Method for synthesizing D(+)beta -(3,4-Dihydroxyphenyl) lactic acid by enzymic method

A technology of enzymatic synthesis and danshensu, applied in microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve the problems of high efficiency, shortage of raw materials, low cost, etc., to solve the problems of high price and high molar conversion rate. Effect

Active Publication Date: 2018-08-21
SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to provide a new method of enzymatically synthesizing Danshensu to solv

Method used

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  • Method for synthesizing D(+)beta -(3,4-Dihydroxyphenyl) lactic acid by enzymic method
  • Method for synthesizing D(+)beta -(3,4-Dihydroxyphenyl) lactic acid by enzymic method
  • Method for synthesizing D(+)beta -(3,4-Dihydroxyphenyl) lactic acid by enzymic method

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Experimental program
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Embodiment 1

[0048] Embodiment 1 A method for enzymatically synthesizing Danshensu, the specific steps are as follows:

[0049] 1. Heterologous expression of tyrosine ammonia lyase TAL-made, phenylpyruvate reductase PPR-made and glucose dehydrogenase GDH-made:

[0050] 1. Heterologous expression and enzyme activity determination of tyrosine ammonia lyase TAL-made:

[0051] (1) Obtain TAL-made gene fragment

[0052] According to the tyrosine ammonia lyase (TAL) gene sequence (Genbank accession number: CP015288, SEQ ID NO.1), the TAL-made gene (SEQ ID NO. 2) The amino acid sequence of the gene is completely identical to that of the TAL gene, and BamHI and XhoI restriction enzyme sites are introduced upstream and downstream of the gene sequence to facilitate subsequent vector construction.

[0053] (2) Construction of pET28a-TAL-made expression vector

[0054] The synthesized TAL-made gene and the pET28a vector were digested with BamHI and XhoI respectively, and after ligation and transfor...

Embodiment 2

[0074] Except that the following steps are different, all the other steps are the same as in Example 1.

[0075] Obtain the expression cells of TAL, PPR and GDH according to the method in step 1, and add the following drug preparation catalyst systems in turn to the 250ml Erlenmeyer flask: levodopa 1.0mol, amino acid deaminase wet cells 20g / L, phenylpyruvate Reductase wet cell 20g / L, glucose dehydrogenase wet cell 20g / L, adjust the pH of the reaction system to 8.0, transform at 30°C for 16h, the obtained Danshensu content is 58.0g / L, and the molar conversion rate is greater than 58.1 %.

Embodiment 3

[0077] Except that the following steps are different, all the other steps are the same as in Example 1.

[0078] Obtain the expression cells of TAL, PPR and GDH according to the method in step 1, and add the following medicine preparation catalyst system successively in the 250ml Erlenmeyer flask: Levodopa 1.5mol, amino acid deaminase wet cell 30g / L, phenylpyruvate Reductase wet cell 30g / L, glucose dehydrogenase wet cell 30g / L, adjust the pH of the reaction system to 8.0, transform at 30°C for 16h, the content of Danshensu obtained is 60.7g / L, and the molar conversion rate is greater than 60.8 %.

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Abstract

The invention discloses a novel synthesis method for D(+)beta -(3,4-Dihydroxyphenyl) lactic acid. The method is characterized in that the beta-(3,4-dihydroxyphenyl) lacticacid is produced by catalyzing levodopa through tyrosine ammonia-lyase, phenylpyruvate reductase and glucose dehydrogenase and specifically comprises the following steps: putting 0.5 to 1.5 mol of the levodopa (3,4-dihydroxyphenyl alanine), 10 to 30 g/L of tyrosine ammonia-lyase wet cell mass, 10 to 30 g/L of phenylpyruvate reductase wet cell mass and 10 to 30 g/L of glucose dehydrogenase wet cell mass into a reactor for uniformly mixing; and adjusting the pH value to be 7.0, and carrying out catalytic reaction at the temperature of 30 DEG C for 16 to 20 hours to obtain the beta-(3,4-dihydroxyphenyl) lacticacid. Accordingto the method, the D(+)beta -(3,4-Dihydroxyphenyl) lactic acid is produced by utilizing the tyrosine ammonia-lyase, the phenylpyruvate reductase and the glucose dehydrogenase for the first time and taking the levodopa as a substrate through an enzymic method; and raw materials are saved and the pollution of chemical synthesis is avoided.

Description

technical field [0001] The invention belongs to the technical field of biosynthesis, and in particular relates to a method for enzymatically synthesizing danshensu. Background technique [0002] Danshensu, whose chemical name is β-(3,4-dihydroxyphenyl) lactic acid, is named Danshensu because it is an active ingredient of the Chinese herbal medicine Danshen. Pharmacological studies have found that Danshensu has the ability to reduce the size of myocardial infarction and relieve At the same time, it has a protective effect on myocardial ischemia and reperfusion injury, can remove exogenous oxygen free radicals, and protect the function of mitochondria; and Danshensu can significantly inhibit the aggregation of platelets, which is effective for coronary heart disease and helps the body tissue recovery and correction of serious complications such as adult respiratory distress syndrome; Clinical studies have also found that Danshensu can inhibit the synthesis of endogenous chole...

Claims

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Application Information

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IPC IPC(8): C12P7/42C12N15/70C12N1/21C12R1/19
CPCC12N9/0006C12N9/88C12N15/70C12P7/42C12Y101/01047C12Y403/01025
Inventor 张娟冯志彬段春利
Owner SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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