from Bacillus sp. mainly produces a mixture of ß- and cyclodextrin (10.4 to 18.7%) when adding EtOH (JP 63,133,998).
• the enzyme is not characterized in terms of its kinetic properties, so that it cannot be assigned to a specific type.
• the object of the invention was therefore to produce enzymes which predominantly produce -CD.
• Bacteria were cleaned and characterized biochemically.
• 7 -Cyclodextringlycosyltransferasen purposes of the invention are primarily cyclodextrin producing 7 - cyclodextrin glycosyl preferably those which are primarily formed with the substrate thickness of 7-cyclodextrin, and as a result products almost exclusively cyclic oligosaccharides.
• starch-degrading particularly preferably alkalophilic, starch-degrading bacteria are preferably used.
• the shape, width, length and mobility of the bacteria are determined. Furthermore, their colorability in the gram reaction, their ability to form catalase and their GC content are determined. In addition, their growth behavior at different temperatures, different pH and different NaCl concentrations is determined.
• the enzyme is characterized biochemically. For example, molecular weight, pH optimum, pH stability, temperature optimum and temperature stability of the enzyme are determined.
• the coding gene is genetically modified and expressed in secretor mutants.
• the gene is first cloned and sequenced.
• the gene is preferably cloned in E. coli.
• the identified gene is then placed under the control of a promoter, preferably a regulatable promoter (eg APL, trp, lac, trc promoter), particularly preferably the lactose inducible tac promoter.
• a promoter preferably a regulatable promoter (eg APL, trp, lac, trc promoter), particularly preferably the lactose inducible tac promoter.
• a vector in addition to the 7 -CGTase encoding gene and a leader sequence, and regulatory elements such as, for example, containing the tac-Pro otor is, for the production of 7 - CGTase in microorganisms, preferably E. coli, especially preferably introduced a secretory mutant of E. coli.
• Suitable mutant secretors can be prepared by the process disclosed in EP-A-338 410.
• By inducing the promoter in the case of the tac promoter with lactose or IPTG, the overproduction and secretion of the 7 -CGTase in the culture medium can be achieved.
• the protein according to the invention can be purified in a known manner from the supernatant of the secretory mutant.
• the enzymes described here are the first to produce 7 -cyclodextrin-producing CGTases.
• the enzymes described can be used to obtain the enzymes in larger quantities than is possible from their natural microorganisms of origin. With the help of the enzymes, the production times of 7- cyclodextrins can be greatly shortened by the method described in US Pat. No. 4,822,874.
• 7- cyclodextrins increase the solubility of hydrophobic substances in aqueous solution, they stabilize labile substances (eg UV protection, oxidation protection) and bind volatile substances.
• Cyclodextrin can also be used as a formulation agent.
• the 7- cyclodextrins are used in the following, among others
• Fig. 1 Kinetics of the production of ⁇ - and 7- cyclodextrin of the 7 -CGTase of the alkalophilic bacterial strain Bacillus 290-3
• Fig. 2 Open reading frame of the 7 -CGTase from Bacillus 290-3
• Fig. 4 The synthetic oligonucleotides M8 and M9
• Fig. 5 The expression plasmid pCM750
• Fig. 6 The synthetic oligonucleotides MIO, Mll, M12 and Ml3
• Example 1 Screeninq for -CGTase-producing alkalophilic bacteria
• Soil samples from various regions of the world were collected. 0.1-0.2 g of earth was weighed out and slurried in sterile vessels with 1 ml of sterile physiological saline. After the coarse portions had settled, 0.1 ml each was placed on a starch agar plate (medium 1:10 g / 1 soluble starch; 5 g / 1 peptone; 5 g / 1 yeast extract; 1 g / 1 KH 2 PO; 0.2 g / 1 MgS0 x 7 H 2 0; 10 g / 1 Na 2 C0 3 ; 15 g / 1 agar; pH 10.4) plated. The agar plates were incubated at 30 ° C. for 2-3 days.
• Colonies of starch-degrading bacteria showed a cloudy halo, which was created by retrograding low-molecular starch molecules.
• the colonies were isolated and cleaned twice on starch agar plates. Then cultivation was carried out in 2 ml of liquid medium of the above composition. After incubation at 30 ° C. for 48 h, the cells were centrifuged off and the supernatant was tested for CGTase activity. 200 ⁇ l of supernatant were mixed with 200 ⁇ l of 10% starch solution in 20 mM Tris / HCl pH 9.0; 5 M
• Example 2 Taxonomic characterization of the Bacillus 290-3 strain
• the taxonomic classification showed that it is a gram-positive, spore-forming bacterium that can be assigned to the so far not exactly characterized Bacillus firmus / lentus complex (see Table 1).
• Table 1 Taxonomic characteristics of isolate 290-3
• the Bacillus 290-3 strain was grown in medium 1 (see Example 1). After a growth period of 48 h, the cells were separated by centrifugation and the culture supernatant (NH4) 2 SO 4 was added until a saturation of 66% was reached. The mixture was kept at 4 ° C. for 1 hour and the precipitate was then separated off by centrifugation (10,000 ⁇ g; 20 min). The precipitate was resuspended in 1/100 of the starting volume in buffer A (20 M Tris / Cl pH 8.5, 5 mM CaCl 2 , 0.05% 7- CD) and dialyzed against the same buffer.
