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Genetic engineering bacterium of expression high-specificity beta cyclodextrin glycosyltransferase and construction method and application of genetic engineering bacterium

A technology of glycosyltransferase and genetically engineered bacteria, which is applied in the direction of transferase, bacteria, and the use of vectors to introduce foreign genetic material, etc. It can solve problems such as large uncertainty, inapplicability of β-CD, and high cost

Pending Publication Date: 2019-05-03
HEFEI UNIV OF TECH
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Problems solved by technology

[0005] At present, Ling Guoqing's published master's thesis "Bacillus cereus β-CGTase Fermentation, Gene Cloning and Expression and Enzymatic Properties Research" discloses a high-yielding β-CGTase strain, which uses nitrogen ions to stimulate Bacillus cereus Obtained by mutagenesis selection, although it has obtained a strain with high β-CGTase production, but the uncertainty is large and the cost is high
Ling Kai’s published master’s thesis "Cyclodextrin Glycosyltransferase Molecular Modification, Product Specificity and Enzymatic Properties Research" found that the amino acid at position 43 of CGTase derived from Bacillus cereus plays an important role in the specificity of CGTase products. The plasmid pET22b / cgt was used as a template, and the 43-position histidine was saturated by the mutation method of the whole plasmid, and finally the mutant enzyme with the best specificity for γ-CD was obtained. It can be seen that although Ling Kai adopted the vertex mutation method to obtain the mutation enzyme, but it is suitable for γ-CD specificity, and is not suitable for β-CD (ie β-cyclodextrin glycosyltransferase) for industrial applications. It can be seen that there is an urgent need for a method that can be directly obtained for β-CD The engineering bacterium and method for the production of

Method used

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  • Genetic engineering bacterium of expression high-specificity beta cyclodextrin glycosyltransferase and construction method and application of genetic engineering bacterium
  • Genetic engineering bacterium of expression high-specificity beta cyclodextrin glycosyltransferase and construction method and application of genetic engineering bacterium
  • Genetic engineering bacterium of expression high-specificity beta cyclodextrin glycosyltransferase and construction method and application of genetic engineering bacterium

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Embodiment 1

[0077] The acquisition of embodiment 1 mutant

[0078] 1. Acquisition of β-CGTase target gene

[0079] The present invention uses the bacterial strain of β-CGTase disclosed in Ling Guoqing's published master's thesis "Bacillus cereus β-CGTase fermentation, gene cloning expression and enzymatic properties research" as the source of target gene acquisition. GeneBank login The number is Accession No.KF269705.1 ,Specific steps are as follows:

[0080] 1.1. Take out the above-mentioned glycerol-preserved bacteria liquid of Bacillus cereus from the refrigerator at -80°C, and inoculate it into LB liquid medium at an inoculum size of 1% for overnight culture;

[0081] 1.2. Take 4mL of the bacteria solution cultured for 12h, centrifuge for 1min, and keep the precipitated bacteria;

[0082] 1.3. Add 250uL RB solution (containing RNase A), and shake fully with a vortex shaker to suspend E. coli in RB until there are no visible bacterial clumps;

[0083] 1.4. Add 250uL LB solution, ...

Embodiment 2

[0111] The acquisition of embodiment 2 genetically engineered bacteria

[0112] 1. Escherichia coli plasmid transformation into cloning bacteria

[0113] 1.1 Take out E.coli DMT competent cells from the -80°C refrigerator, melt them in an ice bath, and divide them into six groups;

[0114] 1.2 After the competent cells melted, add 5 μL PCR amplification products of A47S, A47M, A47Y, and A47R mutants to E.coli DMT competent cells in each group, flick and mix well, and bathe in ice water for half an hour;

[0115] 1.3 Heat shock: Heat shock in a water bath at 42°C for 45 seconds, then quickly place it in an ice bath for 2 minutes, avoid violent shaking during the period;

[0116] 1.4 Activated bacteria liquid: In a sterile environment, preheat the liquid LB medium without resistance to 37°C, add 250uL liquid LB medium to each group, and incubate for 1 hour at 200rpm and 37°C on a shaker;

[0117] 1.5 Bacteria solution resuspension: Centrifuge the activated bacteria solution at...

Embodiment 3

[0129] Example 3 Obtaining of β-CGTase Enzyme

[0130] Inoculate genetically engineered bacteria E.coli BL21(DE3) / A47S, E.coli BL21(DE3) / A47M, E.coli BL21(DE3) / A47Y, E.coli BL21(DE3) / A47R to 50-100μg / mL ampicillin in 50mL LB medium, culture at 250r / min, 35-40℃ for 16-19h, transfer 50μL seed culture solution to 50-100μg / mL ampicillin in 50mL LB medium , Cultivate at 250r / min, 35-40℃ to bacterial concentration OD 600 When the concentration reaches 1.8, add IPTG inducer with a concentration of 0.2mmol / L, ferment and induce at 25°C for 16 hours, and centrifuge to obtain the bacteria. After separation, the supernatant is the crude enzyme solution, and the enzyme activity of β-CGTase is detected by the iodine value method, reaching 9241U / mL.

[0131] It should be noted that, at a fermentation temperature of 15-35°C, OD 600A value of 0.2-2.2, an IPTG concentration value of 0.1-1.0mM, and an induction time of any value within the range of 4-20h can achieve the technical purpose of...

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Abstract

The invention discloses a genetic engineering bacterium of expression high-specificity beta cyclodextrin glycosyltransferase and a construction method and application of the genetic engineering bacterium, and relates to the genetic engineering bacterium. The genetic engineering bacterium adopts plasmid cgt / pET-22b as a formwork. Through fixed-point mutation, mutants A47S, A47M, A47Y and A47R are obtained, and transfer is performed in a host bacterium E.ColiBL21 to obtain BL21(DE3) / A47S, BL21(DE3) / A47M, BL21(DE3) / A47Y and BL21(DE3) / A47R. The engineering bacterium effectively improves the product specificity of beta-CGTase enzymes of Bacillus cereus, and a certain foundation is provided for application of the engineering bacterium to industrial production of beta-cyclodextrin.

Description

technical field [0001] The invention relates to a genetically engineered bacterium, in particular to a genetically engineered bacterium expressing a highly specific β-cyclodextrin glucose glycosyltransferase and its construction method and application. Background technique [0002] Cyclodextrins (CDs) are cyclic glucans composed of 6 to more than 100 glucose units, the common ones are α-, β-, γ-CD (the corresponding numbers of glucose units are 6, 7, 8 respectively) . CDs contain unique hydrophobic cavities that can form clathrates with hydrophobic small molecules. This unique property enables CDs to form water-soluble inclusion complexes with many insoluble drugs, enhance the water solubility of drugs, and improve the stability and bioavailability of drugs. They are ideal drug excipients and are used in water-based parenteral 35 pharmaceutical products including solutions, nasal sprays and eye drops. CDs are also widely used in fields such as food, agriculture, and cosme...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/11C12N15/70C12N1/21C12P19/18C12P19/04
Inventor 张洪斌花敬涵杨静文胡雪芹
Owner HEFEI UNIV OF TECH
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