Short-chain dehydrogenase as well as mutant and application thereof
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A short-chain dehydrogenase and mutant technology, applied in the field of medicine, can solve the problem of not using adenine, etc., and achieve the effects of high conversion rate, stereoselectivity, green environmental protection, and high stereoselectivity
Pending Publication Date: 2022-06-03
SHENYANG PHARMA UNIVERSITY
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Problems solved by technology
[0008] In the prior art, there is no relevant report of using the short-chain dehydrogenase of the present invention to prepare (R)-(+)-9-(2-hydroxypropyl)adenine
Method used
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Embodiment 1
[0053] Example 1 Construction of mutants
[0054] Using the plasmid containing the short-chain dehydrogenase wild-type gene as a template, the mutant gene was obtained by overlapping extension PCR method.
[0061] Example 2 Construction of mutant gene recombinant plasmid
[0062] The mutant gene obtained by overlapping extension PCR was double digested with the vector plasmid pET22b, and the reaction system was as follows:
[0063]
[0064] After 4-8 hours of incubation in a 37°C incubator, use a common DNA product gel recovery kit to recover the digested nucleic acid fragments; use T4 DNA ligase to double-digest the mutated gene fragments and vectors with sticky ends The plasmid pET22b fragment was ligated (16°C for 2-6h) to obtain the recombinant plasmid.
Embodiment 3
[0065] Example 3 Preparation and transformation of competent cells E. coli Rosetta (DE3)
[0066] a) Take 1 mL of seed medium and inoculate it into 100 mL of LB liquid medium, and inoculate it with shaking at 37°C and 200 r / min for 3 hours;
[0067] b) 3000r / min, 10min to enrich the bacteria, discard the supernatant;
[0068] c) Add 400μL of pre-chilled 0.1M CaCl 2 solution, resuspend the bacterial cells, aliquot into 4 EP tubes, and take an ice bath for 30 min to obtain a competent cell suspension;
[0069] d) Add 20 μL of recombinant plasmid ligation solution to the competent cell suspension, mix gently, and take an ice bath for 30 minutes;
[0070] e) Heat shock at 42°C for 90s, ice-water bath for 3min, add 800μL of LB liquid medium, incubate at 37°C for 1-2h, shake at 200r / min for 1-2h;
[0071] f) Take 150 μL of culture solution and spread it on LB solid medium with kanamycin or ampicillin resistance respectively, and pick positive transformants after culturing at 37°C...
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Abstract
The invention belongs to the technical field of medicines, relates to short-chain dehydrogenase as well as a mutant and application thereof, in particular to application of the short-chain dehydrogenase and the mutant thereof in synthesis of (R)-(+)-9-(2-hydroxypropyl) adenine, and particularly relates to application of the short-chain dehydrogenase and the mutant thereof in catalytic reduction of a substrate 6-amino-9-(2-acetonyl) purine, synthesis of (R)-(+)-9-(2-hydroxypropyl) adenine and synthesis of (R)-(+)-9-(2-hydroxypropyl) adenine. The invention also relates to the application of the compound in the preparation of (R)-(+)-9-(2-hydroxypropyl) adenine with extremely high chiral purity. The amino acid sequence of the short-chain dehydrogenase is as shown in SEQ ID NO: 1, and mutants of the short-chain dehydrogenase are as shown in SEQ ID NO: 2-34. The short-chain dehydrogenase and the mutant thereof are used as biocatalysts, 6-amino-9-(2-acetonyl) purine (1) is used as a substrate, a coenzyme regeneration system is carried, and NAD (P) < + > is used as a coenzyme to realize enzymatic synthesis of (R)-(+)-9-(2-hydroxypropyl) adenine (2). Compared with the existing chemical synthesis process of (R)-(+)-9-(2-hydroxypropyl) adenine, the method has the advantages of high product yield, good stereoselectivity and environmental protection. The conversion rate and stereoselectivity of the compound are high, and the highest conversion rate and stereoselectivity can reach 99%.
Description
technical field [0001] The invention belongs to the technical field of medicine, relates to a short-chain dehydrogenase, a mutant thereof and applications thereof, in particular to a short-chain dehydrogenase and a mutant thereof in (R)-(+)-9-(2-hydroxypropyl ) application in adenine synthesis, be specifically related to utilizing a kind of short-chain dehydrogenase and its mutant in catalytic reduction substrate 6-amino-9-(2-acetone) purine, obtain extremely high chiral purity (R )-(+)-9-(2-hydroxypropyl)adenine. Background technique [0002] (R)-(+)-9-(2-hydroxypropyl)adenine is a key chiral intermediate for the antiviral drugs tenofovir disoproxil and tenofovir alafenamide, tenofovir Verdipiramide and tenofovir alafenamide are novel nucleotide reverse transcriptase inhibitors developed by Gilead, which are effective against a variety of viruses, mainly for the treatment of AIDS (HIV) and Chronic hepatitis B (HBV). In multiple synthetic routes of tenofovir, (R)-(+)-9-(2...
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