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Bacillus subtilis for high yield of recombinant nitrilase and application of bacillus subtilis

A technology of Bacillus subtilis and nitrilase, which is applied in the fields of genetic engineering and enzyme engineering, can solve problems such as increasing process steps and production costs, achieves good application prospects, improves process efficiency, and reduces production costs.

Active Publication Date: 2017-10-17
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] As a traditional, safe and reliable expression host, Bacillus subtilis has been widely used in the fields of industry, agriculture, and medicine. Most of the traditional recombinant expression of nitrilase needs to be induced in vitro, which increases the process steps and production costs.

Method used

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  • Bacillus subtilis for high yield of recombinant nitrilase and application of bacillus subtilis
  • Bacillus subtilis for high yield of recombinant nitrilase and application of bacillus subtilis
  • Bacillus subtilis for high yield of recombinant nitrilase and application of bacillus subtilis

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Experimental program
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Effect test

Embodiment 1

[0020] Example 1: Construction of Bacillus subtilis NIT-2 producing recombinant nitrilase

[0021] 1) Amplification of the NIT sequence of the nitrilase gene: Genomic DNA was extracted from Pseudomonas putida CGMCC3830, used as a template, and upstream and downstream primers 2PU and A5D were designed, and Nde I and MluI restriction sites were added at both ends , for PCR amplification, primers:

[0022] 2PU: 5'-GGAATTC CATATG ATGGTTACGTACACGAATAAGTT-3'

[0023] A5D: 5'-CG ACGCGT TCAGCTCTCTTCATGGACCTTAA-3'

[0024] PCR amplification system:

[0025]

[0026]

[0027] PCR reaction program: cycle after pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 90 s, a total of 30 cycles; final extension at 72°C for 5 min; incubation at 12°C to obtain Nde at both ends Nitrilase gene NIT at I and Mlu I restriction sites.

[0028] 2) Preparation of pMA5 plasmid containing Nde I and Mlu I restriction enzymes:...

Embodiment 2

[0034] Embodiment 2: Preparation of Bacillus subtilis NIT-2 free cells

[0035] Centrifuge the fermentation broth cultured in TB medium for 8-10 hours at 12,000×g for 1 min, discard the supernatant, resuspend the bacteria with pH 7.2, 100 mM sodium phosphate buffer (PBS) and wash 2-3 times , to obtain the cells after removing the culture medium residue, and finally resuspend the cells with the same sodium phosphate buffer to obtain a free cell suspension, measure the concentration of the cells and store them in a refrigerator at 4°C for later use.

Embodiment 3

[0036] Embodiment 3: biotransformation synthetic nicotinic acid

[0037] Mix the prepared free-cell bacterial suspension (100mL, the dry weight of the bacteria is 2.87g / L) with 1.04g 3-cyanopyridine (the initial concentration of 3-cyanopyridine is 100mM), and carry out at 30°C and 220rpm. In the conversion reaction, the remaining substrate in the conversion solution is detected by HPLC, and when the substrate is completely converted, the conversion solution containing niacin is obtained.

[0038] The free cells of Bacillus subtilis NIT-2 can completely convert 100mM substrate within 13min, the conversion rate of 3-cyanopyridine can reach 100%, and the by-product nicotinamide is not detected by HPLC. The average conversion rate of pyridine was 16.72 g / h.

[0039] SEQ ID NO:1

[0040]Composition 262 A; 293 C; 276 G; 282 T;

[0041] Percentage: 23.5% A; 26.3% C; 24.8% G; 25.3% T;

[0042] Molecular Weight (kDa): ssDNA: 343.21 dsDNA: 686.18

[0043] ORIGIN

[0044] 1 TCAGCTC...

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Abstract

The invention discloses bacillus subtilis for high yield of recombinant nitrilase and application of the bacillus subtilis in nicotinic acid synthesis and belongs to the technical field of biological engineering. A method comprises the following steps: performing PCR (polymerase chain reaction) amplification on a Pseudomonas putida CGMCC 3830 nitrilase gene coding sequence, connecting with a pMA5 plasmid, establishing a recombinant plasmid pMat-NIT, and transforming into Bacillus subtilis WB600, thereby obtaining a recombinant bacterium, namely B.subtilis WB600 (pMA5-NIT) which is capable of efficiently expressing nitrilase, wherein the recombinant bacterium is named as Bacillus subtilis NIT-2 and is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.14255. With recombinant bacillus subtilis free cells as a catalyst, 3-cyanopyridine can be completely transformed to generate nicotinic acid. The invention provides a bacillus subtilis strain for high yield of recombinant nitrilase, and the bacillus subtilis strain has outstanding nitrilase activity and is short in recombinant bacterium fermentation period, high in catalysis efficiency and good in application prospect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and enzyme engineering, and specifically relates to the construction of a high-yield recombinant nitrilase Bacillus subtilis NIT-2 and its application in the synthesis of nicotinic acid. Background technique [0002] Niacin, also known as vitamin PP or vitamin B3, is one of the 13 essential vitamins for the human body. As a drug intermediate, it can be used to synthesize a variety of drugs for the treatment of skin diseases, hypertension and coronary heart disease, such as isoniazid , nicothamide and inositol nicotinate, etc., can also be used as feed or food additives, and have a wide range of domestic and foreign markets. According to statistics, the world consumes more than 80,000 tons of nicotinic acid each year, which are currently produced by chemical methods, and the production capacity in Europe and the United States is about 40,000 tons. At present, my country basically relie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/55C12P17/12C12R1/125
CPCC12N9/78C12P17/12C12Y305/05001
Inventor 龚劲松许正宏史劲松张强李恒蒋敏
Owner JIANGNAN UNIV
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