Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Patent
1995-02-07
1997-02-11
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
435 691, 43525411, C12N 904, C12N 114, C12P 2106
Patent
active
056020180
DESCRIPTION:
BRIEF SUMMARY
The invention concerns a hypoglycosylated recombinant glucose oxidase (GOD EC 1.1.3.4) as well as the production thereof and its use in diagnostic tests.
There are three ways in which a protein can be provided posttranslationally with carbohydrates. A distinction is made between:
N-glycosidic linking of the carbohydrate chain to Asn
O-glycosidic linking of the carbohydrate chain to Thr or Ser
component of some membrane proteins,
the GPI anchor serves to embed them in the phospholipid membrane.
The glycosylation of proteins is described for example in:
Kukuruzinska, M. A. et al., Ann. Rev. Biochem. 56 (1987) 915-944;
Paulson, C. P., TIBS 14 (1989) 272-276;
Warren, C. E., BFE 7 (1990) 392-395;
Ballou, C. E., In: Strathern, J. N., et al., The Molecular Biology of the Yeast Saccharomyces, Cold Spring Harbor Laboratory, New York, pp. 355-360 (1982).
Kornfeld, R.; Kornfeld, S., Ann. Rev. Biochem 54 (1985) 631-664;
Tanner, W.; Lehle, L., Biochim. Biophys. Acta 906 (1987) 81-99;
Innis, M. A., In: Barr, P. J. et al., Yeast genetic engineering, Butterworths, Stoneham, Mass., pp. 233-246 (1989).
The O-glycosidic carbohydrate structures of yeast proteins consist of an unbranched mannose chain of 1-5 mannose residues. The O-glycosylation begins in the ER (transfer of the first mannose residue) and is completed in the Golgi apparatus.
N-glycosylation takes place in two steps. A core unit of N-acetylglucosamine, mannose and glucose is built on a lipid carrier intermediate and this is transferred in the ER onto Asn residues of glycoproteins. After the protein-bound core unit has been processed (cleavage of the glucose residues and a specific mannose residue in the ER), the sugar structure is elongated in the Golgi apparatus ("outer chain" glycosylation). The structure of the outer chain glycosylation is organism-specific.
Glucose oxidase (GOD EC 1.1.3.4) from Aspergillus niger is a naturally secreted N-glycosylated homodimer with a molecular weight of ca. 80 kDa per subunit (SU), 1 FAD as a cofactor/SU and one disulfide bridge/SU. GOD from A. niger has a relatively uniform carbohydrate structure (core glycosylation).
The technical production of glucose oxidase from Aspergillus niger is, however, difficult. GOD in Aspergillus niger is evidently transported into the peroxisomes (Dijken, J. P. van and Veenhuis, M., Eur. J. Appl. Microbiol. 9 (1980) 275-283) which impedes the processing. However, under certain conditions the enzyme can also be secreted into the medium (Mischak, H. et al., Appl. Microbiol. Biotech. 21 (1985) 27-31). The yield of GOD is only low in this case. For these reasons many attempts have already been made to recombinantly produce the glucose oxidase from Aspergillus niger in Saccharomyces cerevisiae. The recombinant GOD from Saccharomyces cerevisiae is more thermostable and pH-stable than the native enzyme from Aspergillus (De Baetselier A. et al., Biotechnology 9 (1991) 559-561). Although high yields of enzyme were obtained in the recombinant production, it turned out, however, that the recombinant enzyme is heterogeneous with regard to the portion of carbohydrate and to the molecular weight of 80 to 140 kDa/SU (SU=subunit) due to a non-uniform "outer chain glycosylation" of up to 150 mannose residues. The recombinant enzyme is hyperglycosylated (carbohydrate portion ca. 70% instead of 16% as in the native enzyme) (De Baetselier et al., Biotechnology 9 (1991) 559-561) and therefore has a substantially higher molecular weight (ca. 80-140 kDa/SU) than the native enzyme. This is disadvantageous for the application of GOD especially in diagnostic tests e.g. the hyperglycosylated recombinant enzyme has a lower specific activity in units per unit of weight (ca. 65 U/mg compared to 178 U/mg for the native enzyme from Aspergillus).
Kriechbaum, M. et al., FEBS Lett. 255 (1989) 63-66;
Frederick K. R. et al., J. Biol. Chem. 265 (1990) 3793-3802;
De Baetselier, A. et al., Biotechnology 9 (1991) 559-561;
Whittington, H. et al., Curr. Genet. 18 (1990) 531-536;
Rosenberg, S., WO 89/12675;
The sequence of the glucose
REFERENCES:
patent: 5094951 (1992-03-01), Rosenberg
Ballou et al. (1991) Proc. Natl. Acad. Sci. USA 88:3209-3212.
Kopetzki Erhard
Lehnert Klaus
Boehringer Mannheim GmbH
Hobbs Lisa J.
Wax Robert A.
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