Method for increasing soluble expression quantity of 4, 6-alpha-glucosyltransferase

A technology of glucosyl and transferase, which is applied in the fields of protein engineering and fermentation engineering, and achieves the effect of remarkable effect, simple operation process, and improvement of soluble expression amount.

Inactive Publication Date: 2020-12-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Facing these difficulties and aiming at the current existing problems, the present invention proposes a strategy of adding small molecular substances to the medium to increase the soluble expression of exogenous proteins

Method used

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  • Method for increasing soluble expression quantity of 4, 6-alpha-glucosyltransferase
  • Method for increasing soluble expression quantity of 4, 6-alpha-glucosyltransferase
  • Method for increasing soluble expression quantity of 4, 6-alpha-glucosyltransferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1: Construction of the expression vector derived from the 4,6-α-glucosyltransferase gene (gtfB) of Lactobacillus fermentum (Lactobacillus fermentum)

[0054] Using a synthetic plasmid containing the gtfB gene (nucleotide sequence shown in SEQ ID NO.1) as a template, the target gene was obtained by PCR, and then connected to the pET15b plasmid to finally obtain the expression vector pET15b-gtfB. The specific process is as follows:

[0055] gtfB-F: 5'- CATATG CAGGCCAACGATGGTCATTG-3' (the underline is the NdeI restriction site, SEQ ID NO.3)

[0056] gtfB-R: 5'- GGATCC TTAATCATCTTCAATATTG-3' (the underline is the BamHI restriction site, SEQ ID NO.4) artificial sequence

[0057] PCR reaction system: 10xSuper PfxMasterMix 5 μL, GtfB-F (10 μM) 1 μL, gtfB-R (10 μM) 1 μL template 0.5 μL, add sterile water to make up to 50 μL. PCR amplification program: 98°C 5min pre-denaturation; (98°C 15S, 55°C 30S, 72°C 3.5min) x 30 cycles; 72°C extension 10min.

[0058] The P...

Embodiment 2

[0061] Example 2: Transformation of BL21(DE3) with the recombinant plasmid pET15-gtfB to obtain a recombinant strain

[0062] Take 2-5 μL of recombinant plasmid pET15-gtfB, add BL21(DE3) competent cells, incubate on ice for 30 min, then heat shock at 42°C for 90 s, add 800 μL LB to recover for 40 min, and coat with LB containing ampicillin (100 μg / mL) for solid culture culture base at 37°C for 12 hours, select the correct transformants into LB liquid medium, culture at 37°C and 200rpm for 10 hours, add glycerol, store the strain at -80°C, and ferment the strain BL21 / pET15-gtfB .

Embodiment 3

[0063] Example 3: Adding small molecular substances to TB medium increases the enzyme production of recombinant Escherichia coli BL21(DE3) / pET15-gtfB shake flask fermentation

[0064] (1) Seed activation stage

[0065] From the glycerol tube at -80°C, take 10 μL of the recombinant bacteria constructed in Example 2 and insert it into a 100mL Erlenmeyer flask containing 10mL of liquid LB medium, and cultivate it on a rotary constant temperature and speed-adjustable shaker, controlling the temperature at 37°C and rotating speed 200rpm, initial pH 7.0, cultured for 10h.

[0066] (2) Shake flask fermentation stage

[0067] Set up different fermentation media: add betaine, maltose, β-cyclodextrin, trehalose, and sorbitol to the TB medium.

[0068] The seed culture solution (OD 600 About 3) Inoculate 2mL / 100mL inoculum into the above-mentioned fermentation medium respectively, at a rotation speed of 200rpm and a temperature of 37°C, after culturing for 2 hours, add inducers with a...

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Abstract

The invention discloses a method for increasing the soluble expression quantity of 4, 6-alpha-glucosyltransferase, belongs to the technical field of protein engineering and fermentation engineering, and particularly relates to a method for increasing the soluble expression quantity of 4, 6-alpha-glucosyltransferase (GtfB) by adding betaine, maltose, beta-cyclodextrin, trehalose and sorbitol smallmolecular substances into a fermentation medium in a process of expressing 4, 6-alpha-glucosyltransferase (GtfB) derived from lactobacillus fermentum by respectively taking escherichia coli and bacillus subtilis as hosts. The total enzyme activity of fermentation is improved. An effective strategy is provided for efficient expression of 4, 6-alpha-glucosyltransferase (GtfB), the operation processis simple, the cost is low, the effect is remarkable, and a foundation is laid for industrial production.

Description

technical field [0001] A method for increasing the soluble expression level of 4,6-α-glucosyltransferase belongs to the technical fields of protein engineering and fermentation engineering. Background technique [0002] 4,6-α-glucosyltransferase belongs to the glycoside hydrolase (GH) 70 family, and the enzymes of this family can use sucrose or starch as substrates to synthesize α-glucans of various structures, so they are widely used in food field. [0003] 4,6-α-glucosyltransferase is mostly derived from Lactobacillus (Lactobacillaceae). Due to the low fermentation level of wild bacteria, the expression level was increased through heterologous expression. Escherichia coli (Escherichia coli) is the most widely used host because of its clear genetic background and mature genetic engineering technology. In general, there are three ways for E. coli to express foreign proteins, intracellular expression, periplasmic space expression, and extracellular secretion. With the imp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N15/74C12N1/21C12R1/19C12R1/125
CPCC12N9/1051C12N15/70C12N15/74
Inventor 吴敬王蕾饶德明陈晟刘展志霍润甜杨卫康盛露菲
Owner JIANGNAN UNIV
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