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A bacillus amyloliquefaciens nitrite reductase, a gene and applications

A technology for decomposing starch spores and nitrite, applied in the field of bioengineering

Inactive Publication Date: 2016-12-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports on the types and properties of nitrite reductase contained in Bacillus amyloliquefaciens at home and abroad.

Method used

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  • A bacillus amyloliquefaciens nitrite reductase, a gene and applications
  • A bacillus amyloliquefaciens nitrite reductase, a gene and applications
  • A bacillus amyloliquefaciens nitrite reductase, a gene and applications

Examples

Experimental program
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Effect test

Embodiment 1

[0037]Embodiment 1: Cloning and expression of Bacillus amyloliquefaciens H47 nitrite reductase gene, the steps are as follows:

[0038] Bacillus amyloliquefaciens H47 bacterial genome total DNA was extracted according to its operation using a bacterial genomic DNA extraction kit (TaKaRa Company), its quality and purity were identified by agarose gel electrophoresis, and its concentration was determined by an ultraviolet spectrophotometer. Design upstream and downstream primers based on whole gene sequencing: m1 (SEQ ID NO: 3,5'-GCCGCATATGATGGGAAAAAAACAGCTAGTC-3') and m2 (SEQ ID NO: 4,5'-GCCGGGATCCTCAATACAGGATGTATACATTCTC-3'), using the PCR method, using the above-mentioned Bacillusamyloliquefaciens The H47 genome was used as a template, and the above-mentioned m1 and m2 were used as specific primers to amplify the full-length coding frame sequence of the Bacillusamyloliquefaciens H47 nitrite reductase gene. The PCR reaction conditions are: Genomic DNA is used as a template, in...

Embodiment 3

[0049] Example 3: Extraction, purification and enzyme activity determination of crude protein in recombinant bacteria

[0050] The recombinant thallus obtained in Example 2 and the control thallus are washed twice with sterile water, add 10 ml per 100 ml of culture solution, and pre-cool 0.02M phosphate buffer (pH7.0) in an ice bath at 4°C Ultrasonic crushing for 5 minutes, after crushing, centrifuge at 8000rpm at 4°C for 5 minutes, take the supernatant as NiR crude protein solution, and perform SDS-PAGE detection at the same concentration, the results are as follows: image 3 As shown, lane 1 is the crude enzyme solution of BL21 group containing empty plasmid induced by adding IPTG, lane 3 is the crude enzyme solution of BL21 group containing recombinant plasmid induced by adding IPTG, and lane 5 is the crude enzyme solution of BL21 group containing recombinant plasmid induced by adding IPTG The crude enzyme solution shows that there are two obvious specific expression bands ...

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Abstract

The invention discloses a bacillus amyloliquefaciens nitrite reductase, a gene and applications, and belongs to the field of biological engineering. The nitrite reductase gene of bacillus amyloliquefaciens H47 is cloned by utilizing a PCR technique and subjected to inducible expression in escherichia coli, and a high-purity recombinant protein is obtained by purification utilizing affinity chromatography. A foundation is laid for researching enzymatic properties of the reductase, researching the nitrite degrading route of the bacillus amyloliquefaciens H47 and improving the nitrite degrading rate of the bacillus amyloliquefaciens H47. NiRBD in the bacillus amyloliquefaciens H47 is one of nitrite reductases, reduces nitrites into ammonium, can be applied for degrading nitrites in ensilage for ruminants and can be used for degrading nitrites in kitchen waste fermented fertilizers.

Description

technical field [0001] The invention relates to a nitrite reductase gene, in particular to the cloning, expression and application of a Bacillus amyloliquefaciensH47 nitrite reductase gene, which belongs to the technical field of bioengineering. Background technique [0002] Nitrite can cause methemoglobinemia in infants, congenital malformations in infants, goiter, and react with secondary amines, products of protein decomposition, to generate strong carcinogen nitrosamines. Standards for the limited amount of nitrite in water and food. Nitrite reductase is widely found in microorganisms and plants. It is a key enzyme in the nitrogen cycle process in nature. It can degrade nitrite into NO or ammonium, thereby reducing the accumulation of nitrite nitrogen in the environment and reducing the accumulation of nitrite. resulting in toxic effects on organisms. [0003] Nitrite reductases can be divided into three types according to the different pathways involved and reaction p...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/06C12N15/70C12N1/21C12R1/19
CPCC12N9/0044C12Y107/01004
Inventor 廖祥儒王爽蔡宇杰管政兵梁金溪赵思洁
Owner JIANGNAN UNIV
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