• buffer A (20 M Tris / Cl pH 8.5, 5 mM CaCl 2 , 0.05% 7- CD
• the enzyme solution was applied to an affinity column ( 7- cyclodextrin coupled to Sepharose 6B via butyl diglycidyl ether). Elution was carried out with buffer A, which was treated with 1% 7 -cyclodextrin.
• the eluted protein material was concentrated by ammonium sulfate precipitation and dialyzed again. The purity of the protein was checked by SDS polyacrylamide gel electrophoresis.
• Chromosomal DNA from the Bacillus 290-3 strain was partially cleaved with the restriction enzyme Sau 3A and after size fractionation of the fragments by agarose gel electrophoresis, these DNA fragments were cloned into pUC18 (Ba HI cleaved).
• DNA fragments were subcloned into the plasmid pUC18 or pUC19. With exonuclease III, deletions were generated in the inserts of these plasmids in such a way that DNA sequencing according to the "Sanger dideoxy chain termination method" led to overlapping sequences (DNA, 1985, 4, 165-170).
• the open reading frame encoding 7 -CGTase spans 2097 Nucleotides (Fig. 2).
• the protein derived from this consists of 699 amino acids with a molecular weight of 78,000 Da. After the signal peptide has been split off, the molecular weight is 75,000 Da.
• Example 6 Expression and secretion of the i-CGTase in E. coli
• the "tac" promoter plasmid pJFll ⁇ u (FIG. 3) was used for the overexpression of the 7 -CGTase.
• the plasmid was split with the restriction enzymes EcoRI and HindIII and the small DNA fragment was separated from the "poly-linker” by agarose gel electrophoresis.
• the plasmid pl9 (see Example 5) was cleaved with AccI and HindIII and a 2.4 kb DNA fragment was isolated by agarose gel electrophoresis, which carries the structural gene of the 7 -CGTase almost completely. A short region coding for the signal peptide is missing at the 5 'end of the gene (see “AccI site” in FIG. 2). This region is replaced by two synthetic oligonucleotides M8 and M9 (FIG. 4).
• the expression plasmid pCM750 (FIG. 5) was obtained by ligation of the EcoRI-HindIII fragment from pJFll ⁇ u with the oligonucleotide pair M8, M9 and the Accl-HindIII fragment from pl9.
• the oligonucleotide pairs MIO, Mll and M12, M13 were synthesized by the phosphoramidite method (FIG. 6).
• the DNA sequence of the oligonucleotides codes for the N-terminal amino acid sequence of the ⁇ -CGTase from Bacillus 1-1 (Proc. 4th Int. Symposium on Cyclodextrins (Huber O., Szejtly J., eds) 1988, pp 71-76, Kluwer Acade ic) and is homologous to the N-terminal region of the -CGTase, which encompasses the DNA region up to the Sspl site (see FIG. 2).
• the plasmid pCM750 was partially digested with the restriction enzyme AccI. Subsequent cleavage was carried out with Sspl and a 7.3 kb DNA fragment was isolated by agarose gel electrophoresis. Ligation of this DNA fragment with the double-stranded oligonucleotides MIO, Mll and M12, M13 gave the plasmid pCM720 (FIG. 7), which codes for a chimeric 7 -CGTase.
• Transformation of the secretion mutant E. coli WCM100 and cultivation in accordance with Example 6 resulted in a two-fold increase in the 7 -CGTase yield compared to the results in Example 6.
• Example 5 The experiment was carried out as described in Example 5, except that 100 U a-CGTase from Bacillus macerans was used instead of 7 -CGTase. After 32 hours, a maximum yield of 7- cyclodextrin of 43% was reached.

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• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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• Transition And Organic Metals Composition Catalysts For Addition Polymerization (AREA)
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• 1990
  • 1990-03-27 DE DE4009822A patent/DE4009822A1/de not_active Withdrawn
• 1991
  • 1991-03-22 KR KR1019920702261A patent/KR960013463B1/ko not_active Expired - Fee Related
  • 1991-03-22 JP JP3506310A patent/JPH0630575B2/ja not_active Expired - Fee Related
  • 1991-03-22 DE DE59105733T patent/DE59105733D1/de not_active Expired - Fee Related
  • 1991-03-22 ES ES91906466T patent/ES2073743T3/es not_active Expired - Lifetime
  • 1991-03-22 HU HU922951A patent/HUT63190A/hu unknown
  • 1991-03-22 US US07/927,316 patent/US5409824A/en not_active Expired - Lifetime
  • 1991-03-22 AT AT91906466T patent/ATE123808T1/de not_active IP Right Cessation
  • 1991-03-22 EP EP91906466A patent/EP0521948B1/de not_active Expired - Lifetime
  • 1991-03-22 WO PCT/EP1991/000560 patent/WO1991014770A2/de not_active Ceased
  • 1991-03-22 FI FI924294A patent/FI924294L/fi unknown
  • 1991-03-22 CA CA002078992A patent/CA2078992A1/en not_active Abandoned
  • 1991-04-08 TW TW080102607A patent/TW234146B/zh active

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