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Engineering bacteria with high tolerance to acrylamide and acrylonitrile for producing nitrile hydratase and application thereof

A technology of acrylamide and nitrile hydratase, which is applied in the direction of bacteria, biochemical equipment and methods, and the use of vectors to introduce foreign genetic materials. Increased, high-concentration effects

Active Publication Date: 2010-11-24
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the process of microbial production of acrylamide, another major problem that restricts production efficiency is the poor tolerance of the product of the enzyme-producing cells. The product concentration at the end of the hydration reaction is usually 280-330g / L, which must be further concentrated through a high-temperature concentration process. 400g / L or 500g / L aqueous acrylamide product and 600g / L crystallization mother liquor can be obtained

Method used

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  • Engineering bacteria with high tolerance to acrylamide and acrylonitrile for producing nitrile hydratase and application thereof
  • Engineering bacteria with high tolerance to acrylamide and acrylonitrile for producing nitrile hydratase and application thereof
  • Engineering bacteria with high tolerance to acrylamide and acrylonitrile for producing nitrile hydratase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Cloning of the sigA gene and upstream sequence encoding sigma 70 in Rhodococcus ruber TH3 (amdA-)

[0052] (1) Cultivation of Rhodococcus ruber TH3(amdA-) and extraction of total genomic DNA

[0053] Pick Rhodococcus ruber TH3 (amdA-) with a sterile inoculation loop (preserved in this laboratory, and preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee on February 27, 2008, and the strain preservation registration number is CGMCC No.2381) single colony, inoculated in 50mL LB liquid fermentation medium (the composition and ratio of LB liquid fermentation medium are: glucose 10g / L; yeast extract 5g / L; peptone 10g / L; sodium chloride 10g / L L, the rest is water) in a conical flask; then cultivated in a shaker at a temperature of 28°C and a rotation speed of 200rpm for 72 hours, and then centrifuged at 10000rpm for 5min to collect bacterial precipitates, resuspended in 10ml sterile water, and placed at 10000rp...

Embodiment 2

[0080] Example 2 Construction of sigA gene random mutation library

[0081] (1) Construction of recombinant plasmids for random mutation

[0082] According to the sequence information of the sigA gene fragment obtained in Example 1, primers EsigAup and HsigAdown whose amplified product contains the full-length and upstream sequence of the sigA gene were designed. The above primer sequences are as follows:

[0083] EsigAup: 5′-TCA GAATTC TCGTTACAATGGTGCACAG-3' (the underline is the EcoRI restriction site); (SEQ ID No: 12)

[0084] HsigAdown: 5′-ACA AAGCTT CGCGTTCAGTCCAGGTAGT-3' (the underline is the HindIII restriction site). (SEQ ID No: 13)

[0085] Using Rhodococcus R. ruber TH3 (amdA-) genomic DNA as a template, and using EsigAup and HsigAdown as upstream and downstream primers respectively, PCR amplification was carried out. The reaction system and amplification conditions were the same as step (2) of Example 1. The amplified product (the product contains the sigA g...

Embodiment 3

[0088] Example 3 Screening of Propionamide Growth Tolerance Enhanced Mutants

[0089]The sigA gene random mutation plasmid library constructed in Example 2 was transformed by electroporation into the competent cells of the engineered Rhodococcus R.ruber TH3 (amdA-) whose amidase gene was knocked out, with kanamycin resistance and lethal acrylamide The acrylamide tolerance enhanced mutants were screened under the condition of concentration (4mg / ml). At the same time, the recombinant plasmid pNV18A containing the endogenous natural sigA gene prepared in step (2) of Example 2 was also transferred into R. ruberTH3 (amdA-) to construct strain TH3A as a control reference strain. Wherein, the preparation of Rhodococcus competent cells adopts the preparation method of Gram-positive bacteria competent cells in "Molecular Cloning Guidelines" (J. Sambrook, D.W. Russell). Take 1 μl of the purified plasmid in a 1.5ml centrifuge tube, and place it together with a 0.1CM electroporation cup ...

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Abstract

The invention discloses an engineering bacteria with high tolerance to acrylamide and acrylonitrile for producing nitrile hydratase, a construction method for the engineering bacteria and application of the engineering bacteria in producing the acrylamide by a microorganism method, and belongs to the technical field of industrial microbiology. A bacterial strain for producing the acrylamide from the nitrile hydratase with high yield is used as a parent strain; the parent strain additionally expresses mutational RNA polymerase sigma factor gene in the engineering bacterium; and the engineering bacteria are Rhodococcus ruber TH-5(amdA-) / pNV18AM CGMCC No.3725 preferably. The tolerance of the engineering bacteria to the acrylonitrile and the acrylamide is increased by 1 time compared with that of the parent strain; and the half-life period of the nitrile hydratase is prolonged by 1.3 times when the engineering bacteria are immersed in 40 percent solution of acrylamide. The invention also discloses the construction method for the engineering bacteria and the application of the enginnering bacteria in producing the acrylamide. In the invention, the acrylamide is produced from the bacterial strain, the concentration of a batch hydration product is high, reaction batches are increased, and concentration load is reduced, so that the production cost is reduced obviously.

Description

technical field [0001] The invention belongs to the technical field of industrial microbes, and in particular relates to a nitrile hydratase-producing engineering bacterium with high tolerance to acrylamide and acrylonitrile, a construction method of the engineering bacterium and its application in the production of acrylamide by a microbial method. Background technique [0002] Polyacrylamide (PAM) is known as "all-industry additive", and it has a very wide range of applications in industrial production fields such as tertiary oil recovery, water treatment, papermaking, mining, metallurgy, coal washing, and manufacturing superabsorbent resins. The monomer of polyacrylamide-acrylamide (Acrylamide, the molecular formula is C 3 h 5 NO, the structural formula is H 2 C=CHCONH 2 ) is generally produced with acrylonitrile as raw material, which is formed by catalytic hydration. The nitrile hydratase produced by microorganisms can efficiently catalyze the hydration of acrylonit...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63C12N1/21C12P13/02C12R1/01
Inventor 于慧敏马玉超沈忠耀
Owner TSINGHUA UNIV
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