Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Nattokinase and its coding gene and clone and expressing method

A technology of Bacillus subtilis and microbial strains, applied in the direction of bacteria, transferase, biochemical equipment and methods, etc., can solve the problems of low yield of active products, many purification steps, time-consuming and labor-consuming denaturation and renaturation, etc., to achieve Broad industrial application prospects, high fibrinolytic activity, and good stability

Active Publication Date: 2008-11-26
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, nattokinase is mainly derived from the fermentation of wild Bacillus subtilis, the yield is low, the culture period is long, the medium composition is complicated, the purification steps are many, the yield is low, and the production cost is high
However, the existing nattokinase expressed in Escherichia coli is inactive inclusion body, denaturation and renaturation is time-consuming and labor-intensive, and the yield of active product is extremely low

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Nattokinase and its coding gene and clone and expressing method
  • Nattokinase and its coding gene and clone and expressing method
  • Nattokinase and its coding gene and clone and expressing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, the separation and identification method of Bacillus subtilis YF38 CGMCC No.1308

[0037] 1. Isolation and identification of nattokinase-producing bacteria

[0038] 1. Isolation of nattokinase-producing bacteria

[0039] Select five kinds of fermented soybeans produced by Yunnan Yimen Food Factory, Guangdong Heshan Xingqiu Food Factory, Sichuan Yongchuan Rongmei Food Factory and Sichuan Fushun Shunding Food Factory Shunyi Branch, and separate natto from different products as follows Kinase-producing bacteria: Grind an appropriate amount of tempeh, dilute with sterile water, centrifuge, spread the supernatant on an LB plate, incubate at 37°C for 8-12 hours, pick a single colony, and streak it on the plate Store it, and at the same time inoculate it in a test tube containing LB liquid medium and culture it with shaking at 37°C for 8-12 hours. Finally, the culture supernatants of single colonies isolated from the five products were tested for fibrinolytic a...

Embodiment 2

[0047] Embodiment 2, the cloning of nattokinase whole gene

[0048] 1. PCR amplification of the whole gene of nattokinase

[0049] According to known nattokinase gene sequence design primer, primer sequence is as follows (primer sequence both ends comprise restriction endonuclease Eco RI and Sal I recognition site):

[0050] Primer 1: 5′-gcg gaa ttc gta tga aaa tag tta-3′

[0051] Primer 2: 5′-gta gtc gac tcc ggt gct tgt gaa-3′

[0052]Using the total DNA of YF38 CGMCC No.1308 strain as a template, under the guidance of primer 1 and primer 2, the nattokinase gene was amplified by PCR. The PCR reaction conditions were: 94°C for 1min, 54°C for 1min, 72°C for 1.3min, A total of 30 cycles. After the reaction, 2 μL of the PCR product was taken for 1% agarose gel electrophoresis detection, and there was an obvious band at 1900 bp in ultraviolet detection, and its molecular weight was consistent with the size of the whole gene of nattokinase.

[0053] 2. Construction and identifi...

Embodiment 3

[0055] Embodiment 3, the expression of nattokinase of the present invention in escherichia coli

[0056] 1. Construction of Escherichia coli secretory expression vector pET26b-nkp

[0057] According to the coding sequence design primer of the leader peptide of nattokinase and mature peptide, the primer sequence is as follows: the two ends of the primer sequence comprise restriction endonuclease EcoRI and Sal I recognition site):

[0058] Primer 3: 5′-gac gaa ttc gat ggc cgg aaa aag cagt-3′

[0059] Primer 4: 5′-gta gtc gac tcc ggt gct tgt gaa-3′

[0060] Using the vector pUC18-nkl containing the entire nattokinase gene as a template, PCR amplification was carried out under the guidance of primers 3 and 4. The amplification conditions were: 94°C for 1 min, 54°C for 1 min, and 72°C for 1 min, a total of 30 samples cycle. After the reaction, the PCR product was precipitated and digested, and then the digested product was purified and then digested with the same enzyme and puri...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

This invention discloses a natto kinase, its coding gene and its application, in which, said natto kinase is a protein including one of the sequences of the following amino acid residues, 1), SEQ ID No: 1in the list, 2), the amino acid residue sequence of the SEQ ID No:1 in the list is replaced, deleted and added by 1-10 amino acid residues and has fibrinolysis activity. The expression method is to set up a secretory expression carrier of colibacillus with the natto kinase gene to be introduced into a colibacillus to get a re-structured colibacillus to be induced to express the natto kinase gene, which has high fibrinolysis activity.

Description

technical field [0001] The invention relates to a strain of bacillus subtilis producing nattokinase, the amino acid sequence of the nattokinase produced by the strain, its coding gene, and a method for cloning and expressing the gene. Background technique [0002] Nattokinase is a serine protease derived from natto, a traditional fermented food. Recent studies have shown that it has strong fibrinolytic activity and is a potential thrombolytic drug. It not only has a good thrombolytic effect, but also achieves the purpose of fibrinolysis through oral administration, and can promote the increase of endogenous t-PA at the same time. And it has the advantages of long drug effect time, no antigenicity, and can activate the organism's own fibrinolytic enzyme system. It can gently and continuously improve the fibrinolytic activity of blood, and achieve convenient, safe and effective treatment for cardiovascular and cerebrovascular embolism diseases. Prevention and treatment, its c...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/12C12N15/54C12N15/70
Inventor 钟瑾梁小波贾士芳孙玉芳陈美玲陈秀珠还连栋
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com