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A kind of expression vector of β-glucosidase mutant protein, expression engineering bacteria and expression method

A technology of glucosidase and expression vector, which is applied in the field of expression vector of β-glucosidase mutant protein, can solve the problems of increasing purification steps, difficulty in purification, expensive cytotoxicity, etc., and achieves improved pH and temperature stability, The effect of high expression

Active Publication Date: 2019-12-06
ANHUI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the commonly used E. coli induced expression method has many problems, such as the high price of the inducer IPTG and certain cytotoxicity; a large number of proteins are mostly expressed in the cell, and special instruments are required to break the cell wall and cell membrane, which increases the subsequent purification steps and Difficulty of purification

Method used

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  • A kind of expression vector of β-glucosidase mutant protein, expression engineering bacteria and expression method
  • A kind of expression vector of β-glucosidase mutant protein, expression engineering bacteria and expression method
  • A kind of expression vector of β-glucosidase mutant protein, expression engineering bacteria and expression method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Construction of Engineering Bacteria Expressing β-Glucosidase Mutant Protein

[0039] The cloning vector Trans1T1-bgl1A (A24S / F297Y) containing the bgl1A (A24S / F297Y) gene coding sequence (as shown in SEQ ID NO.1) and the light-sensitive regulatory unit pD coding sequence (as shown in SEQ ID NO.2) The cloning vector Trans1T1-pD was used as a template, and the primers shown in Table 1 below were designed:

[0040] Table 1

[0041]

[0042] The above primers were paired, pD-F and pD-R, bgl-F and bgl-R were used as PCR primer pairs, and the templates used were the cloning vector Trans1T1-bgl1A (A24S / F297Y) and the photosensitive regulatory unit pD The cloning vector Trans1T1-pD of the coding sequence was amplified by PCR according to the system shown in Table 2 below, and the amplified products were named pD and bgl1A(A24S / F297Y) in turn, and recovered for later use;

[0043] Table 2

[0044]

[0045] Use restriction endonucleases Xba I and PshA I to carry out dou...

Embodiment 2

[0054] Application of engineering bacteria expressing β-glucosidase mutant protein in shake flask low-density fermentation to produce β-glucosidase mutant protein

[0055] Inoculate the engineered bacteria BL21(DE3) / pET-22b-pD-bgl1A(A24S / F297Y) confirmed by sequencing into 5ml LB liquid medium with an inoculum size of 1%, and culture at 28°C and 220rpm in the dark for 8-12h;

[0056] Insert the above-mentioned cultivated engineering bacteria into 400ml TB medium according to the inoculum amount of 1%, and culture at 28°C and 200rpm in the dark, when the fermentation broth OD 600 When it reaches 0.6, use a white light source to irradiate, the illumination intensity is 3000lx, and induce expression for 12-14h;

[0057] After the induced expression was completed, the bacteria were collected by centrifugation at 4°C and 8000 g, and 0.1 times the volume of the bacteria liquid was added to the disruption buffer (Na 2 PO 4 - citric acid), under 350W ice bath conditions ultrasonic 4...

Embodiment 3

[0061] Application of engineering bacteria expressing β-glucosidase mutant protein in high-density fermentation of fermenter to produce β-glucosidase mutant protein

[0062] The initial fermentation medium is TB medium, the inoculum size is 5%, the pH is controlled at 6.9-7.1 with 25% (v / v) ammonia water, the DO is controlled at 25-30% by adjusting the ventilation and stirring speed, and the temperature is controlled at 28-30°C, when the glycerol is exhausted and the dissolved oxygen DO of the fermentation broth rises by more than 30%, the rate is 10mL·L -1 h -1 Add feed medium by flow;

[0063] When the fermentation broth OD 600 When it is 10, add light to induce the expression of β-glucosidase mutant protein, the light intensity is 30,000lx, the color is white, and the induction ends after 10-18h;

[0064] After the induced expression was completed, the bacteria were collected by centrifugation at 4°C and 8000 g, and 0.1 times the volume of the bacteria liquid was added t...

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Abstract

The invention discloses an expression vector of a β-glucosidase mutant protein, which is constructed according to the following method: (1) cloning the DNA sequence of the β-glucosidase mutant protein; (2) cloning the DNA of a photosensitive regulatory unit Sequence; (3) connecting the DNA fragmentation of the β-glucosidase mutant protein and the DNA fragmentation of the light-sensitive regulatory unit to the pET22b (+) carrier to obtain the β-glucosidase mutation Body protein expression vector. The present invention constructs a light-inducible expression vector by inserting a photosensitive regulatory unit, and the expression vector can induce expression of the β-glucosidase mutant protein by means of light. Compared with the traditional induction expression method, the light induction method has the ability to express In addition, the intensity of protein expression in the light-induced method can be controlled by light intensity without affecting the subsequent purification steps.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to an expression vector of a beta-glucosidase mutant protein, an expression engineering bacterium and an expression method. Background technique [0002] β-glucosidase (EC 3.1.2.21, β-glucosidase) belongs to the class of fiber hydrolase, which mainly hydrolyzes the β-1,4-glucosidic bonds in glycosides or oligosaccharides, and at the same time releases terminal non-reducing glucose and aglycosides. It has important application value in modern industrial biotechnology such as food, medicine, feed processing, energy refining and other fields. At present, bacterial β-glucosidase generally adopts the production method of heterologous expression, and Escherichia coli is used as the main expression host bacterium. However, the commonly used E. coli induced expression method has many problems, such as the high price of the inducer IPTG and certain cytotoxicity; a large number of proteins are ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/66C12N1/21C12N9/42C12R1/19
CPCC12N9/2445C12N15/66C12N15/70C12Y302/01021
Inventor 肖亚中常飞方泽民房伟
Owner ANHUI UNIVERSITY
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