A method for enzymatically synthesizing Danshensu
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Embodiment 1
[0048] Embodiment 1 A kind of method for enzymatic synthesis of Danshensu, concrete 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 assay of tyrosine ammonia lyase TAL-made:
[0051] (1) Obtaining TAL-made gene fragments
[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.1) was synthesized by the company after codon optimization suitable for expression in E. coli. 2), the amino acid sequence of the gene is exactly the same as that of the TAL gene, and restriction sites of BamHI and XhoI 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 pET28a vector ...
Embodiment 2
[0074] Except that the following steps are different, the remaining steps are the same as those in Example 1.
[0075] According to the method in step 1, the expression cells of TAL, PPR and GDH were obtained, and the following drugs were added to the 250ml conical flask to prepare the catalytic system: 1.0 mol of levodopa, 20 g / L of amino acid deaminase wet cells, phenylpyruvate Reductase wet cell 20g / L, glucose dehydrogenase wet cell 20g / L, adjusting the pH of the reaction system to 8.0, and transforming at 30°C for 16h, the obtained Danshensu content was 58.0g / L, and the molar conversion rate was greater than 58.1 %.
Embodiment 3
[0077] Except that the following steps are different, the remaining steps are the same as those in Example 1.
[0078] According to the method in step 1, the expression cells of TAL, PPR and GDH were obtained, and the following drugs were added to the 250ml conical flask to prepare the catalytic system: 1.5mol of levodopa, 30g / L of amino acid deaminase wet cells, phenylpyruvate Reductase wet cell 30g / L, glucose dehydrogenase wet cell 30g / L, adjusting the pH of the reaction system to 8.0, and transforming at 30°C for 16h, the obtained Danshensu content was 60.7g / L, and the molar conversion rate was greater than 60.8 %.
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