Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1995-09-12
1999-07-06
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
4352523, 43525231, 43525233, 43525235, 43525411, 43525421, 4352543, 536 232, C12N 940, C12N 120, C12N 114, C07H 2104
Patent
active
059196904
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a DNA construct encoding an .alpha.-galactosidase enzyme and variants thereof having .alpha.-galactosidase activity, a recombinant expression vector and a cell harbouring said DNA construct, and a method of preparing an .alpha.-galactosidase enzyme preparation by use of recombinant DNA techniques. The .alpha.-galactosidase enzyme encoded by the DNA construct of the invention may, inter alia, be used for the degradation of .alpha.-galactosides present in various plant products, or as a digestive aid.
BACKGROUND OF THE INVENTION
.alpha.-galactosidase is a well-known enzyme involved in the hydrolysis of .alpha.-galactosides present in, for instance, various important plants or plant parts used for nutritional purposes such as legumes, vegetables, grains, cereals and the like. .alpha.-galactosidase enzymes are produced by various microorganisms, plants and animals. Mammals, however, are deficient in intestinal .alpha.-galactosidase production and, consequently, are incapable of decomposing ingested .alpha.-galactosides by themselves. Instead, ingested .alpha.-galactosides are decomposed by microorganisms present in the intestine. This microbial decomposition normally results in flatulence and further confers a digestive discomfort to the mammal upon ingestion of .alpha.-galactoside-containing food or feed. The physiological effects of .alpha.-galactosides are discussed in detail by Rackis, J. J., 1975.
In order to overcome the problem associated with mammalian .alpha.-galactosidase deficiency, .alpha.-galactosides contained in food or feed have been modified prior to ingestion, for instance enzymatically by the action of .alpha.-galactosidase. Alternatively, .alpha.-galactosidase has been suggested as a digestive aid, cf. WO 90/14101.
The production of .alpha.-galactosidase has been reported from bacteria, e.g. Bacillus stearothermophilus (U.S. Pat. No. 3,846,239), yeasts, e.g. Saccharomyces cereviciae (U.S. Pat. No. 4,431,737), fungi, e.g. strains of the genii Neurospora and Rhizopus (Worthington and Beuchat, 1974), Aspergillus oryzae (Cruz and Park, 1982), A. ficuum (morphologically similar A. niger) (Zapater et al., 1990) and A. niger (Bahl and Agrawal (1969 and 1972), Christakopoulos et al. (1990), Chun and Lee (1988), Jung and Lee (1986), Lee and Wacek (1970), Adya and Elbein (1976), Kaneko et al. (1991)). All of these references, however, describe the .alpha.-galactosidase production by conventional fermentation of naturally occurring or mutated microbial strains.
Overbeeke et al., 1990, describes the production of .alpha.-galactosidase from guar in Bacillus subtilis and Aslandis et al, 1989, describes an .alpha.-galactosidase from E. coli.
An A. niger .alpha.-galactosidase enzyme preparation (Alpha-Gal.TM.) produced by conventional fermentation is available from Novo Nordisk A/S, Denmark. One drawback associated with the production of .alpha.-galactosidase by fermentation of A. niger is that substantial amounts of oxalic acid, an undesired by-product, are produced by A. niger simultaneously with the production of .alpha.-galactosidase.
It would be desirable to be able to produce an A. niger .alpha.-galactosidase enzyme preparation with reduced or without simultaneous production of oxalic acid, and further to increase the yield and the purity of the .alpha.-galactosidase preparation so produced.
The object of the present invention is to device means and methods for the production of .alpha.-galactosidase enzymes by recombinant DNA techniques. By use of such techniques it is contemplated to be possible to produce .alpha.-galactosidase in substantially larger amounts and more economical than what is possible by use of conventional fermentation technology and at the same time avoid or reduce the amount of oxalic acid formed.
BRIEF DESCRIPTION OF THE INVENTION
Accordingly, in a first aspect the present invention relates to a DNA construct comprising a DNA sequence encoding a polypeptide having .alpha.-galactosidase activity, wherein the DNA seque
REFERENCES:
Bahl et al., J. of Biological Chem., vol. 244, No. 11 pp. 2970-2978, 1969.
Lee et al., Archives of Biochem. & Biophysics, vol. 138, pp. 264-271, 1970.
Agnantiari et al., Acta Biotechnol., 11(5):479-484, 1991.
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989, pp. 11.3-11.19.
Suggs et al., PNAS, 78(11):6613-6617, Nov. 1981.
Halkier Torben
Hjort Carsten M.
Knap Inge Helmer
Kofod Lene Venke
Gregg, Esq. Valeta
Novo Nordisk A S
Slobodyansky Elizabeth
Wax Robert A.
Zelson Esq. Steve T.
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