Taxol biosynthesizing process and special culture medium

A technology for biosynthesis and paclitaxel, applied in the field of bioengineering, can solve the problems of complex large-scale culture process, failure to realize industrialized production, slow cell proliferation, etc., and achieve the effects of low cost, resolving conflicts and broad application prospects.

Inactive Publication Date: 2010-09-29
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 1991, the U.S. Department of Agriculture approved the patent (United States Patent 5,019,504.) of Christen and Gibson et al. to produce paclitaxel and its analogues by cell culture, but because of its slow cell proliferation, low paclitaxel content and complex large-scale cultivation process and other issues, has not achieved industrial production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1, the acquisition of highly active taxus cells and the biosynthesis of paclitaxel

[0025] 1. Acquisition of highly active taxus cells

[0026] Taxus basal medium: MgSO 4 ·7H 2 O 300mg, KNO 3 200mg, KH 2 PO 4 400mg, Ca(NO 3 ) 2 4H 2 O450mg, FeSO 4 27 mg, C 10 h 14 N 2 o 8 Na 2 2H 2 O 38mg, MnSO 4 ·H 2 O 1.2mg, ZnSO 4 ·7H 2 O 0.6mg, KI 0.2mg, CoCl 2 ·6H 2 O 0.03mg, H 3 BO 3 0.3mg, Thiamine Hydrochloride 8mg, Choline 0.4mg, L-cysteine ​​12mg, H 2 NCONH 2 220mg, Inositol 80mg, dilute to 1L with water, pH 5.6.

[0027] Callus induction medium: 0.5 mg / L of 6-benzyl adenine, 0.5 mg / L of α-naphthaleneacetic acid (NAA), 20 g / L of glucose, and 5 g / L of agar were added on the basis of Taxus basic medium , pH 5.6.

[0028] Subculture medium: 0.5 mg / L of 6-benzyl adenine, 0.5 mg / L of α-naphthaleneacetic acid, 20 g / L of glucose, 5 g / L of agar, and pH 5.6 were added to the basic medium of taxus.

[0029] Expansion medium: 0.5 mg / L of 6-benz...

Embodiment 2

[0048] Embodiment 2, the biosynthesis and identification of paclitaxel

[0049] Paclitaxel synthesis medium: MgSO 4 ·7H 2 O 250mg, KNO 3 250mg, KH 2 PO 4 375mg, Ca(NO 3 ) 2 4H 2 O500mg, FeSO 4 27.8 mg, C 10 h 14 N 2 o 8 Na 2 2H 2 O 37.2 mg, MnSO 4 ·H 2 O 1mg, ZnSO 4 ·7H 2 O 0.5mg, KI 0.25mg, CoCl 2 ·6H 2 O 0.025 mg, H 3 BO 3 0.25mg, Thiamine Hydrochloride 10mg, Choline 0.5mg, L-Cysteine ​​10mg, H 2 NCONH 2 200mg, Inositol 100mg, 6-benzyladenine 0.1mg / L, α-naphthaleneacetic acid 0.5mg / L, sucrose 40g / L, maltose 20g / L, combined precursor substances, combined metabolic regulator substances and combined elicitors, water Dilute to 1L, pH 5.6;

[0050] Combination precursor substances: 100mL / L tomato juice, 10mL / L pyruvic acid, 20mL / L benzoic acid, 40mL / L β-phenylalanine and 30μl / L acetic anhydride;

[0051] Combination metabolism regulating substances: composed of 5mL / L ornithine, 20mL / L tryptophan, 100mL / L glutamine and 1mL / L coenzyme A;

[0052] Comb...

Embodiment 3

[0054] Embodiment 3, the biosynthesis and identification of paclitaxel

[0055] Paclitaxel synthesis medium: MgSO 4 ·7H 2 O 200mg, KNO 3 300mg, KH 2 PO 4 350mg, Ca(NO 3 ) 2 4H 2 O550mg, FeSO 4 29 mg, C 10 h 14 N 2 o 8 Na 2 2H 2 O 36mg, MnSO 4 ·H 2 O 0.8mg, ZnSO 4 ·7H 2 O 0.4mg, KI 0.3mg, CoCl 2 ·6H 2 O 0.02mg, H 3 BO 3 0.2mg, Thiamine Hydrochloride 12mg, Choline 0.6mg, L-cysteine ​​8mg, H 2 NCONH 2 180mg, Inositol 120mg, 6-benzyladenine 0.1mg / L, α-naphthaleneacetic acid 0.5mg / L, sucrose 50g / L, maltose 50g / L, combined precursor substances, combined metabolic regulator substances and combined elicitors, water Dilute to 1L, pH 5.7;

[0056] Combination precursor substances: composed of 100mL / L tomato juice, 10mL / L pyruvic acid, 20mL / L benzoic acid, 40mL / L β-phenylalanine and 30μl / L acetic anhydride;

[0057] Combination metabolism regulating substances: composed of 5mL / L ornithine, 20mL / L tryptophan, 100mL / L glutamine and 1mL / L coenzyme A;

[0058] ...

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Abstract

The present invention discloses toxol biosynthesizing process and special culture medium. The special culture medium consists of MgSO4 .7H2O, KNO3, KH2PO4, Ca(NO3)2 .4H2O, FeSO4, C10H14N2O8Na2 .2H2O 36-38mg, MnSO4 .H2O 0.8-1.2mg, ZnSO4 .7H2O 0.4-0.6mg, KI 0.2-0.3mg, CoCl2 .6H2O 0.02-0.03mg, H3BO3, thailamine hydrochloride , Choline , L-cysteine, H2NCONH2, Inositol, 6-benzyladenine, alpha-naphthylacetic acid, cane sugar or glucose, maltose, composite precursor material, composite metabolic matter and composite inducer in certain weight proportion; and has pH 5.6-5.8. The present invention has wide application foreground in the industrial production of toxol.

Description

technical field [0001] The invention relates to a biosynthesis method of natural products and a special culture medium thereof in the field of bioengineering, in particular to a biosynthesis method of paclitaxel and a special culture medium thereof. Background technique [0002] Paclitaxel (trade name taxol) is a diterpene compound with unique anticancer activity first extracted from the bark of Pacific yew (Taxus brevifolia, Taxus.brevifolia) by American chemists Wall and Wani in 1967. . The drug prepared with this compound entered Phase III clinical trials in 1990, and was approved by the US FDA at the end of 1992 for the treatment of ovarian cancer and breast cancer that are ineffective to conventional chemotherapy. In 1995, my country's Peking Union Medical College Pharmaceutical Factory and Haikou Pharmaceutical Factory respectively obtained the approval number of the second-class new drug, becoming the second country in the world to produce paclitaxel and its injectio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/04
Inventor 郭志刚
Owner TSINGHUA UNIV
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