Method for preparing 2'-deoxyadenosine pure product by utilizing enzyme catalysis

Abstract

The invention relates to a method for preparing a 2'-deoxyadenosine pure product by utilizing enzyme catalysis, which comprises the following steps: inoculating an N-deoxyribosyltransferase recombinant escherichia coli strain into a fermentation culture medium, and carrying out induced protein expression fermentation to obtain an N-deoxyribosyltransferase crude product; weighing a deoxyribose donor and a basic group donor in proportion, adding the weighed components into a reaction system, adding an N-deoxyribose transferase crude product, and carrying out a stirring reaction to obtain a 2'-deoxyadenylase catalytic reaction solution; heating the 2'-deoxyadenosine enzyme catalytic reaction solution, centrifuging to remove insoluble substances, and taking the supernatant to obtain a 2'-deoxyadenosine-containing product crude solution; adjusting the pH value of the 2 '-deoxyadenosine-containing product crude liquid by using an alkaline solution, crystallizing the crude liquid in a low-temperature environment, and carrying out drying treatment to obtain a 2'-deoxyadenosine crude product; and adding the pre-cooled alkaline water into the 2'-deoxyadenosine crude product, stirring the mixture at low temperature for washing-off impurities, removing liquid to obtain a solid, and drying the solid. The method has effects of saving raw materials, improving preparation efficiency, and reducing production cost.

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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