Production process for producing antiviral medicament ribavirin through bacillus amyloliquefaciens precursor addition fermentation method

Abstract

The invention discloses a production process for producing antiviral medicament ribavirin through a bacillus amyloliquefaciens precursor TCA addition (1, 2, 4-triazole-3-carboxyformamide) fermentation method. The process comprises the following steps: establishing recombinant plasmid pBSA43-Bs816PNP which can be stably replicated in bacillus amyloliquefaciens cells and efficiently secretes and expresses purine nucleoside phosphorylase (PNPase); converting the plasmid into vernine for producing the bacillus amyloliquefaciens TA208, thereby obtaining a genetically engineered bacterium RBVM (pBSA43-Bs816PNP) with the two characteristics of high-efficiency production and high-efficiency expression of PNPase. By adoption of the genetically engineered bacterium, by taking glucose, yeast extract, bean concentrate, corn steep liquor, monopotassium phosphate, magnesium sulfate, ferrous sulfate and running water as raw materials, the precursor TCA is added for fermenting, and high-concentration ribavirin can be detected in the culture medium after the precursor is added within several hours. The process has the advantages of short fermentation period, high yield, high glucoside conversion rate, easy and convenient process, low cost, low energy consumption, slight pollution and the like.

Description

technical field [0001] The invention belongs to the field of biochemical engineering, and relates to a process for producing antiviral drug ribavirin by adding precursor fermentation with genetically engineered bacteria. Background technique [0002] Ribavirin (ribavirin), the chemical name is 1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, also known as ribavirin, is a broad-spectrum antiviral drug , has a significant inhibitory effect on a variety of DNA and RNA viruses, and has been widely used in antiviral clinical practice. [0003] There are three types of ribavirin production methods: chemical synthesis, fermentation and enzymatic methods. At present, the chemical synthesis method implemented in domestic industrial production accounts for the vast majority, which has many by-products, low yield, many operation steps, serious pollution, and high cost. [0004] The enzymatic production of ribavirin was pioneered by Witkowski et al., developed in the 1980s and 199...

AI Technical Summary

Problems solved by technology

At present, the chemical synthesis methods implemented in domestic industrial production account for the vast majority, and there are many by-products, low yields, many operating steps, serious pollution, and high costs.

[0004] The enzymatic production of ribavirin was pioneered by Witkowski et al., developed in the 1980s and 1990s, and then widely used in the production of ribavirin. However, the production cost of enzyme source cells for enzymatic production of ribavirin is relatively high. High, the bacteria must be separated before they can be put into the reaction. The culture process of the bacteria and the ribavirin production process are divided into two stages, the coupling cannot be achieved, and a high reaction temperature needs to be maintained

[0005] Adding precursor fermentation method (Japanese Laid-Open Patent No. 17830 / 1979) has the advantages of low raw material cost, wide range of sources, low energy consumption, bacterial cell culture and ribavirin production process simultaneously, etc., but so far, the yield of this method It is still at the stage of laboratory research and cannot be put into industrial production. The key reason is that due to technical bottlenecks, the current ribavirin production strains cannot efficiently produce nucleosides and efficiently express purine nucleoside phosphorylase These two characteristics

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