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Method for enzymatically producing dl-tyrosine, broad-spectrum amino acid racemase, and application

A technology of tyrosine and racemase, applied in the field of enzyme catalysis, to achieve the effect of good safety, low raw material price and high conversion rate

Active Publication Date: 2022-05-17
绵阳晟氏健康科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Acetic acid and acetic anhydride are used in the chemical racemization preparation process of DL-tyrosine, which are slightly toxic solvents, and their vapors can form explosive mixtures with air, which can cause combustion and explosion when exposed to open flames or high-energy heat, posing a greater safety hazard

Method used

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  • Method for enzymatically producing dl-tyrosine, broad-spectrum amino acid racemase, and application
  • Method for enzymatically producing dl-tyrosine, broad-spectrum amino acid racemase, and application
  • Method for enzymatically producing dl-tyrosine, broad-spectrum amino acid racemase, and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] A method for enzymatically producing DL-tyrosine, comprising the following steps:

[0042] S1. Construct the recombinant expression vector pET32a-PH0138, transform it into Escherichia coli BL21(DE3) / pG-KJE8, and obtain the recombinant engineered bacterium BBAR;

[0043] S11. According to the GenBank report, the gene sequence encoding broad-spectrum amino acid racemase (BAR) derived from Pyrococcus horikoshii OT3 was codon-optimized to be suitable for exogenous expression in E. coli strains, and the PH0138 gene with optimized sequence was synthesized (SEQ ID NO.1), clone and construct pUC57-PH0138 vector, carrying NdeI and XhoI restriction sites at both ends;

[0044] S12. Digest pUC57-PH0138 vector and pET-32a(+) plasmid with Nde I and Xho I, respectively, use 0.8% agarose gel electrophoresis, cut the gel to recover the target gene fragment and the vector backbone, according to the vector backbone: target gene =1:9 to connect and transform Escherichia coli DH5α compete...

Embodiment 2

[0057] This example is based on Example 1, and the difference from Example 1 is that the biotransformation involved in steps S3-S4 is different, specifically:

[0058] S3. Carry out 600L system conversion in a 1000L conversion tank. The amount of wet bacteria added is 5g / L, weigh 3Kg wet bacteria, resuspend the bacteria with 30L 0.1mol / L potassium phosphate buffer (pH=7.0), and use an ultrasonic pulverizer or a high-pressure homogenizer for cell destruction Obtain BAR crude enzyme liquid afterward, standby;

[0059] S4. Add 30Kg of L-tyrosine, 3Kg of potassium dihydrogen phosphate, and 8.4Kg of dipotassium hydrogen phosphate trihydrate into the 1000L conversion tank, add appropriate amount of water, start stirring, so that L-tyrosine is evenly distributed in the water phase , add BAR crude enzyme solution, then add 15.6g PLP, make up the volume with water to 600L, raise the temperature to 80°C and start stirring transformation, take samples during the transformation process t...

Embodiment 3

[0063] This example is based on Example 1, and the difference from Example 1 is that the biotransformation involved in steps S3-S4 is different, specifically:

[0064] S3. Perform 3000L system conversion in a 5000L conversion tank. The added amount of wet bacteria is 1g / L, weigh 3Kg of wet bacteria, resuspend the bacteria with 30L of 0.1mol / L potassium phosphate buffer (pH=7.0), and use an ultrasonic pulverizer or a high-pressure homogenizer for cell destruction Obtain BAR crude enzyme liquid afterward, standby;

[0065] S4. Add 150Kg of L-tyrosine, 15Kg of potassium dihydrogen phosphate, 42Kg of dipotassium hydrogen phosphate trihydrate into the 5000L conversion tank, add an appropriate amount of water, start stirring, so that L-tyrosine is evenly distributed in the water phase, Add BAR crude enzyme solution, then add 78g of PLP, make up the volume with water to 3000L, raise the temperature to 80°C and start stirring transformation, take samples during the transformation pro...

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Abstract

The invention discloses a method for enzymatically producing DL-tyrosine and a broad-spectrum amino acid racemase and its application. The method for enzymatically producing DL-tyrosine comprises the following steps: S1, constructing a recombinant expression vector pET32a-PH0138, Transform into Escherichia coli BL21(DE3) / pG‑KJE8 to obtain the recombinant engineered bacteria BBAR; S2, ferment the recombinant engineered bacteria BBAR expressing BAR enzyme to obtain wet cells; S3, suspend the wet cells and perform crushing treatment, crush The solution is a crude enzyme solution containing a broad-spectrum amino acid racemase; S4, adding L-tyrosine and PLP to the crude enzyme solution obtained in step S3, stirring and converting, until the specific rotation is 0°, the reaction is terminated, and the obtained DL‑Tyrosine in water. The method has the advantages of low raw material price, high conversion rate, simple post-treatment, high product quality, and is suitable for large-scale industrial production.

Description

technical field [0001] The invention relates to the technical field of enzyme catalysis, in particular to a method for enzymatically producing DL-tyrosine, a broad-spectrum amino acid racemase and its application. Background technique [0002] DL-Tyrosine (DL-Tyrosine, DL-Tyr) is a racemate of L-tyrosine, which is widely used in the fields of medicine and chemical industry. In recent years, with the in-depth development and research of drugs, DL-tyrosine has been used as a raw material for drug synthesis, such as therapeutic drugs for liver disease and heart disease, amino acid infusion and medical clinical research. In addition, DL-tyrosine is used as a raw material for preparing D-tyrosine, and D-tyrosine is a non-protein source chiral amino acid, which has a very wide application in the synthesis of chiral drugs. For example, atosiban synthesized with D-tyrosine as a chiral intermediate is an important tocolytic, and the synthesized deacetylanisomycin has antiprotozoal, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/90
CPCC12P13/225C12N9/90C12N15/70C12Y501/0101
Inventor 李晚军曾帅刘鹏张瑞
Owner 绵阳晟氏健康科技有限公司
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