Method for producing tetramethylpyrazine from cheap raw materials

Abstract

The invention discloses a method for producing tetramethylpyrazine from cheap raw materials. The method comprises steps as follows: nucleotide sequences of an alpha-acetolactate synthase gene, an alpha-acetolacetate decearboxylase gene and an NADH oxidase gene are subjected to codon optimization; a gene cluster containing the three genes is obtained through splicing; the gene cluster is inserted into an expression vector, and polycistron recombinant plasmid is obtained; the polycistron recombinant plasmid is introduced into a host strain E.coli, and a gene engineering strain producing acetylmethylcarbinol is obtained; a fermentation medium containing the cheap raw materials is inoculated with the activated strain for fermentation culture, and fermentation broth containing acetylmethylcarbinol is obtained; the fermentation broth is subjected to centrifugal treatment, a supernatant is taken, diammonium hydrogen phosphate is added, acetylmethylcarbinol produced through fermentation reacts with NH4<+>, and tetramethylpyrazine is synthesized. The widely sourced and low-cost raw materials can be effectively utilized by the strain for producing a high-concentration precursor substance, namely, acetylmethylcarbinol, oxidative coenzyme NAD<+> can be regenerated effectively, yield and production efficiency of acetylmethylcarbinol can be increased, so that the production cost of acetylmethylcarbinol is effectively reduced, and production period is shortened.

Description

technical field [0001] The invention belongs to the field of biochemical industry, in particular to a method for producing tetramethylpyrazine by utilizing cheap raw materials. Background technique [0002] Tetramethylpyrazine (2,3,5,6-tetramethylpyrazine), also known as ligustrazine, is the main active alkaloid component of the rhizome of traditional Chinese medicine Chuanxiong. It has good effects such as dilating blood vessels, improving microcirculation, inhibiting platelet adhesion and aggregation and thrombosis. Pharmacological effects have certain curative effects in the treatment of cardiovascular and cerebrovascular diseases, respiratory system diseases and glomerular diseases, and have been widely used in clinical practice as a standing drug. In addition, tetramethylpyrazine occurs naturally in dairy products, soy products, cocoa, coffee, peanuts, hazelnuts, and liquor, and has a characteristic roasted and nutty aroma. Tetramethylpyrazine is also an important arom...

AI Technical Summary

Problems solved by technology

However, due to the relatively late start of research on the use of microbial fermentation to produce acetidine and its conversion into tetramethylpyrazine, the technology for producing tetramethylpyrazine through biotechnology is far from mature

[0004] Chinese patent 201010238685.5 discloses a method for producing tetramethylpyrazine and its production strains. Screened Bacillus subtilis XZ1124, Bacillus licheniformis BL-L1, MT-6 and MT-15 use glucose as carbon source and yeast extract as nitrogen When the source is used, the accumulation of acetoin in the fermenter is 30-44g / L, but the cycle is as long as 72h, and the conversion rate of glucose is too low due to the accumulation of by-products

In addition, this invention needs to use expensive yeast extract as a nitrogen source, and the carbon source glucose is high-purity sugar, which also has the problem of high cost, which further reduces the economic feasibility of this method

The composition of the medium for microbial fermentation has a great influence on the production cost, and the use of high-purity sugar (glucose) will increase the cost of raw materials

In addition, yeast powder / yeast extract is often used as a nitrogen source to ferment acetoin and tetramethylpyrazine because it is rich in nutrients such as amino acids, vitamins, and trace elements, but its use usually greatly increases production costs, such as In some lactic acid fermentations, the cost of yeast powder can account for 30% of the total fermentation cost

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