Group of amino acid sequences capable of improving activity and stability of SOD and application thereof

An amino acid and stability technology, which is applied to a group of amino acid sequences that can improve the activity and stability of SOD and its application fields, and can solve the problems of limited raw materials, difficult operation, and difficulty in meeting the needs of industrialization

An amino acid and stability technology, which is applied to a group of amino acid sequences that can improve the activity and stability of SOD and its application fields, and can solve the problems of limited raw materials, difficult operation, and difficulty in meeting the needs of industrialization

CN110195044APending Publication Date: 2019-09-03NANKAI UNIV
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  • Group of amino acid sequences capable of improving activity and stability of SOD and application thereof
  • Group of amino acid sequences capable of improving activity and stability of SOD and application thereof
  • Group of amino acid sequences capable of improving activity and stability of SOD and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] A clone of the complete sequence gene (sodM1) encoding Fe / Mn-SOD of M1 was constructed, and a clone of the DNA sequence (sodAM1) encoding the domain sodA of SODM1 was constructed, and the optimal activity and stability of the expressed protein were determined.

[0094] 1. Extraction of total DNA from Paenibacillus polymyxa M1

[0095] In this example, Paenibacillus isolated from oil well formation water in Dagang Oilfield, Tianjin, China Paenibacillus polymyxa For M1, take 3 mL of fresh culture cultured overnight, collect the cells by centrifugation, suspend the cells in 250 μL of 50 mM Tris buffer (pH8.0), add 10 μL of 0.4M EDTA (pH8.0), mix well and then incubate at 37 °C After 20min, add 30μL 20mg / L lysozyme, mix well and keep warm at 37℃ for another 20min, then add 5μL 20mg / L proteinase K, mix gently, then add 20μL 10% SDS, keep warm at 50℃ until the solution is clear, use Equal volume of phenol: chloroform: isoamyl alcohol extraction twice, chloroform: isoamyl al...

Embodiment 2

[0123] build code B. subtilis Mn-SOD full sequence gene of BSn5 (sod-BSn5 ), and construct the DNA sequence encoding the domain sodA of SOD-BSn5 (sodA-BSn5) clone, and finally construct the complete sequence gene encoding recombinant SOD (N-terminal sequence of SOD-GTNG_2215 and SODA of B. subtilis BSn5 recombined into recombinant SOD) ( sod- combinant ) clones. And the enzyme activity and thermal stability of the expressed protein were measured.

[0124] 1. B. subtilis Extraction of BSn5 total DNA

[0125] In this example, 3 mL of the fresh culture cultured overnight was collected, and the cells were collected by centrifugation. The cells were suspended in 250 μL of 50 mMTris buffer (pH8.0), added with 10 μL of 0.4M EDTA (pH8.0), and mixed Incubate at 37°C for 20min, then add 30μL 20mg / L lysozyme, mix well and then incubate at 37°C for 20min, then add 5μL 20mg / L proteinase K, mix gently, then add 20μL 10% SDS, keep warm at 50°C until solution For clarification, u...

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Abstract

The present invention relates to a group of amino acid sequences capable of improving stability and activity of SOD and an application thereof, the amino acid sequence is an N-terminal amino acid sequence derived from Paenibacillus Fe / B-SOD. The amino acid sequences have the following characteristics: 1) before the sodA domain of the Fe / Mn-SOD protein, a sequence length is 60 to 296 amino acids; and 2) the amino acid sequences have 75 to 100% homology with each other; and 3) the amino acid sequences specifically bind with the metal ions Fe and Mg, can transfer the bound metal ions to the SODAfunctional domain, and have determination effect on the stability activity of the Paenibacillus genus Fe / Mn-SOD, and the amino acid sequences can be widely used in the modification of other SOD (especially normal temperature SOD), which significantly improves the stability and activity.

Description

technical field [0001] The invention relates to a group of amino acid sequences that can improve the activity and stability of SOD and its application. Background technique [0002] Superoxide dismutase (SOD) is the only enzyme that can specifically scavenge free radical O 2 · ˉ The antioxidant metalloenzyme is the key to the defense of organisms against oxygen toxicity. The enzyme widely exists in organisms in nature. Since it was discovered and officially named from bovine red blood cells in 1969, scientists have isolated it from bacteria, fungi, protozoa, algae, insects, fish, plants and mammals. SOD. Due to the special effects of SOD, it has wide application value in the fields of medicine, daily chemical industry, food, agriculture and environmental protection. At present, the clinical application of SOD is mainly focused on anti-inflammation (mainly rheumatoid and inflammation patients caused by radiation therapy), in addition to some autoimmune diseases (such as l...

Claims

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Application Information

Patent Timeline
03 Sep 2019
Publication
CN110195044A
IPC
C12N9/02
CPC
C12N9/0089; C12Y115/01001
Inventors
王威; 孙林博