Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1992-01-29
1994-05-17
Wax, Robert A.
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
435 11, 435 19, 435 691, 435 712, 4351721, 435197, 935 10, 935 14, 935 29, 935 56, 935 72, 536 232, C12N 1500, C12N 1555, C12N 918, C12Q 144, C12Q 160
Patent
active
053127439
DESCRIPTION:
BRIEF SUMMARY
The invention concerns a process for the preparation of cholesterol esterases with changed substrate specificity, with cholesterol esterases obtained in this way, as well as with their use for the enzymatic determination of cholesterol.
The determination of cholesterol in serum is an important parameter in the diagnosis of arteriosclerosis. The cholesterol present in serum is present in very heterogeneous compounds, whereby about 70 to 80% of the cholesterol is esterified with fatty acids of varying length. Thus, the exact determination of the cholesterol level depends upon how completely the cholesterol esters present are cleaved to free cholesterol. In clinical and medical practice, the enzymatic cleavage of the cholesterol esters by means of cholesterol esterase has proven to be the simplest process. However, it is a disadvantage of this determination that different cholesterol esters are cleaved by particular cholesterol esterases with differing specificity. Therefore, it is an object of the invention to make available cholesterol esterases with changed substrate specificity and improved activity.
According to the invention, this task is solved by a process for the preparation of cholesterol esterases with changed substrate specificity by cloning of a cholesterol esterase gene, the active site of which has the sequence -Gly-His-Ser-X-Gly-, wherein X signifies an amino acid, into a vector, transformation of a micro-organism with this vector and expression of the cholesterol esterase gene, which is characterised in that one exchanges the amino acid X of the active site for another amino acid by mutagenesis.
The invention concerns cholesterol esterases which, as active site (i.e. as region responsible for the enzymatic activity) contains the amino acid sequence -Gly-His-Ser-X-Gly-, whereby X signifies any desired amino acid. Such cholesterol esterases with different amino acid residues X are known (see e.g. Table 1). However, these cholesterol esterases originate from different organisms and, consequently, also differ in the nucleotide sequence of the genes coding therefor. According to the present invention, a process is now made available for obtaining a batch of different cholesterol esterases from a single cholesterol esterase in a simple and rapid manner by mutagenesis of the amino acid X of the active site, which only differ in the amino acid X. However, by the change of the nucleotide sequences in the cholesterol esterase gene, at the same time an additional change of the peptide frame outside of the active site can also take place insofar as this is desired.
Surprisingly, it has been found that the substrate specificity of cholesterol esterases, the active site of which has the amino acid sequence -Gly-His-Ser-X-Gly-, can be changed in that one exchanges the amino acid X in the active site by another amino acid. This is expediently achieved by mutation of the codon which codes for the amino acid X in the active site. The mutagenesis is preferably carried out in an objective manner, namely, with the use of oligonucleotides. One cuts out from the cholesterol esterase gene an oligonucleotide which contains the coding region for the active site and, in place thereof, ligates a new oligonucleotide into the gene which, in the place of X, codes for another amino acid.
The new oligonucleotide is preferably synthesised by chemical means. The synthesis is expediently carried out on a solid phase. Such solid phase methods are described summarily in E.-L. Winnacker, Gene und Klone, VCH Verlagsgesellschaft Weinheim (1985), pages 44 et seq. The new oligonucleotide is expediently prepared according to the amidite process, especially the phosphoramidite process.
Alternatively to the chemical synthesis, it has also proved to be expedient to obtain the new oligonucleotide from genes or gene of building blocks of other lipase/cholesterol esterases. One usually proceeds in a manner such that one selects the gene from such a lipase/cholesterol esterase which already displays the substrate specificity for the desired e
REFERENCES:
G. Gibney et al. "Mutagenesis of Essential Functional Residues in . . . " Proc. Natl. Acad. Sci. 87: 7546-7550 (Oct. 1990).
T. Uwajima et al. "Purification and Properties of Cholesterol Esterase . . . " Agric. Biol. Chem. 40(10) 1957-1964 (1976).
A. J. Poulose et al. "Alteration of Substrate Specificity of a Lipase . . . " Abstract Papers of the Chemical Congress, North America BTEC 47(1988).
W. H. Rastetter "Enzyme Engineering: Applications and Promise" Trends in Biotechnology 1(3) 80-84 (1983).
R. M. Tel et al. "Incomplete Hydrolysis of Cholestryl Estes . . . " J. Clinical Chem., and Clin. Biochem. 18(10) 595-601 (Oct. 1980) (See Medline Abstract).
W. Kugimiya et al. "Molecular Cloning and Nucleotide Sequence . . . " Biochem. Biophys. Res. Commun. 141(1) 185-190 (Nov. 1986).
Fischer Stephan
Hanke Christian
Schumacher Gunther
Boehringer Mannheim GmbH
Prouty Rebecca
Wax Robert A.
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