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A group of amino acid sequences that can improve the heat-resistant temperature and thermostability of SOD and its application

A technology of heat-resistant temperature and thermal stability, applied in the fields of biochemical equipment and methods, enzymes, oxidoreductases, etc., can solve the problems of less obvious effect, difficult to meet the needs of industrialization, difficult to operate, etc.

Active Publication Date: 2017-04-19
北京迈恩泰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thermophilic SOD has excellent characteristics of extremely high thermal stability, resistance to physical and chemical denaturants, and has great application value in industrial and agricultural production. The main source is separated from nature, and the raw materials are limited, so it is difficult to meet the needs of industrialization.
At present, the methods for producing and processing SOD to improve its thermal stability mainly include genetic engineering methods, research on SOD simulants, chemical modification, enzyme immobilization, etc. Not too obvious disadvantages and limitations, these problems have seriously affected the industrialization process of thermophilic SOD

Method used

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  • A group of amino acid sequences that can improve the heat-resistant temperature and thermostability of SOD and its application
  • A group of amino acid sequences that can improve the heat-resistant temperature and thermostability of SOD and its application
  • A group of amino acid sequences that can improve the heat-resistant temperature and thermostability of SOD and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Construct the complete sequence gene of Fe / Mn-SOD encoding NG80-2 ( sod-GTNG_2215 ), and construct the DNA sequence encoding the domain sodA of SOD-GTNG_2215 ( sodA-GTNG_2215 ) clone, and determine the optimum reaction temperature and thermal stability of the expressed protein.

[0061] 1. Extraction of total DNA from Bacillus thermodenitrophilus NG80-2 (CGMCC No. 1228)

[0062] In this example, the thermophilic denitrification bacillus NG80-2 ( Geobacillus thermodenitrificans The strain has been preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures, and its preservation number is CGMCC No.1228). Take 3 mL of fresh culture cultured overnight, collect the bacteria by centrifugation, and suspend the bacteria in 250 μL of 50 mM Tris buffer (pH8.0), add 10μL 0.4M EDTA (pH8.0), mix well and keep warm at 37°C for 20min, then add 30μL 20mg / L lysozyme, mix well and keep warm at 37°C for 20min, then add 5μL 20mg / L proteas...

Embodiment 2

[0085] 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.

[0086] 1. B. subtilis Extraction of BSn5 total DNA

[0087] 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 mM Tris buffer (pH8.0), and 10 μL of 0.4M EDTA (pH8.0) was added, 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 clarificati...

Embodiment 3

[0126] Practical application:

[0127]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 lupus erythematosus, dermatomyositis), emphysema, anti-inflammatory Cancer and oxygen poisoning have certain curative effects; in the food industry, it is mainly used as a food additive and an important functional base; in the cosmetics industry, it is mainly used as an important functional ingredient for anti-inflammatory and anti-aging.

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Abstract

The invention relates to an amino acid sequence capable of improving heat-resistance temperature and heat stability of SOD and application thereof. The amino acid sequence is an N-terminal amino acid sequence of 13 special thermophilic Fe / Mn-SOD from Geobacillus category. The amino acid sequence has the following features: (1) the amino acid sequence is before the sodA functional domain of Fe / Mn-SOD protein, the sequence length is 101-344 amino acids and the function is unknown; (2) 85-100% of homology exists among the amino acid sequence; (3) one or two peculiar consensus repeats Geo-N-repeat are included. The amino acid sequence can play a determinant role on the heat-resistance temperature and the heat stability of Fe / Mn-SOD of the Geobacillus category, can be widely applied to improvement on other SODs (particularly normal-temperature SOD), and can remarkably improve the heat-resistance temperature and the heat stability.

Description

technical field [0001] The invention relates to a group of amino acid sequences capable of improving the heat-resistant temperature and thermal 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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02
CPCC12N9/0089C12Y115/01001
Inventor 王威张宝亮
Owner 北京迈恩泰生物科技有限公司
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