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Method for producing pectinase by fermentation of recombinant Escherichia coli

A technology for recombining Escherichia coli and pectinase, applied in the field of fermentation engineering, can solve the problems affecting the synthesis of target proteins, cell physiology and metabolic changes, etc., and achieves a simple and easy method, improves secretion and expression efficiency, and improves yield and production intensity. Effect

Inactive Publication Date: 2012-03-07
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it should be noted that during exponential feeding, when induction begins, the actual specific growth rate (μ exp ) are often significantly lower than the set specific growth rate (μ set ), the continuous accumulation of nutrients will lead to substantial changes in cell physiology and metabolism, which in turn will affect the synthesis of target proteins

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  • Method for producing pectinase by fermentation of recombinant Escherichia coli
  • Method for producing pectinase by fermentation of recombinant Escherichia coli
  • Method for producing pectinase by fermentation of recombinant Escherichia coli

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Strain: E.coli BL21DE3(pET-20b(+)PGL), expressed by extracellular secretion.

[0037] Medium:

[0038] 1) Seed medium (LB): Yeast powder 5g L -1 , Tryptone 6-14g L-1, NaCl 6-14g L-1, Ampicillin 1mM, pH 7.0

[0039] 2) Fermentation medium (MTB): glycerin 6-14g L-1, yeast powder 24g L -1 , peptone 12g L -1 , KH 2 PO 4 17mM,K 2 HPO 4 72mM, ampicillin 0.1mM

[0040] 3) Solid plate medium: LB medium, 1.5% agar

[0041] 4) Glycerol feeding medium: glycerol 500g L -1 , yeast powder 50g L -1 , peptone 50g L -1

[0042] Training method:

[0043] 1) Seed culture: Inoculate the preserved strains into a 250mL Erlenmeyer flask containing 25mL LB medium, and cultivate for 8 hours at a rotary shaker with a rotation speed of 200r / min and a culture temperature of 37°C.

[0044] 2) Fermentation culture: Inoculate the bacterial liquid in LB into a fully automatic 3L fermenter (BioFlo 110, New Brunswick Scientific Co.) with a 5% inoculum amount.

[0045] MTB medium (containi...

Embodiment 2

[0053] The parameters of the fermentation process are slightly different, and all the other materials and methods are the same as in Example 1, using MTB medium (containing 100 μg mL -1 Ampicillin) is the fermentation medium, and the whole fermentation process can be divided into three stages:

[0054] 1) The initial glycerol concentration is 10g L-1, and the culture temperature is 37°C. When the DO rebound glycerol is exhausted, the second stage begins;

[0055] 2) Carry out exponential feed glycerol medium according to the following formula, and the specific growth rate is 0.2h -1

[0056] F ( t ) = μ set X 0 V 0 exp ( μ set t ...

Embodiment 3

[0061] The parameters of the fermentation process are slightly different, and all the other materials and methods are the same as in Example 1, using MTB medium (containing 100 μg mL -1 Ampicillin) is the fermentation medium, and the whole fermentation process can be divided into three stages:

[0062] 1) The initial glycerol concentration is 14g L-1, and the culture temperature is 37°C. When the DO rebound glycerol is exhausted, the second stage begins;

[0063] 2) Carry out exponential feed glycerol medium according to the following formula, and the specific growth rate is 0.23h -1

[0064] F ( t ) = μ set X 0 V 0 exp ( μ set t ...

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Abstract

The invention provides a method for producing pectinase by fermentation of recombinant Escherichia coli, which belongs to the technical field of fermentation engineering. According to the invention, Escherichia coli BL21DE3 is used as an original strain, and a glycerin medium is fed in batches according to a certain specific growth rate. The method provided in the invention is simple and practicable, substantially improves output, production intensity and secretory expression efficiency of pectinase, is favorable for large scale industrial production, and has important significance in guiding secretory expression level of foreign proteins in other Escherichia coli expression systems.

Description

Technical field: [0001] The invention discloses a method for producing pectinase by recombinant Escherichia coli fermentation, which belongs to the technical field of fermentation engineering. Background technique: [0002] Alkaline pectinase generally refers to polygalacturonic acid lyase (E.C.4.2.2.2, PGL for short), its optimum pH is 8-10, and it can break pectin polymers by trans-elimination under high pH conditions backbone, resulting in Δ4:5 unsaturated oligogalacturonic acids. Alkaline pectinase can be used in the scouring process of textile pretreatment, thereby replacing the traditional alkaline cooking method, which can not only reduce energy consumption and the difficulty of wastewater treatment, but also improve product quality and enhance the international competitiveness of products. It is currently a textile biological A research hotspot in the field of technology. [0003] The yield and secretion efficiency of the target protein depend on many factors, amon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/26C12R1/19
Inventor 陈坚房淑颖李江华堵国成张东旭刘龙
Owner JIANGNAN UNIV
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