Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-04-19
2001-03-06
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S256100
Reexamination Certificate
active
06197566
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a DNA construct encoding an &agr;-galactosidase enzyme and variants thereof having &agr;-galactosidase activity, a recombinant expression vector and a cell harbouring said DNA construct, and a method of preparing an &agr;-galactosidase enzyme preparation by use of recombinant DNA techniques. The &agr;-galactosidase enzyme encoded by the DNA construct of the invention may, inter alia, be used for the degradation of &agr;-galactosides present in various plant products, or as a digestive aid.
BACKGROUND OF THE INVENTION
&agr;-galactosidase is a well-known enzyme involved in the hydrolysis of &agr;-galactosides present in, for instance, various important plants or plant parts used for nutritional purposes such as legumes, vegetables, grains, cereals and the like. &agr;-galactosidase enzymes are produced by various microorganisms, plants and animals. Mammals, however, are deficient in intestinal &agr;-galactosidase production and, consequently, are incapable of decomposing ingested &agr;-galactosides by themselves. Instead, ingested &agr;-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 &agr;-galactoside-containing food or feed. The physiological effects of &agr;-galactosides are discussed in detail by Rackis, J. J., 1975.
In order to overcome the problem associated with mammalian &agr;-galactosidase deficiency, &agr;-galactosides contained in food or feed have been modified prior to ingestion, for instance enzymatically by the action of &agr;-galactosidase. Alternatively, &agr;-galactosidase has been suggested as a digestive aid, cf. WO 90/14101.
The production of &agr;-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 &agr;-galactosidase production by conventional fermentation of naturally occurring or mutated microbial strains.
Overbeeke et al., 1990, describes the production of &agr;-galactosidase from guar in
Bacillus subtilis
and Aslandis et al, 1989, describes an &agr;-galactosidase from
E. coli.
An
A. niger
&agr;-galactosidase enzyme preparation (Alpha-Gal™) produced by conventional fermentation is available from Novo Nordisk A/S, Denmark. One drawback associated with the production of &agr;-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 &agr;-galactosidase.
It would be desirable to be able to produce an
A. niger
&agr;-galactosidase enzyme preparation with reduced or without simultaneous production of oxalic acid, and further to increase the yield and the purity of the &agr;-galactosidase preparation so produced.
The object of the present invention is to device means and methods for the production of &agr;-galactosidase enzymes by recombinant DNA techniques. By use of such techniques it is contemplated to be possible to produce &agr;-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 &agr;-galactosidase activity, wherein the DNA sequence a) encodes a polypeptide comprising the amino acid sequence shown in the appended SEQ ID No. 3, or b) is an analogue of the DNA sequence of a), which
i) hybridizes with the DNA sequence shown in the appended SEQ ID No. 1 or 2 or an oligonucleotide probe prepared on the basis of said DNA sequence or on the basis of the amino acid sequence shown in SEQ ID No. 3 under the conditions defined below, and/or
ii) encodes a polypeptide reactive with an antibody reacting with at least one epitope of a polypeptide comprising the amino acid sequence shown in the appended SEQ ID No. 3, and/or
iii) encodes a polypeptide being at least 50% identical with the polypeptide having the amino acid sequence shown in the appended SEQ ID No. 3.
The nucleotide sequence shown in SEQ ID No. 1 illustrates an entire &agr;-galactosidase gene (including introns) isolated and characterized from a strain of
Aspergillus niger,
and the nucleotide sequence shown in SEQ ID No. 2 is the corresponding cDNA sequence. The nucleotide sequences are further described in the examples hereinafter. The amino acid sequence shown in SEQ ID No. 3 is deduced from the DNA sequence shown in SEQ ID No. 2 and illustrates the amino acid sequence of the
A. niger
&agr;-galactosidase enzyme including its signal peptide.
In a further aspect the present invention relates to a recombinant expression vector harbouring the DNA construct of the invention and a cell which either harbours the DNA construct or the expression vector of the invention.
A still further aspect of the present invention is a process for the production of a polypeptide exhibiting &agr;-galactosidase activity, which process comprises culturing a cell as described above harbouring a DNA construct of the invention in a suitable culture medium under conditions permitting expression of the polypeptide, and recovering the resulting polypeptide from the culture.
The polypeptide exhibiting &agr;-galactosidase activity may comprise the amino acid sequence shown in SEQ ID No. 3. or be a variant thereof. The variant may be a naturally-occurring variant derived from any source or organism, and in particular from a naturally-occurring microorganism or a mutant or derivative thereof. Furthermore, the “variant” may be a genetically engineered variant, e.g. prepared by suitably modifying a DNA sequence of the invention resulting in the addition of one or more amino acid residues to either or both the N- and C-terminal end of the polypeptide encoded by the unmodified DNA sequence, substitution of one or more amino acid residues at one or more different sites in the amino acid sequence, deletion of one or more amino acid residues at either or both ends of the polypeptide or at one or more sites in the amino acid sequence, or insertion of one or more amino acid residues at one or more sites in the amino acid sequence.
By use of the process of the invention it is possible to produce enzyme preparations having a higher content of &agr;-galactosidase than what is possible by conventional fermentation of a parent microorganism, such as
A. niger,
inherently producing the &agr;-galactosidase. Furthermore, the resulting &agr;-galactosidase preparations are essentially free from any other components derived from the parent microorganism, in particular components giving rise to undesirable enzymatic side-activities. Accordingly, by use of the process of the invention it is possible to optimize the production of &agr;-galactosidase enzyme components thereby producing an enzyme preparation with a higher specific &agr;-galactosidase activity at lower cost than what is possible by methods known in the art. At the same time the undesirable production of oxalic acid may be substantially reduced or avoided.
DETAILED DISCLOSURE OF THE INVENTION
In the DNA construct of the invention, the analogue of the DNA sequence encoding a polypeptide having &agr;-galactosidase activity may, for instance, be a subsequence of said DNA sequence, a genetically engineered modification of said sequence which may be prepared by well-known procedures, e.g
Halkier Torben
Hjort Carsten M.
Knap Inge Helmer
Kofod Lene Venke
Gregg Valeta
Novo Nordisk A S
Slobodyansky Elizabeth
Zelson Steve T.
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