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

A glucose-based and cyclodextrin technology, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of inconvenient separation and purification of products and increase costs, and achieve the effect of improving specificity and facilitating industrial production

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

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Problems solved by technology

This not only brings great inconvenience to the separation and purification of the product, but also increases the cost

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: This example illustrates the preparation of mutant enzymes K47R, K47H and K47T.

[0026] 1) Site-directed mutation

[0027] Utilize fast PCR technology, carry out site-directed mutation with the expression vector cgt / pET-20b(+) as template, and introduce the site-directed mutation primer of Arg47 codon as follows:

[0028] Forward primer: 5'-CGATCCAATTTG CGG CTCTATTTCGG-3' (the underline is the mutant base),

[0029] Reverse primer: 5'-CCGAAATAGAG CCG CAAATTGGATCG-3' (the underline is the mutated base), the site-directed mutagenesis primer for introducing the His47 codon is:

[0030] Forward primer: 5'-CGATCCAATTTG CAC CTCTATTTCGG-3' (the underline is the mutant base),

[0031] Reverse primer: 5'-CCGAAATAGAG GTG CAAATTGGATCG-3' (the underline is the mutated base), the site-directed mutagenesis primer for introducing the Thr47 codon is:

[0032] Forward primer: 5'-CGATCCAATTTG ACG CTCTATTTCGG-3' (the underline is the mutant base),

[0033] Reverse...

Embodiment 2

[0040] Example 2: This example illustrates an enzyme activity assay.

[0041] 1) Enzyme activity assay method

[0042] Method for measuring α-cyclization activity by methyl orange method: Take 0.1 mL of appropriately diluted enzyme solution, add 0.9 mL of 3% (w / v) soluble starch solution prepared in advance with 50 mM phosphate buffer (pH 6.5) After reacting at 40°C for 10min, add 1.0mL of 1.0N hydrochloric acid to stop the reaction, then add 1.0mL of 0.1mM methyl orange prepared with 50mM phosphate buffer, incubate at 16°C for 20min, and measure the absorbance at 505nm. One enzyme activity unit is defined as the amount of enzyme required to generate 1 μmol α-cyclodextrin per minute under the above conditions.

[0043] The method for the determination of β-cyclization activity by phenolphthalein method: 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 advance with 50 mM phosphate b...

Embodiment 3

[0049] Example 3: This example illustrates the HPLC method for analyzing the amount of cyclodextrin produced.

[0050] Prepare 5% (wet basis, water content 8%, w / v) soluble starch solution as substrate, dissolve 5g starch in 90mL sodium phosphate buffer (pH 6.0), dilute to 100mL, boil in boiling water for 30min. Add a certain amount of wild CGTase, mutant enzyme K47R, K47H, or K47T to make the enzyme activity in the reaction system 0.2U / mL, place it at 40°C for 40h, take 600μL of samples at intervals, centrifuge at 12000rpm for 10min, and take 500μL of supernatant , add 5 μL of glucoamylase (70 U / mL), saccharify at 30°C for 1 hour, inactivate by boiling for 10 minutes, centrifuge at 12,000 rpm for 30 minutes, and take 20 μL of the supernatant for HPLC analysis after filtering through a 0.45 μm ultrafiltration membrane.

[0051] The concentration of α-, β-, and γ-cyclodextrin in the reaction solution is measured by HPLC, and the chromatographic conditions for product analysis b...

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Abstract

The invention relates to a mutant of a cyclodextrin glucosyltransferase with the capability of highly yielding beta-cyclodextrin and a mutation method, which belong to the fields of gene engineering and enzyme engineering. The invention improves the capability of the cyclodextrin glucosyltransferase (CGT enzyme for short) for producing the beta-cyclodextrin by a rite-directed mutagenesis method, provides a mutant proposal for improving the capability of CGT enzyme from Peanibacillus macerans JFB05-01 (CCTCC NO: M 208063) for producing the beta-cyclodextrin, and substitutes Lys on the 47 position of the CGT enzyme for Arg, His and Thr respectively; the beta-cyclodextrin production capacity of the obtained three mutant enzyme of K47R, K47H and K47T is improved compared with wild type CGT enzymes, wherein the mutant enzyme K47T is particularly obvious. The mutant enzymes are more favorable for industrial production of the beta-cyclodextrin than the wild type CGT enzymes.

Description

technical field [0001] The invention relates to a mutant of cyclodextrin glucosyltransferase with high ability to produce β-cyclodextrin and a mutation method, and the invention belongs to the fields of genetic engineering and enzyme engineering. Specifically, the present invention is a technique for improving the product specificity of cyclodextrin glucosyltransferase (CGTase) by site-directed mutagenesis of protein engineering. Background technique [0002] Cyclodextrin is a non-reducing compound series of oligosaccharides with a ring-shaped hydrophobic conical structure formed by more than six glucose links, among which the most commonly used are α-, β-, and γ-cyclodextrin, which are composed of 6, 7, 8 glucose units. At present, the industrial production of cyclodextrin is synthesized by enzymatic method, that is, under the catalysis of CGT enzyme, cyclodextrin is synthesized by converting starch and related substrates through cyclization reaction. Because 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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