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Application of a dehydrogenase in the preparation of (r)-9-(2-hydroxypropyl)adenine

A technology of dehydrogenase and adenine, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of unstable catalytic rate, unstable salt-forming catalytic rate, etc., and achieve the effect of efficient and economical production

Active Publication Date: 2021-05-07
NANJING OCEAN PHARMA TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In this route, compound IV (that is, compound II in this application) increases the solubility to achieve a catalytic rate of 1, and the catalytic rate of salt formation is still unstable, and the catalytic rate is unstable.

Method used

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  • Application of a dehydrogenase in the preparation of (r)-9-(2-hydroxypropyl)adenine
  • Application of a dehydrogenase in the preparation of (r)-9-(2-hydroxypropyl)adenine
  • Application of a dehydrogenase in the preparation of (r)-9-(2-hydroxypropyl)adenine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction of Dehydrogenase Recombinant Strain

[0029] Design the upstream and downstream primers F and R of the dehydrogenase gene (TAA_AEL) (as shown in Table 1), use the pUC57-TAA_AEL constructed by whole gene synthesis as a template, and amplify the gene containing XhoI and BamHI restriction sites by PCR. TAA_AEL. The PCR conditions were: 98°C for 3 min, 98°C for 30 s, 55°C for 90 s, 72°C for 90 s, 30 cycles. PCR amplification system: template 1.5 μL, upstream and downstream primers 1.5 μL each, sterilized double distilled water 20.5 μL, PrimerSTAR Mix 25 μL. The gel recovery kit was used to purify and recover the PCR product, and the concentration of the recovered product was checked by electrophoresis. XhoI, BamHI enzyme-digested gel recovery product (target gene TAA) and pET-30a plasmid (expression vector), gel recovery kit to purify and recover the enzyme-digested gel recovery product, gel recovery kit to digest the plasmid Purify and recover, an...

Embodiment 2

[0034] Example 2 Fermentative production of (R)-9-(2-hydroxypropyl)adenine by recombinant bacteria

[0035] (1) Preparation of dehydrogenase crude enzyme solution

[0036] The recombinant bacteria constructed in Example 1 were inoculated in 5 mL of LB medium, cultured overnight at 37 °C with shaking, and transferred to LB medium the next day according to the inoculum size of 1%, and cultured at 37 °C until OD600=0.6, adding 50 μl of 0.5 mol / L IPTG was induced at 18°C ​​for 14 h, and the bacterial cells were collected by centrifugation, washed with normal saline, and crushed to obtain the crude dehydrogenase enzyme solution.

[0037] (2) Fermentative production of (R)-9-(2-hydroxypropyl)adenine by recombinant bacteria:

[0038] Add 50 mL of dehydrogenase crude enzyme solution obtained by crushing wet cells containing 5 g of dehydrogenase and 10 g of substrate compound II into a 250 mL Erlenmeyer flask, and shake well. Subsequently, 300 μL of 100 mg / mL NADP+coenzyme mother sol...

Embodiment 3

[0041] Example 3 Fermentative production of (R)-9-(2-hydroxypropyl)adenine by recombinant bacteria

[0042] Add 50 mL of the dehydrogenase crude enzyme solution obtained by crushing wet cells containing 5 g of dehydrogenase prepared in step (1) of Example 2 and 10 g of substrate compound II into a 250 mL Erlenmeyer flask, and shake well. Subsequently, 300 μL of 100 mg / mL NADP+coenzyme mother solution (final concentration of the reaction system 0.2 mg / mL) and 5 mL of IPA (isopropanol) 5 g of isopropanol dehydrogenase enzyme powder were added. Finally, dilute the reaction system to 100 mL with 0.2M pH 6.0 PB buffer and shake well. The 100 mL reaction system was placed on a shaker at 35 °C and 220 rpm for reaction.

[0043]

[0044] Monitor the reaction process and add IPA, add 1mL IPA every 6 hours, at the same time, take a sample to monitor the reaction, take 10 μL of the reaction solution, add 200 μL of ethyl acetate to extract, centrifuge, take the supernatant for TLC det...

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Abstract

The present invention relates to the technical field of biocatalysis, especially the application of a dehydrogenase in the preparation of (R)-9-(2-hydroxypropyl) adenine, the dehydrogenase gene sequence is shown in SEQ ID NO: 1 Show. In application, the crude enzyme solution of dehydrogenase is used as a catalyst to catalyze the reduction of compound II to the target product compound I(R)‑9‑(2‑hydroxypropyl) in the presence of NADP+, coenzyme cycle enzymes and buffer Adenine, the synthetic route is:. This route obtains products with high catalytic rate, and the catalytic efficiency is as high as 2, which has high economic value.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and enzyme engineering, and the specific field is a dehydrogenase and its preparation method and application. Background technique [0002] Tenofovir disoproxil (trade name: Viread) is a very successful star product of Gilead, and (R)-9-(2-hydroxypropyl)adenine is a key intermediate for the production of tenofovir disoproxil. Tenofovir disoproxil, the full name of tenofovir disoproxil fumarate, is a new type of nucleotide reverse transcriptase inhibitor, developed by Gilead and listed in the United States in 2001, the domestic market Exclusively marketed by GlaxoSmithKline, it is mainly used to treat AIDS (HIV) and chronic hepatitis B (HBV). [0003] [0004] The following is the molecular structural formula of tenofovir disoproxil (trade name: Viread): [0005] [0006] At present, there are few reports on the preparation of (R)-9-(2-hydroxypropyl)adenine by biosynthesis. The...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12P17/18
CPCC12N9/0006C12N15/70C12P17/182
Inventor 陈本顺石利平李大伟徐春涛尹斌钱若灿叶金星董经纬刘轩豪
Owner NANJING OCEAN PHARMA TECH
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