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
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, the formula of the existing self-induction medium ca

Method used

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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...

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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

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Application Information

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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
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