The method of obtaining baccatin and/or derivatives baccatin 10 - hydroxylase-about-acetyltransferase for its implementation, the method of obtaining this acetyltransferase and method for producing taxol and/or derivatives of taxol

 

The invention relates to biotechnology. The method of obtaining baccatin and/or derivatives baccatin provides for the conversion of 10-deacetylbaccatin or derived 10-deacetylbaccatin in the presence of 10-hydroxylase-About-acetyltransferase and donor of acetyl groups. As the enzyme uses acetyltransferase with a molecular mass 70-72 kDa, determined by electrophoresis in polyacrylamide body in the presence of sodium dodecyl sulfate, which is obtained from cell cultures of Taxus chinensis. The invention provides a high regiospecificity in respect of acetylation at position 10, make up more than 99%. 4 C. and 9 C.p. f-crystals, 5 PL.

Description text in facsimile form (see graphic part))

Claims

1. The method of obtaining baccatin and/or derivatives baccatin, characterized in that the 10-deacetylbaccatin or derived 10-deacetylbaccatin transform in the presence of the selected enzyme and donor of acetyl groups, and as enzyme use 10-hydroxylase-O-acetyltransferase isolated from Taxus chinensis, characterized by the following properties: substrate specificity; acetylase 10-deacetylbaccatin the mass; 70-72 kDa when determining the method of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; isoelectric point; at pH of 5.4 to 5.8; constant Michaelis-Menten;m55-65 μm to acetyl-COA; the pH optimum; from 5 to 11 (in Tris buffer at 35°C for 25 min, the substrate 10 desacetyldiltiazem).

2. The method according to p. 1, characterized in that baccatin-W obtained from 10-deacetylbaccatin-W.

3. The method according to p. 1, characterized in that 14-hydroxyacetic-W obtained from 14-hydroxy-10-deacetylbaccatin-W.

4. The method according to p. 1, characterized in that taxonomy To get from 10-deacetyltaxol C.

5. The method according to any of paragraphs.1-4, characterized in that the transformation is carried out in the presence of acetyl-COA as the acetyl donor group.

6. 10-hydroxylase-About-acetyltransferase isolated from Taxus chinensis, characterized by the following properties: substrate specificity; acetylase 10-deacetylbaccatin-sh selectively at the position 10 in the presence of the donor of the acetyl group, in particular acetyl-COA; molecular weight; 70-72 kDa when determining the method of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; isoelectric point; at pH of 5.4 to 5.8; constant Michaelis-Menten; Taxowneny).

7. The method of producing the enzyme in p. 6, characterized in that it is isolated from cell culture of Taxus chinensis by cleaning and after each cleaning faction, which is the enzyme by adding 10-deacetylbaccatin or derived 10-deacetylbaccatin and donor of acetyl groups and find the resulting product acetylation.

8. The method according to p. 7, characterized in that the cleaning methods include the use of HighQ-column.

9. The method according to p. 7 or 8, characterized in that to detect containing the enzyme fractions using 10-deacetylbaccatin-III or 10-deacetyltaxol C.

10. The method according to any of paragraphs.7-9, characterized in that use acetyl-COA as the acetyl donor group.

11. The method according to any of paragraphs.7-10, characterized in that the detection of the formed product of the acetylation is carried out by radioactive labeling.

12. The method according to any of paragraphs.7-10, characterized in that the detection of the formed product of the acetylation is carried out by labeling with heavy isotope.

13. The method of producing Taxol and/or derivatives of Taxol, wherein the first receive baccatin or derived baccatin according to any one of paragraphs.1-5 and by the esterification of guy who proizvodnje Taxol.

 

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< / BR>
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< / BR>
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< / BR>
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< / BR>
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