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

Lysobacter enzymogenes mutant strain and preparation method thereof

A technology for producing lysobacteria and mutant strains, applied in biochemical equipment and methods, methods based on microorganisms, bacteria, etc., can solve the problems of long production cycle and low expression

Active Publication Date: 2015-03-11
GENERAL HOSPITAL OF PLA +1
View PDF2 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical methods reported above all have the problem of too long production cycle or low expression level. Therefore, it is of great research and application value to screen a Lys-C producing strain with high yield and short fermentation cycle.

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
  • Lysobacter enzymogenes mutant strain and preparation method thereof
  • Lysobacter enzymogenes mutant strain and preparation method thereof
  • Lysobacter enzymogenes mutant strain and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] The optimization of embodiment 1 culture medium

[0017] Medium A: 0.5% casein, 1% sucrose, 1% beef extract, 0.01% dipotassium hydrogen phosphate, 0.01% potassium dihydrogen phosphate, 0.02% magnesium sulfate, adjust the pH to 7.2±0.1.

[0018] Using medium A as the initial medium, simple optimization of nitrogen and carbon sources was carried out. The optimization process is as follows:

[0019] Nitrogen source investigation: other components in medium A remained unchanged, respectively with 1% polypeptone, 1% Yeast extract, 1% Tryptone, 1% hydrolyzed casein, 1% casein and 0.5% Casein + 1% beef extract was used as nitrogen source for shake flask fermentation. Samples were taken on days 3, 4, 5 and 6 for enzyme activity assays. The results are as follows figure 1 As shown, 1% polypeptone was determined to be the optimum nitrogen source.

[0020] Carbon source investigation: Other components in medium A remain unchanged, nitrogen source is 1% polypeptone, and 1% suc...

Embodiment 2

[0023] Example 2 Preliminary screening of lysine endopeptidase high-producing strains

[0024] Purchase L.enzymogens from ATCC, the strain number is ATCC27796, after recovering the strain, perform single colony isolation, take 10-5, 10-6, 10-7 and other appropriate dilutions to coat 3 nutrient agar medium plates, 30 Static culture at ±1°C for 2-3 days; then transferred to the slant of nutrient agar medium, and cultured at 30±1°C for 2-3 days; then inoculated with 5%-10% inoculum in the fermentation medium for fermentation, loaded The volume of the bottle was 16%, and high-throughput screening was carried out, and the fermentation culture was carried out for 5 days, and then the enzyme activity was determined. The fermentation medium is: 1% (W / V) polypeptone, 1% (W / V) sucrose, 0.01% (W / V) dipotassium hydrogen phosphate, 0.01% (W / V) potassium dihydrogen phosphate, 0.02 % (W / V) magnesium sulfate, the balance is purified water, pH7.2±0.1. Inoculate the first-screened strains in ...

Embodiment 3

[0025] Example 3 Screening of lysine endopeptidase mutant high-yielding strains

[0026](1) Inoculate the strain L.enzymogens PGJZC30 with the highest enzyme activity obtained by natural selection in LB liquid medium, and shake it in a shaker at a temperature of 30±1°C and a rotation speed of 215-225rpm for 20 hours for recovery; use one end of a toothpick After wetting the culture solution, puncture into a space tube filled with 220 μL of LB semi-solid medium, set up one tube for the control group and one tube for the experimental group, and culture at 30±1°C for 2-3 days, then separate the experimental group and the control group The experimental group was sent to the Beijing Aerospace Administration respectively. The experimental group carried the "Shenzhou 10" into space in June 2013, and was subjected to mutagenesis by various space factors such as space cosmic rays, microgravity, and alternating magnetic fields. The return capsule returns to the ground. During this peri...

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 provides a lysobacter enzymogenes mutant strain TGJZC-041. The method comprises the following steps: inoculating the bacteria into a nutrient agar culture-medium slope; standing and culturing at 30+ / -1 DEG C for 2-3 days; inoculating into the fermentation medium at the inoculum size of 5-10%; carrying out shake culturing in a constant temperature shaker at 30+ / -1 DEG C at the rotating speed of 215-225rpm for 3 days, so as to prepare an incision enzyme for lysine peptide. The lysobacter enzymogenes mutant strain has the beneficial effects that the Lys-C generated by the mutant strain is relatively high in expression quantity and shorter in fermentation period.

Description

technical field [0001] The invention belongs to the field of mutation breeding, in particular to a method for space mutation breeding of lysobacterium enzymogenes and a mutant strain of lysobacterium enzymogenesis obtained by space mutation breeding. Background technique [0002] Lysine endopeptidase Lys-C (Endoproteinase Lys-C) is an alkaline protease with a molecular weight of about 30kD and belongs to a member of the serine protease family. It can specifically hydrolyze the C-terminus of lysine residues in proteins or peptide chains, and can also be used for protein identification by peptide mass fingerprinting or MS / MS spectral matching, so it has a good application in proteomics analysis prospect. In addition, Lys-C can also be used for the cleavage of precursors in the industrial production of recombinant proteins (such as insulin and its analogues). Compared with commonly used trypsin, Lys-C has several significant advantages: 1. Strong specificity, enzyme High acti...

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): C12N1/20C12N9/52C12R1/01
Inventor 刘长庭常德方向群王俊峰苏龙翔张学林严凌斌林树珊李利佳
Owner GENERAL HOSPITAL OF PLA
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