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Method for extracellularly producing Q-enzyme in signal-peptide-free manner

A technology of starch branching enzyme and signal peptide, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of low enzyme activity and low protein expression of starch branching enzyme, and achieve the effect of high-efficiency extracellular secretion expression and increased production intensity

Active Publication Date: 2016-09-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The outstanding problems of heterologous expression of starch branching enzyme are low protein expression and low activity of starch branching enzyme

Method used

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  • Method for extracellularly producing Q-enzyme in signal-peptide-free manner
  • Method for extracellularly producing Q-enzyme in signal-peptide-free manner
  • Method for extracellularly producing Q-enzyme in signal-peptide-free manner

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction of recombinant Escherichia coli

[0026] The full length of the thermoglucosidase Geobacillus starch branching enzyme gene (Gene Bank) is 1938bp. as per figure 1 In the construction method shown, first obtain a gene fragment whose sequence does not contain a signal peptide is the sequence shown in SEQ ID NO.1, connect the target gene to pMD18-T simple vector, and obtain the plasmid pMD18-T simple / gbe. Plasmids pMD18-T simple / gbe and pET-20b(+) were digested with double enzymes and ligated with ligase to obtain recombinant plasmid pET-20b(+) / gbe. The recombinant plasmid pET-20b(+) / gbe was transformed into BL21(DE3) competent cells by heat shock method. The transformation solution was spread on LB plates containing ampicillin (100 μg / mL), and the plasmids were extracted and sequenced to verify the constructed recombinant plasmids. The sequencing work was completed by Shanghai Sangong.

Embodiment 2

[0027] Embodiment 2 Recombinant Escherichia coli shake flask culture

[0028] Pick the single clone of E.coli BL21(DE3) containing the recombinant plasmid in LB medium containing 100μg / mL ampicillin, and culture it at 37°C and 200r / min for 8-12h, with an inoculum size of 2% by volume Inoculate into TB medium containing 100 μg / mL ampicillin, and ferment at 30°C and 200r / min for 48h; centrifuge the fermentation broth at 4°C and 10000rpm for 15min to remove bacteria, and collect the supernatant.

Embodiment 3

[0029] Example 3 Enzyme Activity Determination Analysis

[0030] Prepare 0.25% (w / v) potato amylopectin standard solution with 0.9mL 50mmol / L phosphate buffer solution (pH 7.5), add 0.1mL enzyme solution, and react at 50°C for 15min. After the reaction, the enzyme was inactivated in a boiling water bath for 10 minutes, and centrifuged at 10,000 r / min for 2 minutes for color development. The chromogenic system is 0.3mL reaction supernatant and 5.0mL chromogenic solution (0.05% (w / v) KI, 0.005% (w / v) I 2 , pH 7.5) were mixed in a 7mL centrifuge tube, and the absorbance was measured at 530nm after 15min of color development. Definition of enzyme activity: at 530nm, the amount of enzyme required to reduce the absorbance value by 1% per minute is one enzyme activity unit.

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PUM

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Abstract

The invention discloses a method for extracellularly producing Q-enzyme in a signal-peptide-free manner and belongs to the fields of genetic engineering and enzyme engineering. The method is characterized in that Q-enzyme genes from Geobacillus thermoglueosidan and without self-signal-peptide sequences are expressed in escherichia coli, and some culture strategies are used to achieve the signal-peptide-free extracellular production of the Q-enzyme. Compared with the intracellular production of the Q-enzyme, the method has the advantages that the yield is increased evidently, the separation and purification procedures are simplified, and the extracellular expression of recombinant Q-enzyme in E. coli is beneficial to the industrial large-scale production of the Q-enzyme.

Description

technical field [0001] The invention relates to a method for extracellular production of starch branching enzyme without signal peptide, and belongs to the fields of genetic engineering and enzyme engineering. Background technique [0002] Starch branching enzyme (1,4-α-glucan branching enzyme; GBE; EC 2.4.1.18) is a glycosyltransferase belonging to the glycoside hydrolase family 13, and is a key enzyme for the synthesis of glycogen and amylopectin. It first hydrolyzes an α-1,4 glycosidic bond on the substrate molecule, cuts off a linear glucan fragment, and then transfers the fragment to a glucose residue on the remaining substrate molecule through transglycosylation On the 6th carbon atom, an α-1,6 glycosidic bond is formed. Because the starch modified by starch branching enzyme, that is, highly branched starch, has special physical and chemical properties, physiological functions and higher safety, it has broad application prospects in industries such as food and medicin...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N1/21C12N15/54C12N15/70C12R1/19
CPCC12N9/107C12Y204/01018
Inventor 李兆丰顾正彪刘艺婷李才明程力洪雁
Owner JIANGNAN UNIV
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