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A kind of method for producing proline aminopeptidase by cultivating recombinant escherichia coli at high density

A technology of recombinant Escherichia coli and proline aminopeptidase, which is applied in the field of microbial fermentation and can solve problems such as the application of restricted feedback feeding strategies

Active Publication Date: 2017-03-08
江苏博扬生物制品有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Feedback feeding strategy can reflect the changes of various parameters in the fermentation system online and in a timely manner, but the extremely complex interaction between cells and the external environment and cells during the microbial growth process, and the constant changes in parameters during the cultivation process, this has a great impact on the online environment. The detection equipment puts forward the requirements of high precision, high sensitivity and high reliability, which undoubtedly limits the application of feedback feeding strategy in large-scale industrial production

Method used

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  • A kind of method for producing proline aminopeptidase by cultivating recombinant escherichia coli at high density
  • A kind of method for producing proline aminopeptidase by cultivating recombinant escherichia coli at high density
  • A kind of method for producing proline aminopeptidase by cultivating recombinant escherichia coli at high density

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Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1 fermentation medium and fermentation condition optimization

[0037] (1) Optimization of fermentation medium

[0038] Determination of glucose, yeast extract, peptone, Mg by single factor test2+ The optimal amount of addition, and the specific enzyme activity as the response value, glucose, yeast extract, peptone and Mg 2+ As a factor, choose 4 factors and 3 levels L 9 (3 4 ) orthogonal table to test, determine the best enzyme-producing medium as 5g / L glucose (sterilized separately at 115 ℃), 20g / L tryptone, 30g / L yeast extract, 12.54g / LK 2 HPO 4 , 2.31g / LKH 2 PO 4 , adding Mg after sterilization 2+ The final concentration is 7.5mmol·L -1 , pH7.2.

[0039] (2) Optimization of fermentation conditions

[0040] The amount of inducer added, the timing of induction, the induction time, the initial pH of the fermentation broth, the amount of inoculum and the amount of liquid were optimized. the result shows( Figure 1-6 ): The optimal enzyme production...

Embodiment 2

[0041] Embodiment 2 Recombinant bacteria are fermented at a high density on a 7L fermenter

[0042] (1) batch fermentation

[0043] Add 4L high-density fermentation medium to 7L Zhenjiang Dongfang GBJT-7C fermenter. After sterilization, add antibiotics and a final concentration of 0.75 mmol L -1 Mg 2+ . Inoculate 80 mL of seed culture in logarithmic phase with 2% inoculum size, culture at 37° C., and maintain rotation speed at 500 rpm and ventilation at 3 vvm. When it is detected that the glucose in the fermentation broth is exhausted, start to add the final concentration of 3g L -1 induced by lactose. Such as Figure 7 As shown, 8h after inoculation, glucose was exhausted, and at this time, lactose was added for induction. After 12h of induction, the protein expression reached the highest value of 91.89U / mg, and the cell density (OD 600 ) reached 15.60.

[0044] (2) Fed-batch fermentation

[0045] When the fermentation culture is carried out in a batch fermentation m...

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Abstract

The invention discloses a method for producing proline aminopeptidase by culturing recombinant escherichia coli in high density, and belongs to the field of microbial fermentation. Proline aminopeptidase is produced by fermenting escherichia coli, namely recombinant bacteria, by utilizing fermentation mediums and a fermenting method, and the bacteria density (OD600) and the recombinant protein expression quantity are respectively 142 and 211U / mg.

Description

technical field [0001] The invention relates to a method for high-density culture of recombinant Escherichia coli to produce proline aminopeptidase, which belongs to the field of microbial fermentation. Background technique [0002] High-density culture (HCDC) is high-density fermentation, which generally refers to the growth state or culture technology of microorganisms when the cell population density in liquid medium exceeds 10 times that of conventional culture. Modern high-density culture technology is mainly used for high-density culture of genetically engineered bacteria, especially E.coli engineered bacteria. Compared with traditional culture, high-density culture has many advantages, mainly reflected in: (1) higher biomass; (2) the space required for the reaction system is reduced, and the investment cost is reduced; (3) the production cycle is shortened and The cost is reduced; (4) it is more conducive to the separation and purification of the target product. Alt...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/48C12R1/19
CPCC12N9/485C12Y304/14012
Inventor 田亚平丁国伟
Owner 江苏博扬生物制品有限公司