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Genetically engineered bacterium for efficiently expressing high-molecular weight nitrile hydratase and application of genetically engineered bacterium

A nitrile hydratase, high molecular weight technology, applied in bacteria, lyase, microorganism-based methods, etc., can solve the problem of not being used in a balanced or random manner, and achieve the effects of reducing production costs, high-efficiency expression, and improving product purity.

Active Publication Date: 2015-08-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Escherichia coli does not use every codon equally to encode amino acids. For a certain amino acid, E. coli usually tends to use more of one or more of its corresponding synonymous codons, Rather than using each synonymous codon equally or randomly to encode it, codon usage in E. coli is biased

Method used

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  • Genetically engineered bacterium for efficiently expressing high-molecular weight nitrile hydratase and application of genetically engineered bacterium
  • Genetically engineered bacterium for efficiently expressing high-molecular weight nitrile hydratase and application of genetically engineered bacterium
  • Genetically engineered bacterium for efficiently expressing high-molecular weight nitrile hydratase and application of genetically engineered bacterium

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Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1: Acquisition of high molecular weight nitrile hydratase gene nhhB(rbs)A(rbs)G

[0036] (1) Firstly, the original high molecular weight nitrile hydratase gene (bag) (Genbank ID: X86737.1) derived from Rhodococcus rhodochrous J1 was obtained by searching the GenBank database in NCBI.

[0037] (2) Among the sequences obtained in the database, the sequences between B and A of the nitrile hydratase gene and between A and G were replaced with the SD sequence (AAGGA) and spacer sequence (GATATAGAT) of Escherichia coli.

[0038] (3) The nitrile hydratase gene sequence designed through transformation in the previous step is optimized according to the codon preference of Escherichia coli to obtain the sequence of the nitrile hydratase target gene nhhB(rbs)A(rbs)G, such as SEQ ID NO. 6, and enzyme cutting sites Nde Ⅰ and EcoR Ⅰ were added to the upstream and downstream ends of the gene fragment respectively.

[0039] (4) Send the gene sequence finally obtained in the...

Embodiment 2

[0040] Embodiment 2: Construction of expression vector pET24a(+)-nhhB(rbs)A(rbs)G

[0041] The chemically synthesized target gene fragment was cloned into the Nde I and EcoR I restriction sites of pET24a(+), transformed into E.coli JM109, and recombinants were screened on the 2YT plate containing kanamycin resistance. Extract the recombinant plasmid and perform PCR verification (eg figure 1 shown), a 1645bp electrophoresis band was obtained, which was the same size as the target gene; the recombinant plasmid was digested with EcoR Ⅰ to obtain a 6910bp electrophoresis band (such as figure 2 shown), indicating that the pET24a(+)-nhhB(rbs)A(rbs)G recombinant plasmid was successfully constructed.

Embodiment 3

[0042] Example 3: Induced expression of BL21 / pET24a(+)-nhhB(rbs)A(rbs)G

[0043] The recombinant plasmid pET24a(+)-nhhB(rbs)A(rbs)G was transformed into E. coli BL21 to obtain BL21 / pET24a(+)-nhhB(rbs)A(rbs)G. BL21 / pET24a(+)-nhhB(rbs)A(rbs)G recombinant single colony was inoculated into 5mL of 2YT medium and cultured at 37°C for 10h, then 1mL of the culture solution was transferred to 100mL of 2YT medium and cultured at 37°C until OD 600 When the concentration is 0.6-0.8, add IPTG and 0.1g / L CoCl to a final concentration of 0.4mM 2 ·6H 2 O, after 16 hours of induced expression at 24°C, the bacteria were collected by centrifugation, and the expression was detected by SDS-PAGE. The results are shown in image 3 , which is consistent with the theory that the α subunit is 26KDa and the β subunit is 29KDa.

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Abstract

The invention discloses a genetically engineered bacterium for efficiently expressing high-molecular weight nitrile hydratase and an application of the genetically engineered bacterium, and belongs to the technical field of microbiological genetic engineering. The high-molecular weight nitrile hydratase gene nhhB (rbs) A (rbs) G is obtained from a nitrile hydratase gene bag based on an R.rhodochrous J1 source through codon optimization, express element transformation and chemical synthesis. The whole length of the gene is 1645bp; the gene is encoded with 533 amino acids and can be successfully expressed in escherichia coli; the method is efficient and safe; 96.7U / mL soluble nitrile hydratase can be obtained by inducing at 24 DEG C for 16 hours; the specific enzyme activity can be up to 234U / mg after purification; and the method is beneficial to extraction and purification of the nitrile hydratase and efficient enzymatic synthesis of amide substances in the production process.

Description

technical field [0001] The invention relates to a genetically engineered bacterium for efficiently expressing high-molecular-weight nitrile hydratase and its application, belonging to the technical field of microbial genetic engineering. Background technique [0002] Nitrile hydratase (NHase for short, EC4.2.1.84) is a metalloenzyme capable of hydrating nitriles into more valuable amides. NHase is widely distributed in microorganisms, and has been found in Rhodococcus, Pseudomonas, Pseudonocardia, Bacillus, Nocardia ( The gene sequence of nitrile hydratase can be obtained in Nocardia) and Comamonas. Rhodococcus rhodochrous J1, as the third-generation production strain, has been widely used in the industrial production of acrylamide. The annual output of acrylamide produced by this method is nearly one million tons, nearly one-third of the total output. Rhodococcus is mainly used to convert acrylamide to nitrile hydratase. After induction, two nitrile hydratases with diff...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N9/88C12P13/02C12R1/19
CPCC12N1/20C12N9/88C12N15/70C12P13/02
Inventor 周哲敏张晓欢崔文璟
Owner JIANGNAN UNIV
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