Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Patent
1992-09-23
1995-04-25
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
435101, 435 698, 4352525, 435832, 536 232, C12N 910, C12N 1554, C12P 1918, C12R 107
Patent
active
054098246
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to cyclodextrin glycosyltransferases which primarily produce .gamma.-cyclodextrin and which, with starch as substrate, form primarily .gamma.-cyclodextrin and as secondary products almost exclusively cyclic oligosaccharide.
2. The Prior Art
The enzyme cyclodextrin glycosyltransferase (abbreviation: CGTase) E.C.2.4.1.19 catalyzes the formation of cyclodextrins from starch. Depending on the number of glucose units of which the cyclodextrin (CD) is composed, a distinction is made between .alpha.-CD (6 glucose units), .beta.-CD (7 glucose units) and .gamma.-CD (8 glucose units).
To date, two types of CGTase have been disclosed:
a) CGTases which primarily form .alpha.-cyclodextrin, also called .alpha.-CGTase, such as, for example, the CGTase from Bacillus macerans (U.K. Patent No. 2,169,902), from Klebsiella pneumoniae (EPA 220,714) and from Bacillus stearothermophilus (U.K. Pat. No. 2,169,902).
b) CGTases which primarily form .beta.-cyclodextrin, or .beta.-CGTase, such as, for example, the CGTase from Bacillus circulans (U.S. Pat. No. 4,477,568), from Bacillus megaterium (U.S. Pat. No. 3,812,011), from Bacillus ohbensis (Japan Patent No. 74,124,285), from Micrococcus sp. (EPA No. 017,242) and from alkalophilic Bacillus sp. (J. Gen. Microbiol. 1988, 134, 97-105; Appl. Microbiol. Biotechnol. 1987, 26, 149-153).
Concerning a .gamma.-CGTase, to date there have been two indications in the literature:
a) The cyclodextrin glycosyltransferase from Bacillus sp. produces on addition of EtOH predominantly a mixture of .beta.- and .gamma.-cyclodextrin (10.4 to 18.7%) (Japan Patent No. 63,133,998). The enzyme has not been characterized in terms of its kinetic properties, so that it cannot be assigned to a specific type.
b) Two articles (Agric. Biol. Chem., 1986, 50, (8), 2161=2162 and Denpun Kagaku 1986, 33, 137) describe a .gamma.-CGTase from Bacillus subtilis No. 313. This CGTase is distinguished by the formation of .gamma.-cyclodextrin and linear oligosaccharide. Since CGTases generate only cyclic products from starch, this "CGTase" is a transitional form between an .alpha.-amylase (generates linear oligosaccharide from starch) and a CGTase. This ".gamma.-CGTase" is unsuitable for preparing .gamma.-cyclodextrin because only low yields can be achieved (see EPA 327,099, page 2, lines 40-43).
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to prepare enzymes which predominantly produce .gamma.-CD. The object was achieved by screening bacteria for the secretion of a .gamma.-CGTase, characterizing these bacteria, and purifying and biochemically characterizing the .gamma.-CGTase from the bacteria.
.gamma.-cyclodextrin glycosyltransferases within the meaning of the invention are .gamma.-cyclodextrin glycosyltransferases which primarily produce .gamma.-cyclodextrin and which with starch as substrate form primarily .gamma.-cyclodextrin and as secondary products almost exclusively cyclic oligosaccharide.
Used for screening the bacteria for the production of .gamma.-CGTase are preferably starch-degrading, particularly preferably alkalophilic starch-degrading bacteria.
To characterize the .gamma.-CGTase-producing bacteria, the shape, width, length and motility of the bacteria are determined. Also determined are their stainability by the Gram reaction, their ability to form catalase and their GC content. In addition, their growth behavior at various temperatures, various pH values and various NaCl concentrations are determined.
The enzyme is biochemically characterized after purification of the .gamma.-CGTase. Thus, for example, the molecular weight, pH optimum, pH stability, temperature optimum and temperature stability of the enzyme concentrations are determined.
In order to increase the yield of .gamma.-CGTase, the encoding gene is modified by gene manipulation and expressed in secretor mutants. For this, the gene is initially cloned and sequenced. The gene is preferably cloned in E. coli. The identified gene is then placed
REFERENCES:
Kato, T. et al. Agric. Biol. Chem. 50(8):2161-2162 (1986).
Schmid, G. et al. Chem. Abstracts 112:181767e (1990). (Proc. Int. Symp. Cyclodextrins, 4th (1988), 87-92).
Kato, T. et al. Chem. Abstracts 106:29122j (1987) (Denpun Kagaku 33(2):137-43 (1986)).
Bender, H. Applied Microbiol. Biotechnol. 34:229-230 (1990).
Schmid, G. Trends in Biotechnology 7(9):244-248 (1989).
Consortium fur Elektrochemische Industrie GmbH
Jacobson Dian C.
Wax Robert A.
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