Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

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
View PDF3 Cites 2 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com