Mutant of cyclodextrin glucosyl transferase having highly alpha-cyclodextrin yielding property and mutation method

A technology of glucosyl and cyclodextrin, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of high production cost, limited utilization, expensive commercial use, etc., and achieves the effects of being beneficial to industrialized production and improving specificity

Inactive Publication Date: 2009-08-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Decyl alcohol is generally used for α-cyclodextrin, but this compound is difficult to remove from aqueous solution because its boiling point is as high as 229°C; cyclododexone is generally used for complexation and selective precipitation of γ-cyclodextrin, but this solvent Too expensive for commercial use, and the disadvantages of

Method used

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  • Mutant of cyclodextrin glucosyl transferase having highly alpha-cyclodextrin yielding property and mutation method
  • Mutant of cyclodextrin glucosyl transferase having highly alpha-cyclodextrin yielding property and mutation method
  • Mutant of cyclodextrin glucosyl transferase having highly alpha-cyclodextrin yielding property and mutation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: This example illustrates the preparation of mutant enzymes D372K, Y89D, Y89R, D372K / Y89R.

[0035] 1) Site-directed mutation

[0036] Using rapid PCR technology, single mutant enzymes D372K, Y89D and Y89R site-directed mutations: using the expression vector cgt / pET-20b(+) as a template,

[0037] The primers for site-directed mutagenesis introducing the D372K codon are:

[0038] Forward primer: 5’-GACCGGCGATGGC AAA CCCAACAACC-3' (mutated bases are underlined)

[0039] Reverse primer: 5’-GGTTGTTGGG TTT GCCATCGCCGGTC-3' (mutated base is underlined) The primer for site-directed mutagenesis to introduce the Y89D codon is:

[0040] Forward primer: 5’-CTCCGTCATCAAG GAT TCCGGCGTTA-3' (mutated bases are underlined)

[0041] Reverse primer: 5’-TAACGCCGGA ATC CTTGATGACGGAG-3' (mutated bases are underlined) to introduce the site-directed mutagenesis primer of Y89R codon:

[0042] Forward primer: 5’-CTCCGTCATCAAG CGT TCCGGCGTTA-3' (mutated bases are underlined)

[0043] R...

Embodiment 2

[0055] Example 2: This example illustrates enzyme activity analysis.

[0056] 1) Enzyme activity determination method:

[0057] The method of methyl orange method to determine the activity of α-cyclization: take 0.1mL of a properly diluted enzyme solution and add 0.9mL of a 3% (w / v) soluble starch solution prepared in advance with 50mM phosphate buffer (pH6.5) In, after reacting at 40°C for 10 minutes, add 1.0mL 1.0N hydrochloric acid to stop the reaction, then add 1.0mL 0.1mM methyl orange prepared with 50mM phosphate buffer, incubate at 16°C for 20min, and measure absorbance at 505nm. One unit of enzyme activity is defined as the amount of enzyme required to produce 1 μmol α-cyclodextrin per minute under this condition.

[0058] Phenolphthalein method for determination of β-cyclization activity: take 0.1 mL of appropriately diluted enzyme solution and add it to a test tube containing 0.9 mL of 3% (w / v) soluble starch solution prepared in 50 mM phosphate buffer (pH 6.5) After rea...

Embodiment 3

[0063] Example 3: This example illustrates the HPLC method to analyze the amount of cyclodextrin produced.

[0064] A 5% (wet base, 8% water content, w / v) soluble starch solution was prepared as a substrate, 5 g starch was dissolved in 90 mL sodium phosphate buffer (pH 6.0), the volume was adjusted to 100 mL, and the solution was boiled in boiling water for 30 minutes. Add a certain amount of wild CGT enzyme, mutant enzyme D372K, Y89D, Y89R or D372K / Y89R respectively to make the enzyme activity in the reaction system 0.2U / mL, put it at 40℃ for 40h, sample 600μL every time, centrifuge at 12000rpm for 10min, take 500μL of supernatant, add 5μL of glucoamylase (70U / mL), saccharify at 30°C for 1h, boil for 10min to inactivate, centrifuge at 12000rpm for 30min, filter the supernatant with 0.45μm ultrafiltration membrane and take 20μL for HPLC analysis.

[0065] The concentration of α-, β-, and γ-cyclodextrin in the reaction solution was determined by HPLC. The chromatographic conditions...

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Abstract

The invention relates to a mutant of a cyclodextrin glucosyltransferase with the capability of highly yielding alpha-cyclodextrin and a mutation method, which belong to the fields of gene engineering and enzyme engineering. The invention improves the specificity of products of the cyclodextrin glucosyltransferase (CGT enzyme for short), provides a mutant proposal for improving the capability of CGT enzyme from Peanibacillus macerans JFB05-01 (CCTCC NO: M 208063) for producing the alpha-cyclodextrin, and substitutes Asp on the 372 position of the CGT enzyme for Lys, and Tyr on the 89 position as Asp and Arg to respectively obtain single mutant enzyme D372K, Y89D and Y89R; the alpha-cyclodextrin production capacity of the obtained mutant enzyme is improved compared with wild type CGT enzymes; genetic fragments of the CGT enzyme with Lys 372 are substituted by corresponding genetic fragments of Y89R so as to obtain double mutant enzyme D372K/Y89R; and the yield of the alpha-cyclodextrin of the ouble mutant enzyme D372K/Y89R is improved by 1.5 times compared with the wild type CGT enzyme, while the yield of the beta-cyclodextrin is reduced by 57 percent. The mutants are more favorable for industrial production of the beta-cyclodextrin than the wild type CGT enzymes.

Description

Technical field [0001] The present invention belongs to the field of genetic engineering and enzyme engineering with a mutant of cyclodextrin glucosyltransferase with high alpha-cyclodextrin ability and a mutation method. Specifically, the present invention is a technique for improving the product specificity of cyclodextrin glucosyltransferase (CGTase) by using the site-directed mutagenesis method of protein engineering. Background technique [0002] Cyclodextrin is a series of oligosaccharide non-reducing compounds with cyclic hydrophobic conical structure formed by connecting more than six glucoses. Among them, the most commonly used are α-, β-, and γ-cyclodextrins, which are composed of It is composed of 6, 7, 8 glucose units. At present, the industrial production of cyclodextrin is synthesized by enzymatic method, that is, cyclodextrin is synthesized by converting starch and related substrates through cyclization reaction under the catalysis of CGT enzyme. Since cyclodextrin...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/70C12P19/34C12R1/01
Inventor 吴敬陈坚李兆丰张佳瑜
Owner JIANGNAN UNIV
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