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Nitrilase, gene sequence and application method thereof

A nitrilase and sequence technology, applied in the field of enzyme genetic engineering and enzyme engineering, can solve the problems of cloning, expression, and few reports of enzymatic properties, and achieve the advantages of short fermentation period, high catalytic efficiency and high nitrilase activity. Effect

Active Publication Date: 2013-02-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are not many reports on the cloning, expression, and enzymatic properties of the nitrilase gene of wild bacteria used to catalyze the conversion of 3-cyanopyridine into nicotinic acid.
At present, there is no research on the cloning and expression of Pseudomonas putida nitrilase gene in China

Method used

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  • Nitrilase, gene sequence and application method thereof
  • Nitrilase, gene sequence and application method thereof
  • Nitrilase, gene sequence and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] This example illustrates the method for cloning the partial sequence (Nit M) of the nitrilase gene.

[0039] Pseudomonas putida ( Pseudomonas putida ) XY4 (preservation number CGMCC No.3830) cloning of Nit M sequence: the Pseudomonas The amino acid sequence of nitrilase was homologously compared, and the primers were designed by CODEHOP software. After further analysis by DNAMAN software, a pair of degenerate primers P1 and P2 were designed to target Pseudomonas putida ( Pseudomonas putida ) XY4 (Accession No. CGMCC No.3830) The total DNA was used as a template to clone the Nit M sequence:

[0040] P1: 5'-GGCGGAAGCTGaarccnacnca-3'

[0041] P2: 5'-ACGTCGGGCCTGswrtartgncc-3'

[0042] The PCR reaction was carried out in a 50 μL system, and the reaction conditions were pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 40 s, and extension at 72°C for 40 s, a total of 30 cycles; and final extension at 72°C for 10 min...

Embodiment 2

[0044] This example illustrates the cloning method of the 5' end sequence (Nit 5') of the nitrilase gene.

[0045] Pseudomonas putida ( Pseudomonas putida ) Cloning of the Nit 5' sequence of XY4 (Deposit No. CGMCC No.3830): According to the isolation and purification of Pseudomonas putida ( Pseudomonas putida ) XY4 (preservation number CGMCC No.3830) N-terminal sequence measured by the nitrilase produced by designing degenerate primer P3, and primer P4 was designed according to the Nit M sequence obtained above, and Pseudomonas putida ( Pseudomonas putida ) XY4 (preservation number CGMCC No.3830) total DNA as template for cloning Nit 5' end sequence:

[0046] P3: 5’-ATGGTRACATAYACNAAYAA-3’

[0047] P4: 5'-TCTACATGCGTCGGCTTCAGCTT-3'

[0048] The PCR reaction was carried out in a 50 μL system, and the reaction conditions were pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 40 s, and extension at 72°C for 40 s, a total of 3...

Embodiment 3

[0050] This example illustrates the cloning method of the 3' end sequence of the nitrilase gene.

[0051] Design three specific primers P5, P6 and P7 according to the Nit M sequence obtained above, use the random primers used in the above three primers and the Tail-PCR method, to Pseudomonas putida ( Pseudomonas putida ) XY4 (preservation number CGMCC No.3830) total DNA as a template to clone the Nit 3' end sequence:

[0052] P5: 5'-CGATGGCGGCAGCCTCTACATGAG-3'

[0053] P6: 5’- GACGAAAGCTGAAACCCACTCATGTA-3’

[0054] P7: 5'-CCAGACCCCAACCAAATACGCGATGT-3'

[0055] The PCR reaction was carried out in a 50 μL system, and the reaction conditions were those used in the Tail-PCR method. The first round of PCR was carried out with P5 and non-specific primers AP1, AP2, AP3 and AP4 respectively. The PCR products amplified by P5 and AP2 were selected as templates, and P6 and NAP were used as primers for the second round of amplification. The third round of PCR was perfo...

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Abstract

The invention relates to novel nitrilase, and a gene cloning and expressing method thereof. A nitrilase gene SEQ ID NO: 1 is acquired from total deoxyribonucleic acid (DNA) of pseudomonas putida XY 4 (serial number CGMCC 3830), the total length of the gene is total length of 1113 nucleotides, and the nitrilase is encoded by 370 amino acid with sequences represented as SEQ ID NO: 2. The invention further discloses a method for structuring, efficiently expressing and purifying restructuring nitrilase, an optimum operation temperature and an optimum operation potential of hydrogen (PH) value of purifying the restructuring nitrilase. A plasmid pET28a (+) is used as an expression vector, and E. coli Rosetta-gami (DE3) is used as an expression host to achieve efficient expression of the nitrilase gene. The optimum operation temperature of the restructuring nitrilase is 55 DEG C, the optimum operation PH value is 7.5, the restructuring nitrilase can efficiently convert 3-cyanopyridine into nicotinic acid, and the restructuring nitrilase has large industrial production potential and economic values.

Description

technical field [0001] The present invention relates to be derived from Pseudomonas putida ( Pseudomonas putida ) XY4 (preservation number CGMCC No.3830) of a new type of nitrilase gene sequence, as well as the construction of nitrilase engineering bacteria, high-efficiency expression and separation and purification of recombinant nitrilase, which belong to the field of enzyme genetic engineering and enzyme engineering. Background technique [0002] Nitrilase can catalyze the conversion of nitrile compounds into carboxylic acid compounds with wide application value, such as acrylic acid, mandelic acid, nicotinic acid, isonicotinic acid, iminodiacetic acid, glycine, etc. At the same time, the enzyme can be used to treat waste water containing nitrile and toxic waste water polluted by nitrile. The wide range of substrates and excellent chemical, regio, and stereoselectivity of nitrilase show great application potential in the synthesis of carboxylic acids, and have good appli...

Claims

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Application Information

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IPC IPC(8): C12N9/78C12N15/55C12N15/10C12N15/70C12R1/40
Inventor 许正宏朱小燕李恒史劲松龚劲松钱建瑛
Owner JIANGNAN UNIV
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