Method for improving soluble expression quantity of beta-cyclodextrin glucosyltransferase

A glucose-based and cyclodextrin technology, applied in the field of enzyme engineering and fermentation engineering, can solve the problems of difficulty in improving, low soluble expression of exogenous proteins, and limited large-scale production and application, and achieves increased bacterial concentration and simple operation. Effect

Active Publication Date: 2022-03-01
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] During the fermentation process of β-cyclodextrin glucosyltransferase, it is often encountered that the exogenous protein cannot be folded correctly in the cell to form an insoluble inclusion body in the cell, and the soluble expression of the exogenous protein is low and difficult to increase. The large-scale production and industrial application of β-cyclodextrin glucosyltransferase are limited, therefore, it is necessary to greatly increase the production of β-CGTase

Method used

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  • Method for improving soluble expression quantity of beta-cyclodextrin glucosyltransferase
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  • Method for improving soluble expression quantity of beta-cyclodextrin glucosyltransferase

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Embodiment 1

[0038]Embodiment 1: Containing the construction of the genetically engineered bacteria expressing the β-cyclodextrin glucosyltransferase gene of the present invention

[0039] Specific steps are as follows:

[0040] The construction of the engineering bacteria containing the β-cyclodextrin glucosyltransferase gene is carried out according to the following methods:

[0041] (1) Obtain the target gene by PCR (the nucleotide sequence of β-cyclodextrin glucosyltransferase is shown in SEQ ID NO.1), and the PCR reaction system is: TaKaRa Ex Taq (5U / μL) 0.25μL, 10× Ex Taq Buffer (mg2+plus) 20mM 5μL, Dntp Mixture (2.5mM each) 4μL, Template 1μL, Primer 1 (1μM) 1μL, Primer 2 (1μM) 1μL, sterile water 37μL. Reaction steps: 98°C for 10s, 55°C for 30s, 72°C for 3min 30s, and 30 cycles.

[0042] (2) Perform double enzyme digestion on the PCR product and the extracted plasmid pET-20b(+). The double enzyme digestion system is: 2×10×Buffer Y 20 μL, Nco I / Nde I 0.5 μL, Bam HI 0.5 μL, Plasmid ...

Embodiment 2

[0045] Example 2: Expression of β-cyclodextrin glucosyltransferase

[0046] Specific steps are as follows:

[0047] (1) Configure fermentation medium: yeast powder 24g / L, tryptone 12g / L, KH 2 PO 4 2.32g / L,K 2 HPO 4 ·3H 2 O16.43g / L, glycerin 5g / L.

[0048] (2) Host bacteria activation culture:

[0049] The E.coli BL 21(DE3) / pET-20b(+)-cgt recombinant strain constructed in Example 1 was streaked and isolated on LB solid medium, cultured in a constant temperature incubator at 37°C for 12 hours, and picked The positive single colony was inoculated into a 250mL Erlenmeyer flask containing 50mL of LB liquid medium, placed in a rotary shaker at 200r / min, and cultured at 37°C for 12h; the seed solution was prepared.

[0050] (3) Fermentation culture:

[0051] The seed solution prepared in step (1) was inoculated in the fermentation medium at an inoculation amount of 4% (v / v), and placed in a rotary shaker at a rate of 200r / min, and induced at 30°C for 96h .

[0052] Ampicil...

Embodiment 3

[0054] Embodiment 3: Optimization of fermentation medium

[0055] Specific steps are as follows:

[0056] details as follows:

[0057] (1) Choice of nitrogen source

[0058] The specific embodiment is the same as Example 2, the difference is that the nitrogen source (yeast powder, tryptone) in the fermentation medium is replaced by: corn steep liquor, yeast powder, tryptone, soybean peptone, fish powder peptone, the concentration is: 36g / L , after the end of the fermentation, the enzyme activity was detected respectively, and the results are shown in Table 1:

[0059] Table 1 Enzyme activity under different nitrogen source conditions

[0060] Type of nitrogen source corn syrup yeast tryptone soy peptone Fish Meal Peptone Enzyme activity (U / mL) 15.95 4.48 4.48 11.79 4.96

[0061] The results showed that when corn steep liquor and soybean peptone were used as nitrogen sources, it was beneficial to increase the soluble expression of β-cyclode...

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Abstract

The invention discloses a method for improving soluble expression quantity of beta-cyclodextrin glucosyltransferase, and belongs to the technical field of enzyme engineering and fermentation engineering. In order to improve the yield of beta-cyclodextrin glucosyltransferase, the invention provides a strategy for improving the soluble expression of foreign protein in recombinant escherichia coli by changing the dissolved oxygen level of a fermentation culture medium and adding metal ions. An effective strategy is provided for efficient expression of the beta-cyclodextrin glucosyltransferase, the operation process is simple, the cost is low, the effect is remarkable, a certain foundation is laid for industrial production of the beta-cyclodextrin glucosyltransferase, and potential industrial application value is achieved.

Description

technical field [0001] The invention relates to a method for increasing the soluble expression of β-cyclodextrin glucosyltransferase, belonging to the technical fields of enzyme engineering and fermentation engineering. Background technique [0002] Cyclodextrin Glycosyltransferase (CGTase for short, EC2.4.1.19) can use glucose polymers such as starch and maltooligosaccharides to generate cyclodextrin through cyclization reaction, and is widely used in food, medicine, chemical industry, agriculture, etc. And cosmetics and other fields have a wide range of applications, the most commonly used cyclodextrin mainly has three kinds, namely α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin. Cyclodextrins are mostly produced by enzymatic methods in the industry. According to the type of main cyclodextrins produced in the initial stage of the catalytic reaction, CGTases can also be divided into α-CGTase, β-CGTase and γ-CGTase. Among them, β - CGT enzymes are most widely used in ind...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N1/20C12R1/19
CPCC12N9/1074C12N1/20C12Y204/01019Y02A50/30
Inventor 李才明周媛媛李兆丰陈双娣顾正彪程力洪雁班宵逢
Owner JIANGNAN UNIV
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