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High-density fermentation method of recombinant EK enzyme engineering bacteria

A technology of high-density fermentation and enzyme engineering, applied in the field of high-density fermentation of recombinant EK enzyme engineering bacteria, can solve the problems of low expression efficiency of construction engineering bacteria, high cost of enzyme production, and difficulty in improving product yield, etc. The effect of application value, cost reduction, and yield improvement

Active Publication Date: 2022-07-22
BEIJING HUIZHIHENG BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Moreover, in terms of fermentation, due to the low expression efficiency of engineering bacteria constructed in the existing fermentation system and the large loss of materials, it is difficult to increase the output of the product, and at the same time, the cost of enzyme production remains high

Method used

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  • High-density fermentation method of recombinant EK enzyme engineering bacteria
  • High-density fermentation method of recombinant EK enzyme engineering bacteria
  • High-density fermentation method of recombinant EK enzyme engineering bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] This example is used to illustrate the construction process of recombinant EK enzyme engineering bacteria:

[0094] 1. Synthesis of recombinant bovine enterokinase light chain expression cassette

[0095] Synthesize expression cassettes for expressing the recombinant fusion protein of the present invention, and the expression cassettes respectively express the EK of the present invention L m1 and EK L Recombinant fusion protein of m3 expressing EK L The expression cassette sequence of m1 is shown in SEQ ID No. 3, expressing EK L The expression cassette sequence of m3 is shown in SEQ ID No.4.

[0096] 2. Construction of expression vector of recombinant protein and construction of engineering bacteria

[0097] The expression EK constructed in step 1 above was L m1 and EK L The m3 expression cassette sequence was inserted into the expression vector pET-30a(+) NdeI and Xho The recombinant expression vector was constructed and obtained between the I restriction site...

Embodiment 2

[0140] Adopt the recombinant EK enzyme engineering bacteria constructed in Example 1 to carry out high-density culture:

[0141] 1. Strain activation:

[0142] First-level activation: In the ultra-clean workbench, take 50 μL of frozen glycerol bacteria into a 250-mL conical flask containing 50 mL of activation medium and cultivate in a constant temperature shaker at a temperature of 37.0 ± 1.0 °C and a rotation speed of 220 ± 10 rpm , the incubation time is 12 to 16 hours.

[0143] Two-stage activation: In the ultra-clean workbench, take 40 mL of the above-mentioned first-stage activated seed culture solution and put it into a 1000-mL conical flask containing 200 mL of activation medium and cultivate in a constant temperature shaker at a temperature of 37.0±1.0 °C and a rotation speed of 220 ±10 rpm, incubation time 3 hours.

[0144] 2. Fermentation regulation:

[0145] (1) Initial parameter setting of fermentation tank

[0146] Temperature: 37.0±1.0°C, air flow: 500mL / min...

Embodiment 3

[0158] Based on the method of Example 2, the difference is:

[0159] (1) Reduce the concentration of yeast extract powder in the feed medium;

[0160] (2) The induction OD600 value was adjusted from 160 to 150, and the growth rate of the bacteria before induction was reduced (the OD600 value reached 150 after 12-14 hours of feeding);

[0161] (3) The feed medium adopts formula 2; other conditions are the same as those of embodiment 2.

[0162] Fermentation results: Growth curves such as image 3 As shown, the bacterial collection data and HPLC expression detection results are shown in Table 8:

[0163] Table 8

[0164]

[0165] The contrast photos of the inclusion body pigments expressed in Example 2 and Example 3 are as follows: Figure 4 shown. Wherein A is the color and luster of the expression product of Example 2 after dissolution, and B is the color and luster of the expression product of Example 3 after dissolution.

[0166] According to the experimental data o...

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Abstract

The invention relates to the technical field of biological fermentation engineering, in particular to a high-density fermentation method of a recombinant EK enzyme engineering bacterium, which specifically comprises the following steps: synthesizing and expressing an expression cassette sequence of a recombinant fusion protein containing an EK enzyme, the expression cassette sequence is shown as SEQ ID No.4, constructing the recombinant EK enzyme engineering bacterium, activating a strain, performing high-density fermentation culture, and inducing to express the EK enzyme. According to the high-density fermentation method of the recombinant EK enzyme engineering bacteria, the expression quantity can be increased to 19g / L or above, and meanwhile, the pigment content of inclusion body protein can be remarkably reduced.

Description

technical field [0001] The invention relates to the technical field of biological fermentation engineering, in particular to a high-density fermentation method of recombinant EK enzyme engineering bacteria. Background technique [0002] Serine protease enterokinase (referred to as enterokinase or EK enzyme), also known as enteropeptidase, is a heterodimeric serine protease, a mammalian species that catalyzes the conversion of trypsinogen into active trypsin enzymes. Enterokinase preferentially selects the substrate sequence Asp-Asp-Asp-Asp-Lys (DDDDK) and selectively cleaves after lysine. Since the light chain structure of enterokinase is conserved in human, bovine and porcine, its substrate recognition sequence Asp-Asp-Asp-Asp-Lys is also highly conserved in vertebrates, and almost all sequenced pancreatic sequences Proteases are characterized by acting on four asparagine-linked recognition sequences, which are very rare in other natural proteins. Enterokinase consists o...

Claims

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Application Information

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IPC IPC(8): C12N9/64C12N1/21C12N15/62C12N15/70C12R1/19
CPCC12N9/6424C12N15/70C12N1/20C12Y304/21009C07K2319/50C07K2319/35C12N2800/101
Inventor 曹海燕林兆生连婕妮王惠朱志伟辛瑞
Owner BEIJING HUIZHIHENG BIOTECHNOLOGY CO LTD
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