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Method for preparing beta-cyclodextrin by yeast

A technology of cyclodextrin and yeast, which is applied in the direction of fermentation, etc., can solve the problems of difficult continuous production, easy pollution, and long culture time of strains, and achieve the effect of eliminating purification steps, improving use efficiency, and simple and effective production

Inactive Publication Date: 2006-07-19
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First of all, the preparation of CGTase is cumbersome, the strain culture time is long, the purification of CGTase is troublesome, and the yield is not high; the next conversion process consumes a lot of energy, equipment investment is high, and it is difficult to achieve continuous production; the final cyclodextrin purification Many process steps, high cost, low efficiency, and easy to cause pollution

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] (1) Construction of recombinant expression vector:

[0045] The cyclodextrin glycosyltransferase Cyclomaltodextrin Glucanotransferase (CGTase, ECnumber: 2.4.1.19) gene was cloned, digested with EcoRI and XhoI and inserted into the yeast expression vector pYD1, named pYD1 / cgt.

[0046] The cyclodextrin glycosyltransferase (CGTase) gene is from Bacillus circulans 251, and its reading frame contains 2139 bases. Encodes a protein of 713 amino acids. Contains the signal peptide Genbank[gi:510491] consisting of the first 25 amino acids.

[0047] (2) Construction of recombinant yeast strain:

[0048] After amplifying the expression vector pYD1 / cgt in Escherichia coli DH5α with high transformation titer, it was transformed into Saccharomyces cerevisiae-EBY100 under the conditions of 1.5Kv and 25μF by electroporation to construct a recombinant yeast strain; The colony grown on the YNB-glucose plate without tryptophan is a positive clone engineering strain;

[0049] Among the...

Embodiment 2

[0061] (1) Construction of recombinant expression vector:

[0062] The cyclodextrin glycosyltransferase Cyclomaltodextrin Glucanotransferase (CGTase, ECnumber: 2.4.1.19) gene was cloned, digested with EcoRI and XhoI and inserted into the yeast expression vector pYD1, named pYD1 / cgt.

[0063] The cyclodextrin glycosyltransferase (CGTase) gene is from Bacillus circulans 251, and its reading frame contains 2139 bases. Encodes a protein of 713 amino acids. Contains the signal peptide Genbank[gi:510491] consisting of the first 25 amino acids.

[0064] (2) Construction of recombinant yeast strain:

[0065] After amplifying the expression vector pYD1 / cgt in Escherichia coli Top10 with high transformation titer, it was transformed into Saccharomyces cerevisiae by electroporation under the conditions of 1.5Kv and 25μF to construct a recombinant yeast strain; The colony grown on the YNB-glucose plate of tryptophan is a positive clone engineering strain;

[0066] Among them, the form...

Embodiment 3

[0078] (1) Construction of recombinant expression vector:

[0079] The cyclodextrin glycosyltransferase Cyclomaltodextrin Glucanotransferase (CGTase, ECnumber: 2.4.1.19) gene was cloned, digested with EcoRI and XhoI and inserted into the yeast expression vector pYD1, named pYD1 / cgt.

[0080] The cyclodextrin glycosyltransferase (CGTase) gene is from Bacillus circulans 251, and its reading frame contains 2139 bases. Encodes a protein of 713 amino acids. Contains the signal peptide Genbank[gi:510491] consisting of the first 25 amino acids.

[0081] (2) Construction of recombinant yeast strain:

[0082] After the expression vector pYD1 / cgt was amplified in Escherichia coli JM109 with high transformation titer, it was transformed into Saccharomyces cerevisiae-EBY100 under the conditions of 1.5Kv and 25μF by electroporation to construct a recombinant yeast strain; The colonies grown on the YNB-glucose plate without tryptophan are positive clones;

[0083] Among them, the formul...

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Abstract

The disclosed preparation method for beta-cyclodextrin comprises: with gene engineering technique, fixing the cyclodextrin glycosyl transfer enzyme expression on surface of saccharomyces cerevisiae cell to ferment and prepare the product; centrifugal removing just the thali cell and residue for separation and purification; condensing the supernatant to obtain the final product. This invention keeps enzyme activity for reutilization, improves yield by producing ethanol and consuming the glucose by yeast, and has wide application with unassailable security.

Description

technical field [0001] The invention relates to a method for producing β-cyclodextrin by using yeast and cyclodextrin glycosyltransferase. Background technique [0002] Cyclodextrin (CD for short) is a group of cyclic oligosaccharides produced by the action of cyclodextrin glucotransferase (CGTase) on starch, the most common ones are α-cyclodextrin, β-cyclodextrin Glycosine and γ-cyclodextrin are composed of 6, 7 or 8 glucosyl units connected by α-1, 4 glycosidic bonds respectively, and the molecular shape is slightly conical ring. According to X-ray and NMR measurements, the inner side of cyclodextrin hole is composed of -CH- group and oxygen atom of glucoside bond, which is hydrophobic; the opening at one end of the hole is the 2,3-position hydroxyl, and the other end is The hydroxyl group at the 6-position makes the outside of the cyclodextrin hydrophilic. Due to the special molecular structure of cyclodextrin "hydrophobic inside and hydrophilic outside", cyclodextrin c...

Claims

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Application Information

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IPC IPC(8): C08B37/16C12P19/04
Inventor 祁庆生王占坤王鹏
Owner SHANDONG UNIV
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