Mutation method for enhancing beta-cyclodextrin production capacity of beta-cyclodextrin glycosyltransferase

A glucosyl 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 a single type of cyclodextrin product, and achieve the effect of improving specificity and facilitating industrial production.

Inactive Publication Date: 2014-02-05
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the wild CGTase acts on starch, the products obtained are all mixtures of α-, β- and γ-cyclodextrins, wh

Method used

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  • Mutation method for enhancing beta-cyclodextrin production capacity of beta-cyclodextrin glycosyltransferase
  • Mutation method for enhancing beta-cyclodextrin production capacity of beta-cyclodextrin glycosyltransferase
  • Mutation method for enhancing beta-cyclodextrin production capacity of beta-cyclodextrin glycosyltransferase

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0028] Example 1: This example illustrates the preparation of mutants A31R, A31P and A31T.

[0029] (1) Site-directed mutation

[0030] Using rapid PCR technology, the expression vector pST / cgt containing wild CGT enzyme gene was used as a template for site-directed mutation.

[0031] Site-directed mutagenesis primers introducing Arg31 codon:

[0032] Forward primer: 5’-GACGGCAATCCC CGC AACAATCC-3’, the mutated base is underlined,

[0033] Reverse primer: 5’-GGATTGTT GCG GGGATTGCCGTC-3’, underlined are the mutated bases;

[0034] Site-directed mutagenesis primers introducing the Pro31 codon:

[0035] Forward primer: 5’-GACGGCAATCCC CCC AACAATCC-3’, the mutated base is underlined,

[0036] Reverse primer: 5’-GGATTGTT GGG GGGATTGCCGTC-3’, underlined are the mutated bases;

[0037] Site-directed mutagenesis primers introducing Thr31 codon:

[0038] Forward primer: 5’-GACGGCAATCCC ACC AACAATCC-3’, the mutated base is underlined,

[0039] Reverse primer: 5’-GGATTGTT GGT GGGATTGCCGTC-3', the m...

Example Embodiment

[0046] Example 2: This example illustrates enzyme activity analysis.

[0047] (1) Determination of enzyme activity

[0048] Determination of α-cyclization activity: Take 0.1 mL of a properly diluted enzyme solution and add it to a test tube containing 0.9 mL of a 1% (w / v) soluble starch solution prepared in 50 mM phosphate buffer (pH 6.0). After reacting at 50°C for 10 minutes, add 1.0mL 1.0N hydrochloric acid to stop the reaction, and then add 1.0mL of 0.1mM methyl orange solution prepared with 50mM phosphate buffer solution and keep it at 20°C for 15min, and measure the absorbance at 505nm. Using the inactivated enzyme as a blank, the content of α-cyclodextrin was determined corresponding to the α-cyclodextrin standard curve. One unit of enzyme activity is defined as the amount of enzyme required to generate 1 μmol of cyclodextrin per minute under the above conditions.

[0049] Determination of β-cyclization activity: Take 0.1 mL of appropriately diluted enzyme solution and add i...

Example Embodiment

[0055] Example 3: This example illustrates the use of HPLC to analyze the amount of cyclodextrin produced

[0056] To prepare a 5% (wet basis, 8% water content, w / v) maltodextrin (DE3) solution as a substrate, 5g maltodextrin (DE3) was dissolved in 90mL sodium phosphate buffer (pH 6.0), set Bring to 100mL and boil for 30min in boiling water. Add a certain amount of wild CGT enzyme, mutant A31R, A31P, and A31T to make the enzyme activity in the reaction system 1U / mL, put it at 50℃ for 9h, sample 600μL every time, boil the enzyme for 10min, centrifuge at 12000rpm for 10min, take 500μL of supernatant, add 5μL of glucoamylase (70U / mL), saccharify at 30℃ for 1h, boil for 10min to inactivate, centrifuge at 12000rpm for 30min, take supernatant through 0.45μm ultrafiltration membrane and take 20μL for HPLC analysis.

[0057] HPLC determination conditions: Waters600 high performance liquid chromatograph (with differential refractive index detector), chromatographic column Lichrosorb NH 2 (...

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Abstract

The invention relates to a mutation method for enhancing beta-cyclodextrin production capacity of beta-cyclodextrin glycosyltransferase (short for beta-CGT enzyme), and belongs to the field of gene engineering and enzyme engineering. According to the present invention, a site-specific mutagenesis method is adopted to increase beta-cyclodextrin production capacity of the CGT enzyme, the mutation scheme for enhancing beta-cyclodextrin production capacity of the beta-CGT enzyme derived from Bacillus circulans STB01 is provided, mutation of alanine on the site 31 in the CGT enzyme into arginine (Arg), proline (Pro) or threonine (Thr) is performed to obtain mutants A31R, A31P and A31T, beta-cyclodextrin production capacity of the mutant is significantly enhanced compared with beta-cyclodextrin production capacity of the wild CGT enzyme, and the mutation method is more suitable for beta-cyclodextrin industrial production.

Description

technical field [0001] The invention relates to a mutation method for enhancing the ability of beta-cyclodextrin glucosyltransferase to produce beta-cyclodextrin, which belongs to the fields of genetic engineering and enzyme engineering. The invention is a technique for enhancing product specificity of cyclodextrin glucosyltransferase by site-directed mutation method. Background technique [0002] Cyclodextrin is composed of D-glucopyranose connected by α-1,4-glycosidic bonds, and has a ring-shaped hollow conical structure, in which α-, β- and Gamma-cyclodextrin is the most common. Due to its external hydrophilic and internal hydrophobic properties, cyclodextrin can form inclusion complexes with many hydrophobic guest molecules, thereby changing the physical and chemical properties of the guest molecules. Therefore, it is widely used in food, medicine and other industrial fields. [0003] The industrial production of cyclodextrin adopts enzymatic process, that is, it is sy...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/10C12N15/75C12R1/09C12R1/125
CPCC12N9/1074C12N15/70C12Y204/01019
Inventor 顾正彪李兆丰班宵逢程力洪雁李才明
Owner JIANGNAN UNIV
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