Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving cholesterol
Patent
1996-01-05
1999-06-29
Patterson, Jr., Charles L.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving cholesterol
435 25, 435190, 536 232, C12Q 160, C12Q 126, C12N 904, C12N 1553
Patent
active
059167599
DESCRIPTION:
BRIEF SUMMARY
The invention concerns a cholesterol oxidase from Brevibacterium sterolicum, a process for the production of a recombinant cholesterol oxidase from Brevibacterium sterolicum, a suitable DNA sequence for this process which results in a cytoplasmic expression of the recombinant cholesterol oxidase in the host bacterium as well as the recombinant cholesterol oxidase obtained in this manner.
Cholesterol oxidase is of major importance for the enzymatic determination of cholesterol. It catalyzes the oxidation of cholesterol to cholesten-3-one and H.sub.2 O.sub.2. Cholesterol oxidase from various organisms such as Pseudomonas, Mycobacterium, Nocardia, Arthrobacter and Brevibacterium have already been described (T. Uwajima et al., Agr. Biol. Chem. 37 (1973), 2345-2350). All these known cholesterol oxidases are secreted proteins. The soil bacterium Brevibacterium sterolicum KY 3643 (ATCC 21387) has a particularly high activity of cholesterol oxidase. Three isoenzymes of cholesterol oxidase are known from this bacterium which differ in their isoelectric point, substrate specificity towards various steroids, affinity for cholesterol at the pH optimum and in their DNA and amino acid sequence (EP-A 0 452 112 and EP-A 560 983). Cholesterol oxidase I from Brevibacterium sterolicum has a low affinity for cholesterol (K.sub.M 1.1.times.10.sup.-3 mol/l) and can only be obtained in a low yield from Brevibacterium sterolicum. It has already been attempted to express a complete DNA coding for cholesterol oxidase I in E. coli, but this has not yet succeeded (K. Fujishiro et al., Biochem. Biophys. Res. Com. 172 (1990), 721-727, T. Ohta et al., Gene 103 (1991), 93-96). The expression of special deletion mutants of the DNA coding for cholesterol oxidase I which were fused with parts of the lac z gene also did not lead to a satisfactory expression in E. coli (T. Ohta et al., Biosci. Biotech. Biochem. 56 (1992), 1786-1791). The cloning and expression of further cholesterol oxidases from Brevibacterium sterolicum is described in EP-A 0 452 112. However, expression of these DNAs likewise does not lead to an adequate amount of active cholesterol oxidase.
The object of the invention was to provide a cholesterol oxidase with a high affinity for cholesterol in large amounts and in an active form.
This object is achieved by a cholesterol oxidase which has the amino acid sequence shown in SEQ ID NO 2. This cholesterol oxidase is obtainable from Brevibacterium sterolicum or can also be produced by recombinant means.
It has surprisingly turned out that such a cholesterol oxidase can be produced recombinantly in a large amount and in an active form. This cholesterol oxidase has a molecular weight of 60 kD, an isoelectric point of ca. 5.5 (each measured in the Phast System, Pharmacia LKB) and a K.sub.M value for cholesterol of 1.times.10.sup.-4 mol/l (in 0.5 mol/l potassium phosphate buffer pH 7.5 at 25.degree. C.) and is active in a pH range of 5.5 to 8.0.
It has turned out that this cholesterol oxidase can be obtained in a large amount and in an active form when a DNA is used for a heterologous expression which codes for a peptide with cholesterol oxidase activity and is selected from the group complementary thereto, or with fragments of this DNA sequence, hybridize with the sequences defined in a) or b) and which code for a polypeptide with the same amino acid sequence, A DNA is preferably used which has the sequence shown in SEQ ID NO 1. However, it is also possible to replace degenerated codons by other codons that code for the same amino acid in a manner familiar to a person skilled in the art. Furthermore codons coding for additional amino acids can be added at the 5' end, at the 3' end or also within the sequence shown in SEQ ID NO 1 provided the DNA variants obtained in this way hybridize with the DNA sequence shown in SEQ ID NO 1 under the usual conditions (see T. Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1989). In addition the DNA used should have one of the seque
REFERENCES:
patent: 4374930 (1983-02-01), Snoke et al.
patent: 5602017 (1997-02-01), Fujishiro et al.
Ohta et al., Biosci. Biotech. Biochem. 56:1786-1791, 1992.
Ohta et al., Gene 103:93-96, 1991.
Boehringer Mannheim GmbH
Patterson Jr. Charles L.
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