A kind of method utilizing enzyme catalysis to prepare 2'-deoxyadenosine pure product

Abstract

A method for preparing pure 2'-deoxyadenosine by enzymatic catalysis, inoculating a recombinant Escherichia coli strain of N-deoxyribosyltransferase into a fermentation medium, and inducing protein expression and fermentation to obtain crude N-deoxyribosyltransferase; Weigh the deoxyribose donor and the base donor in proportion and add them to the reaction system, add N-deoxyribosyltransferase crude product and stir the reaction to obtain 2'-deoxyadenosidase catalytic reaction solution; for 2'-deoxyadenosidase Heat the catalytic reaction liquid, then centrifuge to remove insoluble matter, take the supernatant to obtain the crude product containing 2'-deoxyadenosine; adjust the pH value of the crude product containing 2'-deoxyadenosine with an alkaline solution, and place it in Crystallize in a low temperature environment and dry to obtain crude 2'-deoxyadenosine; add pre-cooled alkaline water to the crude 2'-deoxyadenosine, then stir and wash impurities at low temperature, remove the liquid, obtain a solid and dry deal with. The raw materials are saved, the preparation efficiency is improved, and the preparation cost is reduced.

Description

technical field [0001] The invention belongs to the technical field of recombinant enzyme catalysis engineering and separation and purification, and specifically relates to a method for preparing pure 2'-deoxyadenosine by enzymatic catalysis. Background technique [0002] 2'-Deoxyadenosine (2'-Deoxyadenosine, abbreviated dA), also known as "2'-deoxyribose adenine nucleoside", is a natural deoxyribonucleoside, as the genetic material deoxyribonucleic acid (DNA) Organic components that participate in the transmission of genetic information in the cells of living organisms. 2'-Deoxyadenosine can be used as a raw material for gene medicine and genetic engineering, and is also an important intermediate of many antiviral and antitumor drugs, such as didanosine (2,3-bis deoxyinosine), and fludarabine (a fluorinated nucleoside analog of adenosine vidarabine) for the treatment of chronic lymphocytic leukemia and cladribine (2-chloro-2 '-deoxyadenosine), most of the anti-herpes viru...

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Problems solved by technology

[0003] 2'-deoxyadenosine is used as an intermediate of nucleoside analogues, and its synthesis methods mainly include chemical synthesis, biological fermentation and enzyme engineering synthesis. As described in the synthetic method), the adenosine is esterified with a dialkyl oxide as an esterification agent, and the acylation of the acylating agent and the acid-binding agent is used to obtain an acylate, and the acylate is then subjected to reduction and purification treatment to obtain The product 2'-deoxyadenosine has a purity of about 99%, but the entire chemical synthesis depends on harsh reaction conditions, and it is easy to cause pollution to the environment; patent CN104178541B (a method of transforming Escherichia coli to produce 2'-deoxyadenosine method) using nucleotide phosphorylase to realize the synthesis of 2'-deoxyadenosine, but the reaction solution needs buffer salt, the substrate concentration is only 3-5mmol, and a three-step crystallization process is required, which is useful for industrial scale-up production restricted by

Patent CN105754899B (an N-deoxyribose transferase, its coding gene and its high-yield strain and application) provides Lactobacillus helsii ( Lactobacillus hilgardii ) source of N-deoxynucleoside transferase uses 2'-deoxyuridine as ribose donor to synthesize 2'-deoxyadenosine, but the substrate concentration is only 10mmol / L, and the yield is only 40-60% (see Paragraph 0057 of the specification), it is also not enough to meet the expected requirements of industrial scale-up production

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