Cyclodextrin glucosyltransferase for improving product specificity and preparation method thereof

A glycosyltransferase and glucose-based technology, which is applied in the field of cyclodextrin glycosyltransferase and its preparation, can solve problems such as low efficiency, and achieve the effects of improving glycosylation efficiency and facilitating industrial production.

Active Publication Date: 2018-09-14
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, because the glycosylation efficiency (conversion rate) of cyclodextrin glucosyltransferase (CGTase) to genistein is low, therefore, improving the glycosylation effici

Method used

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  • Cyclodextrin glucosyltransferase for improving product specificity and preparation method thereof
  • Cyclodextrin glucosyltransferase for improving product specificity and preparation method thereof
  • Cyclodextrin glucosyltransferase for improving product specificity and preparation method thereof

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[0017] Example 1: Cyclodextrin glucosyltransferase with improved glycosylation efficiency of genistein

[0018] The cyclodextrin glycosyltransferase of the present invention is based on the gene sequence published by GenBank JX412224, replacing the alanine at position 156 of its mature region with other amino acids, and specifically obtained three mutants, namely A156K, A156V, A156N .

[0019] The three positions in the mature region can be substituted with amino acids by chemical total synthesis or PCR.

Example Embodiment

[0020] Example 2: Preparation method of cyclodextrin glucosyltransferase with improved glycosylation efficiency of genistein

[0021] This example takes the PCR method as an example, but the protection of the invention is not limited to the method of obtaining mutations only through the PCR method. The preparation methods of mutant enzymes A156K, A156V, and A156N are as follows:

[0022] 1) Site-directed mutation

[0023] Site-directed mutagenesis of mutant enzymes A156K, A156V and A156N to express vector cgt / pET-20b(+) 1 (1.Han,RZ,Ge,BB,Jiang,MY,Xu,GC,Dong,JJ,and Ni,Y.(2017)High production ofgenistein diglucoside derivative using cyclodextrin glycosyltransferase fromPaenibacillus macerans,J Ind Microbiol Biot 44, 1343- 1354.) is the template, and the primers for site-directed mutagenesis introducing the A156K codon are:

[0024] Forward primer: 5'-GCTTTGCAGAAAATGGT AAA CTGTA-3', the underline is the mutant base;

[0025] Reverse primer: 5'-GAGCCGTTATCATACAG TTT ACCAT-3', underlined...

Example Embodiment

[0038] Example 3: This example illustrates enzyme activity analysis and detection of genistein glycosylation.

[0039] Enzyme activity determination method:

[0040] The method of methyl orange method to determine the activity of α-cyclization: Take 0.1 mL of a properly diluted enzyme solution and add it to 0.9 mL of a 3% soluble starch solution pre-prepared with 50 mM phosphate buffer (pH 6.5) at 40°C After reacting for 10 minutes, add 1.0 mL of 1.0 M hydrochloric acid to stop the reaction, then add 1.0 mL of 0.1 mM methyl orange prepared with 50 mM phosphate buffer, incubate at 16° C. for 20 minutes, and measure the absorbance at 505 nm. One unit of enzyme activity defines the amount of enzyme required to produce 1 μmol α-cyclodextrin per minute under this condition.

[0041] Starch hydrolysis activity determination method: add an appropriate amount of enzyme solution to 50mM phosphate buffer (pH 6.5) containing 1% soluble starch, react at 50°C for 10min, and then measure the redu...

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Abstract

The invention discloses a cyclodextrin glucosyltransferase and a preparation method thereof, and belongs to the field of genetic engineering and enzyme engineering. Through replacement of 156-site Alanine (Ala) of CGTase of P. Macerans strain JFB05-01 (CCTCC NO: M208063) respectively with lysine (Lys), glutamine (Gln) and valine (Val), the genistein glycosylation efficiency is increased by 23%, 44% and 32%, respectively. The mutant enzymes are more conductive to catalysis production of glycosylated genistein compared with wild-type CGTase.

Description

technical field [0001] The invention relates to a cyclodextrin glucosyltransferase and a preparation method thereof, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] Genistein (also known as genistein, genistein, etc.) is considered to be a soybean isoflavone with the highest activity and function. In legumes, genistein often exists in the form of its glucoside derivative, genistin (also known as 4',5,7-trihydroxyisoflavone-7-glycoside). Genistein has a wide range of pharmacological effects in human and animal cells. The main performances are as follows: 1) It has the effect of chemical anti-cancer (breast cancer and prostate cancer, etc.). 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 cells. 2) It can prevent cardiova...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P19/60C12P19/18
CPCC12N9/1074C12N15/70C12P19/18C12P19/60C12Y204/01019
Inventor 韩瑞枝倪晔葛彬彬姚栋董晋军许国超
Owner JIANGNAN UNIV
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