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Application of acetic anhydride serving as acyl donor to participation in DBAT (deacetyl baccatin acetyloxy transferase) enzymatic reaction

An acyl donor and enzymatic reaction technology, applied in the application field of acetic anhydride as a novel acyl donor participating in the DBAT enzymatic reaction, can solve the problem of high cost, no great economic benefit in industrialized production, and natural substrate acetyl coenzyme. A is expensive and other problems, to achieve great economic value and reduce the cost of industrial applications

Active Publication Date: 2018-11-20
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the price of the natural substrate acetyl-CoA is very expensive (505.44 yuan / mg, refer to the price on the official website of sigma company), and the cost of using it and 10-DAB to enzymatically prepare Bakatin III is very high, which is not very economical for industrial production benefit

Method used

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  • Application of acetic anhydride serving as acyl donor to participation in DBAT (deacetyl baccatin acetyloxy transferase) enzymatic reaction
  • Application of acetic anhydride serving as acyl donor to participation in DBAT (deacetyl baccatin acetyloxy transferase) enzymatic reaction
  • Application of acetic anhydride serving as acyl donor to participation in DBAT (deacetyl baccatin acetyloxy transferase) enzymatic reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] 1. Method:

[0022] 1.1 Expression and purification of recombinant protein

[0023] Pick a single colony of TBL21-DBAT transformed with the pET-DBAT plasmid and inoculate it into a 5 mL medium containing 100 μg·mL -1 Incubate overnight at 37°C in LB liquid medium with ampicillin. Pipette 1mL of bacterial liquid into 100mL of LB liquid medium, culture at 37°C and 220rpm for 2.5h to D 600nm After reaching 0.6-0.8, add IPTG with a final concentration of 0.1mmol / L, and induce culture at 20°C and 120rpm for 18h. Centrifuge at 8000rpm at 4°C for 15 minutes to collect the cells, add phosphate buffer (PBS, pH 7.4) to wash the cells twice, and finally add 10 mL of phosphate buffer to each 100 mL of the original fermentation liquid cells to resuspend, and vortex to shake Mix it thoroughly. Use an ultrasonic cell disruptor to crush the resuspended bacteria, set the ultrasonic power to 400W, and the total time is 12min, with an interval of 6s for every 6s. During the ultrasonic...

Embodiment 2

[0032] With reference to the reaction system in method 1.2 in Example 1, utilize high-performance liquid chromatography (HPLC) to detect the DBAT enzyme activity when respectively using acetyl CoA and acetic anhydride as substrates, and the liquid chromatography conditions are: C18 reverse column ( 5μm, 250mm×4.6mm), the detection wavelength is 227nm, the flow rate is 1mL / min, the column temperature is 30°C, the mobile phase is acetonitrile and water (40:60), and the injection volume is 20μL. A standard curve was prepared for the bacatine III standard under the same conditions, and the sample was quantitatively analyzed by the area external standard method.

[0033] Result analysis:

[0034] Comparison of enzyme activities under different substrates

[0035] The DBAT enzyme activity was measured separately when acetyl CoA and acetic anhydride were used as acyl donors to participate in the enzymatic reaction. The results are shown in Table 1. When acetic anhydride was used as ...

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Abstract

The invention discloses application of acetic anhydride serving as acyl donor to participation in DBAT (deacetyl baccatin acetyloxy transferase) enzymatic reaction, and belongs to the field of biomolecular catalysis. The acetic anhydride can serve as the acyl donor to replace an expensive natural substrate acetyl CoA to participate in DBAT enzymatic reaction; when the acetic anhydride serving as the acyl donor to participate in the enzymatic reaction, the enzyme activity of DBAT is increased by 2.2 times compared with that of the acetyl CoA; and important significance in subsequent baccatin III and taxol in-vitro enzymatic synthesis as well as application in a taxol anabolic synthesis channel is achieved, the industrial application cost can be greatly reduced and great economic value is brought.

Description

technical field [0001] The invention belongs to the field of biomolecular catalysis, and particularly relates to the application of acetic anhydride as a novel acyl donor in participating in DBAT enzymatic reactions. Background technique [0002] Paclitaxel is recognized as one of the best broad-spectrum anticancer drugs with high efficiency and low toxicity among existing anticancer drugs. Baccatin III is an important precursor for the chemical semisynthesis and biosynthesis of paclitaxel. 10-Deacetylbaccatin III acetoxytransferase (DBAT) can use the natural acyl donor acetyl CoA to catalyze the substrate 10-deacetyl The C10 hydroxyl group of baccatin Ⅲ (10-DAB) is acetylated to generate baccatin Ⅲ, the key precursor of paclitaxel. The in vitro enzymatic synthesis of paclitaxel using the precursor baccatin Ⅲ is the most promising and efficient method for the large-scale biological preparation of paclitaxel. protection is also of great significance. However, the price of ...

Claims

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Application Information

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IPC IPC(8): C12P17/02
CPCC12P17/02
Inventor 林俊芳欧阳萍兰魏韬郭丽琼黄佳俊
Owner SOUTH CHINA AGRI UNIV
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