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Optimization method for improving secretory expression of cyclodextrin glucosyltransferase and application of optimization method

An optimization method, glucose-based technology, applied in the directions of glycosyltransferase, transferase, biochemical equipment and methods, etc., can solve the problems of difficult secretion and expression, easy formation of inclusion bodies, etc., to achieve inhibition of formation, improve disproportionation activity, The effect of increasing conversion rates

Active Publication Date: 2021-06-25
BLOOMAGE BIOTECHNOLOGY CORP LTD
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Problems solved by technology

[0007] Aiming at the disadvantages that cyclodextrin glucosyltransferase in Escherichia coli is easy to form inclusion bodies and not easy to secrete and express, the present invention provides an optimized method for improving the secretion and expression of cyclodextrin glucosyltransferase. The fermentation process of recombinant Escherichia coli with base transferase gene was optimized, adding glycine, Ca 2+ , β-cyclodextrin, taurine and lactose, the disproportionation activity of the cyclodextrin glucosyltransferase was significantly improved

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  • Optimization method for improving secretory expression of cyclodextrin glucosyltransferase and application of optimization method
  • Optimization method for improving secretory expression of cyclodextrin glucosyltransferase and application of optimization method

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Embodiment 1

[0032] Embodiment 1: Construction of recombinant escherichia coli

[0033] Based on the nucleotide sequence of cyclodextrin glucosyltransferase derived from Bacillus circulans Jordan, and according to the gene sequence of cyclodextrin glucosyltransferase, a enzyme containing XhoI and BamHI restriction sites was designed and synthesized The primers used to amplify the CGTase gene fragment were used to synthesize the CGTase gene without its own signal peptide sequence by chemical synthesis. The plasmid used to construct recombinant E. coli is pET20b(+), with T7 promoter and pelB signal peptide sequence. The pET20b(+) and CGTase gene fragments were subjected to XhoI and BamHI double enzyme digestion respectively. After the digestion products were gel-cut and recovered, they were ligated with T4 DNA ligase, and the ligated products were transformed into E.coli JM109 competent cells, and picked after culture. For positive clones, the expression plasmid cgt / pET20b(+) was obtained i...

Embodiment 2

[0036] Add ampicillin to the LB medium to make the concentration 100 μg / mL, inoculate the recombinant Escherichia coli preserved in Example 1 in the LB medium (containing 100 μg / mL ampicillin), culture and grow at 25°C for 8 hours, and obtain seeds liquid. Add ampicillin to the TB medium to make the concentration 100 μg / mL, add β-cyclodextrin to the concentration of 10 mM, add taurine to the concentration of 0.8 mM, and inoculate the seed solution according to the inoculation amount of 5%. TB medium (supplemented with 10 mM β-cyclodextrin and 0.8 mM taurine, containing 100 μg / mL ampicillin), cultured in a shaker at 25°C and 200 rpm, when the cells were cultured to OD 600 When it is 0.6, add lactose, glycine and calcium chloride, and their concentrations in the culture system are lactose 15 mmol / L, glycine 200 mmol / L, CaCl 2 10 mmol / L, after continuing to ferment for 72 hours, centrifuge a certain volume of fermentation broth at 4°C and 12,000 rpm for 15 minutes, and take the...

Embodiment 3

[0038] Add ampicillin to the LB medium to make the concentration 100 μg / mL, inoculate the recombinant Escherichia coli preserved in Example 1 in the LB medium (containing 100 μg / mL ampicillin), culture and grow at 25°C for 8 hours, and obtain seeds liquid. Add ampicillin to the TB medium to a concentration of 100 μg / mL, add β-cyclodextrin to a concentration of 10 mM, add taurine to a concentration of 1 mM, and inoculate the seed solution at a 5% inoculum volume TB medium (supplemented with 10 mM β-cyclodextrin and 1 mM taurine, containing 100 μg / mL ampicillin), cultured in a shaker at 25°C and 200 rpm, when the cells were cultured to OD 600 When it is 0.6, add lactose, glycine and calcium chloride, and their concentrations in the culture system are lactose 15 mmol / L, glycine 200 mmol / L, CaCl 2 10 mmol / L, after continuing to ferment for 72 hours, centrifuge a certain volume of fermentation broth at 4°C and 12,000 rpm for 15 minutes, and take the supernatant, which is the crud...

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Abstract

The invention discloses an optimization method for improving secretory expression of cyclodextrin glucosyltransferase and an application thereof. The method optimizes the fermentation culture process of recombinant escherichia coli containing a CGTase gene, five components including glycine, Ca<2+>, beta-cyclodextrin, taurine and lactose are added in the fermentation culture process, membrane permeability is improved, the formation of inclusion bodies is inhibited, the heterologous secretory expression of the cyclodextrin glycosyltransferase protein can be effectively promoted, the disproportionation activity of the cyclodextrin glycosyltransferase is improved, and further the conversion rate of the enzyme to linear substrates is improved, thereby the yield of AA-2G produced by the linear substrates such as maltodextrin and the like can be improved. The industrial production of the AA-2G is more facilitated.

Description

technical field [0001] The invention relates to an optimization method for improving the secretion and expression of cyclodextrin glucosyltransferase, which belongs to the technical fields of enzyme engineering and culture medium. Background technique [0002] Vitamin C glucoside, the Chinese name 2-O-α-D glucopyranosyl ascorbic acid (AA-2G), is the product of vitamin C and starch substances through the action of glycosyltransferase, developed by Hayashibara Institute of Biochemistry and Oka Co-discovered by Shan University Department of Pharmacy. Ascorbic acid glucoside is a sugar derivative of vitamin C, which is mainly used in cosmetics as a whitening additive, and has excellent stability and antioxidant properties, and is a better substitute for L-ascorbic acid. [0003] At present, most of AA-2G used in cosmetics is provided by Hayashibara Corporation of Japan, which has a market monopoly position. AA-2G is expensive, about 100 times more than vitamin C. Therefore, o...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12P19/18C12P19/44
CPCC12N9/1074C12Y204/01019C12P19/18C12P19/44
Inventor 李梦娇石艳丽温朝王冠凤郭学平
Owner BLOOMAGE BIOTECHNOLOGY CORP LTD
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