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

Aminopeptidase Amp0279 sourced from lysinibacillus sphaericus C3-41 as well as recombinant strain and application of aminopeptidase Amp0279

A technology of pht43-amp0279 and Bacillus lysinus, which is applied in the field of protein engineering and genetic engineering, can solve the problem of low yield of other proteins and achieve the effect of stable enzyme activity

Pending Publication Date: 2022-02-08
SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
View PDF13 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the mosquito-killing toxin protein expressed in the wild-type strain is dominant, and the production of other proteins is less

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
  • Aminopeptidase Amp0279 sourced from lysinibacillus sphaericus C3-41 as well as recombinant strain and application of aminopeptidase Amp0279
  • Aminopeptidase Amp0279 sourced from lysinibacillus sphaericus C3-41 as well as recombinant strain and application of aminopeptidase Amp0279
  • Aminopeptidase Amp0279 sourced from lysinibacillus sphaericus C3-41 as well as recombinant strain and application of aminopeptidase Amp0279

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Aminopeptidase AMP0279 Structure and Function Prediction and Gene Sequence

[0065] The protein AMP0279 encoded by the AMP0279 gene (300199..301428) may be predicted according to the spherical lysine Bacillus C3-41 genome sequence (CP000817), which may predict the protein AMP0279 encoded by the AMP0279 gene (300199..301428). The sequence alignment has the highest similarity (51%) (51%) (51%) (51%) (51%) (51%) (51%) (51%). figure 1 . 3D modeling analysis found that it may be a dimer structure with four cobalt ion binding sites, which may belong to the M29 family (see figure 2 . figure 2 Is the AMP0279 3D Structure Model (SWSS-Model). AMP0279 is a homologous dimer. The chart is chain a chain A, leaning on the right is chain B, 4CO 2+ For its active center, labeled a polka dot. The six active sites (Glu 249, Glu 315, His 344, TYR 351, HIS 377, and ASP 379) are respectively coupled to two COs around the chain A. 2+ . The primer with enzyme dug bit BamHI and SACI is d...

Embodiment 2

[0066] Example 2: Recombinant expression plasmid PET28A-AMP0279 and recombinant BL21 (PET28A-AMP0279)

[0067] The amino peptidase AMP0279 gene 5 'end and 3' ends are introduced into restriction endonuclease BamHI and SACI, with restriction endonuclease BamHi and SACI to complete the aminopeptidase AMP0279 gene and E. coli induced expression vector PET28A. Digestion, then use the ligase to connect both, construct the recombinant expression plasmid PET28A-AMP0279. The recombinant expression plasmid was transformed into E. coli BL21, screened by PCR verification, and the positive clonal strain BL21 (PET28A-AMP0279) was screened, and plasmid sequence verification was extracted.

Embodiment 3

[0068] Example 3: Nickel pillar and chromatography method Purified aminogenase amp0279

[0069] The recombinant bacteria were seeded in 5 mL lb (50 μg / ml kanamycin), 37 ° C, 220 rpm overnight culture obtained seed fluid. The seed fluid is added to the proportion of 1: 100 (50 μg / ml kanamycin), and the culture is cultured to the OD. 600 When = 0.6-0.8, IPTG (1 mm at a final concentration) was added to low temperature and slow-speed induction (25 ° C, 160 rpm) for 5 h. Centrifugation (5000g, 10min) is bacterial, abandoned. Ultrasonic crushes, 4 ° C, 12000 g, 30 min after centrifugation, reserved supernatant for protein nickel-column affinity chromatography. After dialysis, the protein concentration was detected with a BCA kit. The known concentration of pure ammoniapinamepase AMP0279 can be detected by SDS-PAGE to a single strip of about 55 kD (purified after the detection of AMP0279) image 3 .

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

The invention discloses a gene of aminopeptidase Amp0279 from lysinibacillus sphaericus C3-41 and an amino acid sequence of the aminopeptidase Amp0279, and constructs a recombinant plasmid containing an encoding gene of the aminopeptidase Amp0279, recombinant escherichia coli BL21 (pET28a-amp0279) and recombinant bacillus subtilis WB800N (pHT43-amp0279). The enzyme is subjected to heterologous expression in escherichia coli, the enzyme is purified by a nickel column affinity chromatography method, and the enzymatic property of purified protein is detected by taking Leu-pNA as a substrate. The optimum reaction conditions of the aminopeptidase Amp0279 provided by the invention are as follows: the temperature is 50 DEG C, the pH is 8.0, the enzyme activity is stable in the range of 40-55 DEG C and the pH is 6.0-9.0, the specific enzyme activity can reach 35383 U / mg after 100 [mu]M Co<2+> is added, and the aminopeptidase Amp0279 is suitable for the industrial fields of feeds, foods, brewing, medicines and the like.

Description

Technical field [0001] The present invention belongs to the field of protein engineering and gene engineering, and more particularly to amino peptidase AMP0279 of spherical lysine Bacillus C3-41 and its recombinant strains and applications thereof. Background technique [0002] During fermentation, Chenhua and other food processing, the protein can be used via a protease, peptidase water into small molecular materials such as polypeptide or amino acid, or destroy sensitive surface, reducing sensitization potential, more beneficial to human intake and absorption. Protein hydrolyzes are thus widely used in the food industry. However, the protein hydrolysis process produces a bitter peptide, affecting the flavor of the food, hindering the promotion of protein hydrolyzatics. Aminopeptidase (EC 3.4.11.-) is a class of enzymes that have a strobing of protein hydrolyzates, which can specifically hydrolyze the bitter peptide amino terminal hydrophobine residues, release one to three amin...

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 Applications(China)
IPC IPC(8): C12N9/48C12N15/57C12N15/75C12N15/70C12N1/21A23K10/14A23L29/00C12R1/125C12R1/19
CPCC12N9/485C12N15/75C12N15/70A23K10/14A23L29/06
Inventor 熊海容胡晓敏赵普瑛张萌
Owner SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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