Cyclodextrin glucosyltransferase mutant for high-specificity production of alpha-cyclodextrin

A glucose-based, mutant technology, applied in the field of enzyme engineering, can solve the problems of reducing the yield of CGTase, increasing the ratio of β-cyclodextrin, increasing the difficulty of separation and purification, etc., achieving a wide range of industrial application prospects, enhancing activity, and reducing production cost effect

Active Publication Date: 2014-01-01
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The CGTase derived from Paenibacillus macerans JFB 05-01 (CCTCC NO: M 208063) used in the present invention mainly produces α-cyclodextrin at the initial stage of the enzymatic conversion reaction without using an organic solvent, but as the reaction proceeds, The ratio of β-cyclodextrin, one of the by-products, increases significantly, which reduces the yield of CGTase (producing α-cyclodextrin) to the substrate, and also increases the difficulty of separation and purification

Method used

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  • Cyclodextrin glucosyltransferase mutant for high-specificity production of alpha-cyclodextrin
  • Cyclodextrin glucosyltransferase mutant for high-specificity production of alpha-cyclodextrin
  • Cyclodextrin glucosyltransferase mutant for high-specificity production of alpha-cyclodextrin

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1 This example illustrates the preparation of mutant and wild enzymes.

[0026] 1) Site-directed mutation

[0027] Using the plasmid ompA-PET20b(+) / cgt (see: Li Bin, Wu Jing, Chen Jian. Effect of signal peptide on extracellular expression of Paenibacillus macerans α-cyclodextrin glucosyltransferase in Escherichia coli[J ].Industrial Microbiology, 2011,41(3):54-59; Paenibacillus macerans JFB 05-01CCTCC NO:M 208063) as a template, using synthetic mutant primers, for overlapping PCR.

[0028]The PCR reaction system is: 5×PS buffer 10μL, dNTPs Mix (2.5mM) 4μL, forward primer (10μM) 1μL, reverse primer (10μM) 1μL, template DNA 1μL, Prime STAR HS DNA polymerase (5U / μL) 0.5 μL, add double distilled water to 50 μL. PCR amplification conditions were: pre-denaturation at 94°C for 4 min; followed by 30 cycles (98°C for 10 s, 58°C for 5 s, 72°C for 6 min); 72°C for 10 min. The PCR product was digested with Dpn I (Fermentas company), and transformed into Escherichia coli ...

Embodiment 2

[0033] Example 2 This example illustrates an enzyme activity assay.

[0034] Enzyme activity assay method:

[0035] Method for measuring α-cyclization activity by methyl orange method: Take 0.1 mL of appropriately diluted enzyme solution and add 2.0 mL of 1% (w / v) soluble starch solution prepared in advance with 50 mM phosphate buffer (pH 5.5) Medium (substrate preheated at 45°C for 10min), after reacting at 45°C for 10min, add 0.2mL of 3.0M hydrochloric acid to terminate the reaction, then add 0.2mL of 0.44mM methyl orange, incubate at 16°C for 15min, under 505nm Measure the absorbance. One enzyme activity unit is defined as the amount of enzyme required to generate 1 μmol α-cyclodextrin per minute under the above conditions.

[0036] The method for the determination of β-cyclization activity by phenolphthalein method: take 0.1mL of appropriately diluted enzyme solution and add it to 2.0mL of 1% (w / v) soluble starch solution prepared in advance with 50mM phosphate buffer (p...

Embodiment 3

[0037] Example 3 This example illustrates the HPLC method for analyzing the amount of cyclodextrin produced.

[0038] Prepare 5% (w / v) soluble starch solution (pH5.5) as the substrate. A certain amount of CGTase wild enzyme and mutant enzyme were added respectively, the amount of enzyme added was 4U / g starch, and placed at 45°C and 150rpm to react for 10h. Sampling, boiled in boiling water for 10 minutes to inactivate the enzyme, mixed 500 μL with 95% (V / V) ethanol 1:1, left at room temperature for 2 hours to precipitate large molecular weight dextrin or limit dextrin, centrifuged at 12000 rpm for 20 minutes, and took the supernatant for HPLC analysis.

[0039] The chromatographic condition that adopts HPLC to carry out product analysis is: Agilent 1200HPLC chromatograph, Agilent autosampler, chromatographic column Thermo Aps-2 HYPERSIL (4.6mm * 250mm), Agilent 2410 differential detector; Mobile phase (V / V) is 70 % acetonitrile aqueous solution, flow rate 0.8mL min -1 ; Colu...

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Abstract

The invention discloses a cyclodextrin glucosyltransferase mutant for high-specificity production of alpha-cyclodextrin, belonging to the field of enzyme engineering. The cyclodextrin glucosyltransferase mutant is prepared by transforming CGTase derived from Paenibacillus macerans JFB 05-01 and carrying out site-specific mutation on 145-site Asp,146-site Arg and 147-Asp of CGTase. The production capacity of beta-cyclodextrin of the prepared single mutant enzyme is lowered than that of wild-type CGTase, the production capacity of alpha-cyclodextrin is slightly increased, and the specificity of a principal product alpha-cyclodextrin is improved; double mutants and three mutants are obtained through combined mutation at the mutation sites of the CGTase; compared with the wild-type CGTase, the production capacity of the beta-cyclodextrin of the mutants is remarkably lowered, the production capacity of the alpha-cyclodextrin is slightly improved, the specificity of the principal product alpha-cyclodextrin is improved, and therefore, the industrial production of the alpha-cyclodextrin is facilitated.

Description

technical field [0001] The invention relates to a cyclodextrin glucosyltransferase mutant capable of producing alpha-cyclodextrin with high specificity, and a preparation and application method thereof, belonging to the field of enzyme engineering. Background technique [0002] Cyclodextrin (Cyclodextrin, referred to as CD), is a kind of starch or starch substrate under the action of cyclodextrin glucosyltransferase (abbreviated as CGTase), which is composed of D-glucopyranose unit through α-1,4- Cyclic compounds connected end to end by glycosidic bonds, common cyclodextrins are composed of 6, 7 and 8 glucose units, called α-, β- and γ-cyclodextrins, respectively. The barrel structure of cyclodextrin molecules, which are hydrophilic on the outside and hydrophobic on the inside, allows them to form clathrates with many types of hydrophobic guest molecules, thereby changing the physical and chemical properties of these guest molecules. This unique property makes it widely used...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N1/21C12N1/15C12P19/18
CPCC12N9/1074C12Y204/01019
Inventor 吴敬王蕾
Owner JIANGNAN UNIV
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