Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1993-07-15
1995-12-12
Furman, Keith C.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435201, 435203, 435207, 435208, 435913, 424 9461, 424 9462, C12N 924, C12N 926, C12N 930, C12N 936
Patent
active
054749220
DESCRIPTION:
BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of PCT/DK92/00037 filed Feb. 6, 1992 which is incorporated herein be reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention comprises a .beta.-1,4-galactanase, a corresponding DNA sequence, a vector, a transformed host, a method for production of a .beta.-1,4-galactanase, an enzyme preparation, and a use of the .beta.-1,4-galactanase.
2. Description of Related Art
.beta.-1,4-galactanases (EC no. 3.2.1.89) is a group of carbohydrases which degrade galactanes. The authorized enzyme name is 1,4-.beta.-D-galactan galactohydrolase, but the short term .beta.-1,4-galactanase is used in this specification with claims. Reference can be made to R. F. H. Dekker and G. N. Richards, "Hemicellulases, their Occurence, Purification, Properties and Mode of Action" in R. S. Tipson and D. Horton, Advances in Carbohydrate Chemistry and Biochemistry, Academic Press 32, 277-352 (1976), R. F. H. Dekker, "The Hemicellulase Group of Enzymes", in J. M. V. Blanchard and J. R. Mitchell, Polysaccharides in Food, Butterworths, 93-108 (1979), and A. G. J. Voragen, F. Geerst and W. Pilnik "Hemicellulases in Enzymatic Fruit Processing", in P. Depuy, Use of Enzymes in Food Technology, Technique et Documentation Lavoisier, 497-502 (1982). Galactanes are found in connection with many gums, agar, and fruit pectins, and they are components of cell walls in e.g. fruits and vegetables.
SUMMARY OF THE INVENTION
The present invention relates to .beta.-1,14-galactanases obtainable from A. aculeatus which has a pH-optimum between 3.0 and 5.0, an isoelectric point of 2.5-3.5, a molecular weight of between 30,000 and 50,000, and temperature optimum between 10.degree. and 50.degree. C. The .beta.1,4-galactanases may gave the partial amino acid sequence
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention is further illustrated by the drawings wherein
FIG. 1 graphically illustrates the results of hydrophobic interaction chromatography on a concentrated .beta.-1,4-galactanase-containing broth from Aspergillus aculeatus.
FIG. 2 graphically illustrates the results of ion exchange chromatography performed on the fraction of FIG. 1 which has been further diluted ultrafiltered.
FIG. 3 graphically illustrates the results of pooled galactanase fractions treated by hydrophobic interaction chromatography.
FIG. 4 graphically illustrates the activity of .beta.-1,4-galactanase of the invention as a function of pH.
FIG. 5 graphically illustrates the stability of a .beta.-1,4-galactanase of the invention as a function of pH.
FIG. 6 graphically illustrates the activity of a .beta.-1,4-galactanase of the invention as a function temperature.
FIG. 7 graphically illustrates the stability of a .beta.-1,4-galactanase of the invention as a function temperature.
FIG. 8 graphically illustrates the determination of Michaelis-Menten-Kinetic of a .beta.-1,4-galactanase of the invention.
FIG. 9 graphically illustrates the determination of the Lineweaver-Burk-kinetic or a .beta.-1,4-galactanase of the invention.
FIG. 10 shows a map of plasmid pYHD17.
FIG. 11 shows a map of plasmid pHD414.
FIG. 12 shows a map of plasmid pHD438.
FIG. 13 graphically illustrates the viscosity of a .beta.-1,4-galactanase alone and of the .beta.-1,4-galactanase with a crude Aspergillus aculeatus preparation as a function of time.
The above indicated partial amino acid sequence can be used for construction of DNA probes which can be used for screening a genomic library for organisms expressing such enzyme, or a cDNA library, thereby obtaining DNA sequences, which can be used either for an overproduction of .beta.-1,4-galactanase, if inserted in the microorganism species, from which the parent DNA molecule originated, or for production of .beta.-1,4-galactanase without accompanying closely related enzymes, if inserted in a host microorganism, which in its not-transformed condition does not produce any enzymes closely related to .beta.-1,4-galactanase. The DNA sequences can be established otherwise
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Christensen Flemming M.
Dalboge Henrik
Dorreich Kurt
Mikkelsen Jan M.
Mischler Marcel
Furman Keith C.
Lambiris Elias J.
Novo Nordisk A S
Zelson Steve T.
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