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

Self-induced medium and application thereof

A self-inducing medium and arabinose technology, applied in the direction of microorganism-based methods, microorganisms, hydrolytic enzymes, etc., to achieve strong controllability and high protein expression

Inactive Publication Date: 2013-06-12
ZHEJIANG APELOA TOSPO PHARMA +1
View PDF3 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, the formula of the existing self-induction medium can still be improved to increase the expression of the target protein

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1LB medium IPTG induction

[0028] (1) LB medium: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, prepared with ultrapure water, pH7.0;

[0029] (2) Pick the recombinant Escherichia coli, inoculate it in 5 mL of LB liquid medium containing kanamycin (50 μg / mL) at a ratio of 1:100, and cultivate overnight at 37°C with shaking at 200 rpm;

[0030] (3) Transfer 0.5 mL of the culture solution to 50 mL of LB liquid medium containing ampicillin (50 μg / mL), and culture at 37°C and 200 rpm for 2 to 4 hours with shaking until the OD600 is about 0.6;

[0031] (4) Add inducer IPTG to the culture to a final concentration of 0.1mmol / L, and induce culture at 20-30°C for 12 hours;

[0032] (5) Centrifuge the fermentation broth at 10,000 rpm for 10 minutes to collect the bacterial cells, resuspend the bacterial cells in pre-cooled phosphate buffer solution for ultrasonic disruption, collect the supernatant by centrifugation, and detect the enzyme activity.

[0033] The specif...

Embodiment 2

[0041] Embodiment 2 Lactose self-induction

[0042] (1) Autoinduction medium: α-lactose 2g / L, glucose 0.5g / L, glycerol 5g / L, KH 2 PO 4 3.4g / L, MgSO 4 0.49g / L, peptone 10g / L, Na 2 HPO 4 3.55g / L, Na 2 SO 4 0.71g / L, NH 4 Cl2.67g / L, trace element solution 200μL / L, prepared with ultrapure water;

[0043] Among them, the trace element solution is: 50μM FeCl3, 20μM CaCl 2 2H 2 O, 10 μM MnCl 2 4H 2 O, 10 μM ZnSO 4 ·7H 2 O, 2 μM CoCl 2 ·6H 2 O, 2 μM CuCl 2 , 2 μM NiCl 2 , 2 μM Na 2 Mo 7 o 24 , 2 μM Na 2 SeO 3 , 2 μM H 3 B 4 o 7 , prepared with ultrapure water;

[0044] (2) Inoculate the recombinant Escherichia coli into 5 mL of LB medium containing kanamycin (50 μg / mL), place it in a shaker at 37°C and 200 rpm, and culture until the OD600 reaches about 2.0 to 3.0;

[0045] (3) Inoculate the seed culture solution in (2) into a 250mL Erlenmeyer flask containing 50mL autoinduction medium at an inoculum volume of 1%, and incubate at 25°C for 40 hours;

[0046] (4...

Embodiment 3

[0047] Embodiment 3 Lactose, arabinose joint self-induction

[0048] (1) Autoinduction medium: α-lactose 2g / L, glucose 0.5g / L, glycerol 5g / L, arabinose 2g / L, KH 2 PO 4 3.4g / L, MgSO 4 0.49g / L, peptone 10g / L, Na 2 HPO 4 3.55g / L, Na 2 SO 4 0.71g / L, NH 4 Cl2.67g / L, trace element solution 200μL / L, prepared with ultrapure water;

[0049] The composition of the trace element solution is as shown in step (1) of Example 2;

[0050] (2) Inoculate the recombinant Escherichia coli into 5 mL of LB medium containing kanamycin (50 μg / mL), place it in a shaker at 37°C and 200 rpm, and culture until the OD600 reaches about 2.0 to 3.0;

[0051] (3) Inoculate the seed culture solution with a 1% inoculation amount into a 250mL Erlenmeyer flask containing 50mL of the self-induction medium from step (1), and incubate at 25°C for 40 hours;

[0052] (4) Use the method in step (5) of Example 1 to obtain the fermentation broth and detect the enzyme activity. The activity of penicillin amidase...

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 self-induced medium and an application thereof. The self-induced medium is composed of 2-10g / L of arabinose, 0.5-2.5g / L of glucose, 5-25g / L of glycerol, 8-12g / L of peptone, 6.5-7.4g / L of phosphate, 1.1-1.3g / L of sulfate, 2.6-2.7g / L of NH4Cl and an appropriate amount of trace element solution. The self-induced medium is used in producing penicillin amidase. Compared with the prior art, the self-induced medium disclosed by the invention has the advantages of strong regulation performance and high protein expression index by utilizing the arabinose as a substrate to induce the expression of an interest protein. In one specific embodiment, the enzyme activity of the penicillin amidase reaches 6U / mL, which is much higher than the protein expression index induced by lactose, and thus providing an important guiding significance for a new process of developing and producing penicillin amidase.

Description

technical field [0001] The invention belongs to the technical field of biological fermentation, and in particular relates to an autoinduction medium and its application. Background technique [0002] Penicillin G amidase (EC3.5.1.11) is an important enzyme in the industry of semi-synthetic β-lactam antibiotics. indigo acid) or 7-ADCA (7-aminocephalosporanic acid). [0003] This enzyme belongs to the family of hydrolases that act on carbon-nitrogen bonds other than peptide bonds, especially on straight-chain amide bonds. The system name is penicillin hydrolase, other commonly used names include penicillin acylase, lipase Novozym 217, α-acyl-β-lactam hydrolase, ampicillin acylase. At present, the enzyme has been widely used in industrial production of key intermediates of β-lactam antibiotics and semi-synthetic β-lactam antibiotics. [0004] When expressing penicillin amidase in genetically engineered bacteria, the expression system based on the strong promoter T7 is often ...

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
IPC IPC(8): C12N9/84C12R1/19
Inventor 陈振明周硕赖敦岳李兰杰陈亮
Owner ZHEJIANG APELOA TOSPO PHARMA
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