Engineering bacteria for producing D-tryptophan and construction method and purpose of producing D-tryptophan

A construction method and tryptophan technology, applied in the field of D-tryptophan production, can solve the problems of unstable chemical properties of N-acetyl-L-tryptophan, multiple materials, loss, etc., and improve yield and product quality , The effect of simplifying the production process and saving fermentation costs

Inactive Publication Date: 2018-08-03
天津博瑞威生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

N-acetyl-L-tryptophan can be reused by generating N-acetyl-DL-tryptophan through chemical racemization, but due to the unstable chemical properties of N-acetyl-L-tryptophan, it is easy to be oxidized. The racemization process will generate a large number of by-products, resulting in more material loss, and the quality of the obtained N-acetyl-DL-tryptophan is also difficult to meet the requirements for reuse
N-acetyl-amino acid racemase is an enzyme that specifically racemizes N-acetyl-amino acids. The enzymatic racemization process of N-acetyl-amino acid racemase replaces the chemical racemization process, which can efficiently convert N- Acetyl-L-tryptophan is converted into N-acetyl-DL-tryptophan, but due to the low activity of this enzyme in nature, it is difficult to apply this enzyme to production at present

Method used

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  • Engineering bacteria for producing D-tryptophan and construction method and purpose of producing D-tryptophan
  • Engineering bacteria for producing D-tryptophan and construction method and purpose of producing D-tryptophan
  • Engineering bacteria for producing D-tryptophan and construction method and purpose of producing D-tryptophan

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Cloning of an acylated amino acid racemase gene and an N-acetyl-D-amino acid acyl hydrolase gene.

[0018] 1. First, synthesize the acylated amino acid racemase gene and the N-acetyl-D-amino acid acyl hydrolase gene with reference to the sequence of the gene database and design primers:

[0019] The gene sequence of acylated amino acid racemase was synthesized in Shanghai Sangon Biotechnology Co., Ltd. with reference to GenBank: D30738.1.

[0020] The gene sequence of N-acetyl-D-amino acid acyl hydrolase was synthesized in Shanghai Sangon Biotechnology Co., Ltd. according to GenBank: D45918.1.

[0021] Primers were designed as follows:

[0022] F2: catat catatg tcccaatccgattc (Add ndeI site) SEQ ID NO.3

[0023] R2: catat ggatcc ctatcaggcggccgt (add bamhI site) SEQ ID NO.4

[0024] F1: catat catatg aaactcagcggtgtg (Add ndeI site) SEQ ID NO.5

[0025] R1: catat ggatcc ctactacgaaccga (add bamhI site) SEQ ID NO.6

[0026] Primers F1 and R1 are used to amplify ...

Embodiment 2

[0048] The error-prone PCR product of the acylated amino acid racemase gene and the error-prone PCR product of the N-acetyl-D-amino acid acyl hydrolase gene obtained in Example 1 were respectively linked to the pET28a vector.

[0049] 1. Use the restriction endonucleases ndeI and bamhI to digest the product of step 2 of Example 1 respectively, and the reaction system is as follows:

[0050]

[0051] The reaction conditions were 37°C for 3 hours. Use the Shanghai Sangong SanPrep Column PCR Product Purification Kit to recover the digested product.

[0052] The product of Step 3 of Example 1 was used instead of the product of Step 2 of Example 1 in this example to perform the same operation to recover the enzyme-cleaved product.

[0053] 2. Digest the pET28a vector with restriction enzymes ndeI and bamhI. The reaction system is as follows:

[0054]

[0055]

[0056] The reaction conditions were 37°C for 3 hours. Shanghai Sangong SanPrep Column PCR Product Purification...

Embodiment 3

[0063] Example 3: Expression of acylated amino acid racemase and N-acetyl-D-amino acid acyl hydrolase, and selection of optimal mutant genes.

[0064]The expression vector obtained in step 3 of Example 2 was transformed into competent cells of Escherichia coli BL21 strain and the optimal mutant sequence was selected. Proceed as follows:

[0065] 1.

[0066] 1.1 Take 500ul of BL21 bacteria liquid, inoculate it in 50ml of LB liquid medium, cultivate at 37℃, 180r / min for 2-3h until OD600 reaches about 0.5.

[0067] 1.2. Transfer the bacterial solution to a 50ml centrifuge tube and place it on ice for 10 minutes.

[0068] 1. Centrifuge at 4000rpm for 10min at 3 and 4°C to recover the cells.

[0069] 1.4. Discard the culture medium and invert for 1 min.

[0070] 1.5. Add 10ml of pre-cooled 0.1mol / L CaCl to every 50ml of bacteria produced by the bacteria solution 2 , resuspend the cells, and place on ice for 10 min.

[0071] 1.6, 4°C, centrifuge at 4000rpm for 10min to recover...

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Abstract

The invention discloses engineering bacteria for producing D-tryptophan and a construction method and a purpose of producing D-tryptophan. The construction method comprises the following steps: transferring an acylated amino acid racemase mutant gene and a N-acetyl-D-amino acid acyl hydrolase mutant gene in escherichia coli, and constructing the engineering bacteria for producing D-tryptophan. Theconstruction method optimizes the N-acetyl-D-amino acid acyl hydrolase mutant gene and the acylated amino acid racemase mutant gene, the acyl hydrolysis activity of the N-acetyl-D-tryptophan by the N-acetyl-D-amino acid acyl hydrolase and the racemization activity of the N-acetyl-L tryptophan by the acylated amino acid racemase are increased, and the output and the product quality of the D-tryptophan are increased. Two types of enzymes can be expressed by the same engineering bacterial strain, the fermentation cost is saved, and the production technology is simplified.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to an engineering bacterium producing D-tryptophan, a construction method and an application for producing D-tryptophan. Background technique [0002] As a non-protein optically active amino acid, D-tryptophan has special physiological properties and has certain value in food, feed industry and agriculture. It can be used as non-nutritive sweetener, feed additive and plant growth agent. Especially in the pharmaceutical industry, D-tryptophan is an important synthetic precursor for anticancer agents and immunosuppressants. D-tryptophan is the raw material of tadalafil, a drug for the treatment of erectile dysfunction (ED) in men. Its chemical name is D-α-amino-β-indolylpyruvate. [0003] Existing methods for preparing D-tryptophan include a membrane splitting method, an aminoacylase splitting method, an enzymatic degradation method, an amidase method, and a hydantoinase method. Among t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/22C12N15/70C12N1/21C12R1/19
CPCC12N9/80C12N9/90C12P13/227C12Y305/01014C12Y501/0101
Inventor 闫博田方焦华阳
Owner 天津博瑞威生物医药科技有限公司
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