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Recombinant amidase Dt-Ami 2, encoding gene, vector, engineering strain and applications of recombinant amidase Dt-Ami 2 and engineering strain

A dt-ami2, encoding gene technology, applied in genetic engineering, recombinant DNA technology, applications, etc., can solve the problems of restriction amidase application and limited amidase

Active Publication Date: 2015-01-21
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the reported amidases are very limited, which greatly limits the application of amidases in biosynthesis

Method used

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  • Recombinant amidase Dt-Ami 2, encoding gene, vector, engineering strain and applications of recombinant amidase Dt-Ami 2 and engineering strain
  • Recombinant amidase Dt-Ami 2, encoding gene, vector, engineering strain and applications of recombinant amidase Dt-Ami 2 and engineering strain
  • Recombinant amidase Dt-Ami 2, encoding gene, vector, engineering strain and applications of recombinant amidase Dt-Ami 2 and engineering strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Acquisition of D. tsuruhatensis ZJB-05174 amidase gene

[0030] The DNA extraction kit was used to extract the whole genome DNA of D. tsuruhatensis ZJB-05174 thalline, using the DNA as a template, primer 1 ( CATATG ACCCAAGCCCTCCCCCTGC), Primer 2 ( GAATTC TCAGCCCTGCGCCGAAGCC) was used as primer for PCR amplification reaction. The amount of each component in the PCR reaction system (total volume: 50 μL): 5 μL of 10×Pfu DNA Polymerase Buffer, 1 μL of 10 mM dNTP mixture (2.5 mM each of dATP, dCTP, dGTP, and dTTP), cloning primer 1 and primer 2 at a concentration of 50 μM 1 μL of each, 1 μL of genomic DNA, 1 μL of Pfu DNA Polymerase, and 40 μL of nucleic acid-free water. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 min, followed by a temperature cycle of 94°C for 30 s, 56°C for 30 s, and 72°C for 1.5 min, a total of 30 cycles, and finally an extension at 72°C for 10 min, with a termination temperature of 4°C.

[0031] Take 50 μL ...

Embodiment 2

[0032] Example 2: Construction of recombinant expression vector pET28a-dt-ami 2

[0033] According to the analysis results of Example 1, the plasmid pMD18-T-dt-ami 2 was extracted using a plasmid extraction kit, and double-digested with restriction endonuclease NdeI / EcoRI (Fermentas), and recovered with T4 ligase (Promega ) Ligate the fragment with the commercialized vector pET-28a (Novagen) treated with the same restriction endonuclease overnight to construct the recombinant expression plasmid pET28a-dt-ami2.

Embodiment 3

[0034] Embodiment 3: Construction of engineering bacteria E.coli BL21(DE3) / pET28a-dt-ami 2

[0035] The recombinant expression vector pET28a-dt-ami 2 constructed in Example 2 was transformed into Escherichia coli BL21(DE3), spread on LB plates containing a final concentration of 50 μg / mL kanamycin, cultured overnight at 37°C, and randomly picked The clones were taken for colony PCR identification, and the positive clones were sequenced and verified. The results showed that the recombinant expression vector pET28a-dt-ami 2 was successfully transformed into the expression host E.coli BL21(DE3), and the amidase Dt-Ami 2 gene had been successfully cloned into pET NdeI and EcoRI sites at -28a.

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Abstract

The invention discloses a recombinant amidase Dt-Ami 2, an encoding gene, a vector, an engineering strain and applications of the recombinant amidase Dt-Ami 2 and the engineering strain. The invention provides a Delftia tsuruhatensis (CCTCC No. M 205115)-derived amidase gene, and a recombinant amidase WB encoded by the gene; and the amidase gene can be connected with an expression vector so as to obtain an intracellular expression recombinant plasmid containing the gene, then the intracellular expression recombinant plasmid is transferred to an escherichia coli strain so as to obtain recombinant escherichia coli, the recombinant escherichia coli contains recombinant amidase, and the recombinant amidase can be used as a catalyst for catalyzing the hydrolysis of a series of amide compounds.

Description

(1) Technical field [0001] The present invention relates to an amidase and its application, in particular to a recombinant amidase Dt-Ami 2, a coding gene, a recombinant expression vector containing the gene and a recombinant expression transformant, and the amidase or a recombinant cell containing the enzyme is Application of catalysts in catalyzing a series of amides. (2) Background technology [0002] Amidase (Amidase, EC 3.5.1.4) can catalyze the hydrolysis of amide C-N bond to generate corresponding carboxylic acid and ammonia. When there is an acyl acceptor stronger than water such as hydroxylamine or hydrazine in the reaction system, the corresponding hydroxamic acid and hydrazide are generated. Amidase has a wide range of substrates, and has excellent stereoselectivity for substrates containing chiral centers at the α position. It has great application potential in the preparation of chiral compounds such as carboxylic acid and amide derivatives. Amidase has been s...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12N15/55C12N15/74C12N1/21C12P7/52C12P7/40C12P7/44C12P7/42C12P13/00C12R1/01
CPCC12N9/80C12P7/40C12P7/42C12P7/52C12Y305/01004
Inventor 郑裕国吴哲明郑仁朝沈寅初
Owner ZHEJIANG UNIV OF TECH
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