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Cyclodextrin glycosyltransferase mutant and application thereof

A technology of glucosyl and transferase, applied in the fields of enzyme engineering and microbial engineering, can solve the problems of low efficiency of long-chain glycosyl genistein, and achieve the effect of high product specificity

Active Publication Date: 2020-02-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing cyclodextrin glucosyltransferase has low efficiency in synthesizing long-chain glycosyl genistein and high efficiency in synthesizing short-chain glycosyl genistein. The longer the length, the better the water solubility (see references for details: Li D, Roh SA, Shim JH, Mikami B, Baik MY, Park CS, Park KH.2005. Glycosylation of genistin into soluble inclusion complex form of cyclic glucans by enzymatic modification.J Agric Food Chem53:6516-24)

Method used

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  • Cyclodextrin glycosyltransferase mutant and application thereof
  • Cyclodextrin glycosyltransferase mutant and application thereof
  • Cyclodextrin glycosyltransferase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Preparation and expression of different cyclodextrin glucosyltransferases

[0039] Specific steps are as follows:

[0040] Chemical synthesis coding amino acid sequence is shown in the gene of cyclodextrin glucosyltransferase shown in SEQ ID NO.1 (the nucleotide sequence of gene is shown in SEQ ID NO.2); The gene obtained and pET-20b (+ ) plasmids were ligated after double enzyme digestion (NcoI and XhoI), transformed into Escherichia coli JM109, the transformed product was spread on LB solid medium, cultivated at 37°C for 8h, picked transformants on LB solid medium, and inserted into Cultured in LB liquid medium, cultured at 37°C for 10 hours, extracted the plasmid, sequenced the plasmid, and obtained the recombinant plasmid pET20b-CGT with correct sequencing; transformed the recombinant plasmid pET20b-CG with correct sequencing into Escherichia coli E.coli BL21(DE3 ) to obtain recombinant Escherichia coli pET20b-CGT / E.coliBL21.

[0041] Using the whole pl...

Embodiment 2

[0073] Example 2: Product specificity of different cyclodextrin glucosyltransferases for long-chain glycosylated genistein

[0074] Specific steps are as follows:

[0075] Genistein (purchased from Sigma Company) was dissolved in dimethylsulfoxide (DMSO) to prepare a genistein solution with a final concentration of 7.5g / L; maltodextrin (purchased from Shanghai Sangon Bioengineering Co., Ltd. ) was dissolved in PBS buffer (50mM, pH 6.5) to prepare a maltodextrin solution with a final concentration of 40g / L; the freeze-dried mutants A156V / L174P, A156V / L174P / A166Y, Pure enzymes of A156V / L174P / A166V, A156V / L174P / A166G, A156V / L174P / A166K, A156S, A156L and L174M were dissolved in PBS buffer (50mM, pH 6.5) to prepare CGTase enzyme solution with a final concentration of 15U / L ; Take 300 μL genistein solution, 500 μL maltodextrin solution and 200 μL LCGTase enzyme solution and mix them in a 2 mL small tube with a cover, place on a 40°C, 120 rpm shaker and shake slowly for 20-24 hours ...

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Abstract

The invention discloses cyclodextrin glucosyltransferase mutants and application thereof, and belongs to the technical field of enzyme engineering and microbial engineering. The CGTase mutants have high specificity to long-chain glycosylated genistein products. The yield of long-chain glycosylated genistein prepared from CGTase mutants A156V / L174P, A156V / L174P / A166Y, A156V / L174P / A166V, A156V / L174P / A166G and A156V / L174P / A166K by using maltodextrin as a glycosyl donor and genistein as a glycosyl receptor is increased by 62.5%, 165%, 112.5%, 112.5% and 59.4% respectively, compared with the yieldof long-chain glycosylated genistein prepared from wild type cyclodextrin glucosyltransferase by taking maltodextrin as a glycosyl donor and genistein as a glycosyl receptor.

Description

technical field [0001] The invention relates to a mutant of cyclodextrin glucosyltransferase and its application, belonging to the technical fields of enzyme engineering and microbial engineering. Background technique [0002] Genistein (also known as genistein, genistein, etc.) is considered to be the soybean isoflavone with the highest activity and function, and its glucoside derivative, genistein (also known as 4',5,7 -trihydroxyisoflavone-7-glucoside) in the form of sophora japonicus, mountain bean root and other leguminous plants. [0003] Genistein has a wide range of pharmacological effects in human and animal cells, mainly as follows: (1) It has chemical anti-cancer (breast cancer and prostate cancer, etc.) effects. Genistein has estrogen-like and anti-hormone effects, can inhibit the activity of related enzymes in the synthesis process of tumor cells, inhibit tumor angiogenesis during the formation of tumor cells, and delay or prevent tumors from becoming cancer ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P19/60C12P19/18
CPCC12N9/1074C12N15/70C12P19/18C12P19/60C12Y204/01019
Inventor 韩瑞枝柴宝成倪晔
Owner JIANGNAN UNIV
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