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A method for synthesizing inulin with inulin as a substrate

A substrate, inulin technology, applied in the field of genetic engineering, can solve problems such as inability to catalyze inulin

Active Publication Date: 2020-09-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the current three reports on difructoanhydrolase, the defect is that there is no identification of the ability of difructoanhydrolase to catalyze inulin or the result of the identification is that the corresponding difructoanhydrolase cannot catalyze inulin

Method used

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  • A method for synthesizing inulin with inulin as a substrate
  • A method for synthesizing inulin with inulin as a substrate
  • A method for synthesizing inulin with inulin as a substrate

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Embodiment 1

[0019] Embodiment 1: AcDFA-IIIase clone expression purification

[0020] The number of the double fructoanhydrolase gene in NCBI is Achl_2895, the full length of the gene is 1338 nucleotides, see SEQ ID No: 1 in the sequence listing. The protein number compiled by the gene is ACL40859.1, with a total of 445 amino acids, as shown in SEQ ID No: 2 in the sequence listing.

[0021] The gene construction plasmid of a double fructoanhydrolase derived from Arthrobacter chlorophenolicus A6 was named pET-22b(+)-AcDFA-IIIase, the plasmid vector used for its cloning was pET-22b(+), and the cloning host cell was E. coli DH5α. Nde I and Xho I were used as the restriction endonucleation sites at both ends of the gene fragment, and a histidine tag was added to the C-terminus for separation and purification experiments by nickel ion affinity chromatography. The constructed pET-22b(+)-AcDFA-IIIase was transformed into Escherichia coli BL21(DE3) competent cells. Containing 100μg mL -1 After...

Embodiment 2

[0023] Example 2: AcDFA-IIIase hydrolyzes double fructose to produce inulin

[0024] AcDFA-IIIase continuously catalyzes inulin in two steps, that is, the enzyme first converts inulin into difructoan, and then further converts difructoan into inulin due to the enzyme’s ability to hydrolyze difructoan. 10U g -1 The AcDFA-IIIase that purifies obtains with 10g L -1 The substrate bisfructose anhydride and 50 mM phosphate buffer solution of pH 6.5 were reacted at 45-60° C. for 12 hours, and the reaction was terminated in a boiling water bath for 10 minutes. After centrifugation at 18000×g for 20min at 4°C, the reaction solution was filtered through a 0.22 μm filter membrane into a liquid phase vial, and the high-performance liquid phase was equipped with a sugar column Sugar-Pak I (4.6mm×250mm, Waters, MA, USA) and a differential refraction display. For the detection of inulin, the mobile phase is degassed ultrapure water, and the temperature of the Sugar-Pak column is 85°C. as ...

Embodiment 3

[0025] Example 3: AcDFA-IIIase continuously catalyzes the production of inulin from inulin

[0026] 10U g -1 The AcDFA-IIIase that purifies obtains with 100g L -1 The inulin is mixed in a buffer solution with a pH of 5.5-7.0, and the reaction temperature is 55° C. After 12-24 hours of reaction, the reaction solution is boiled in water for 10 minutes to remove impurities such as proteins. After filtering the reaction solution with a 0.22 μm ultrafiltration micromembrane, the sample was loaded on the high performance liquid phase system, which was equipped with Sugar-Pak I (4.6 mm×250 mm) and a differential refractive index display. The mobile phase was degassed ultrapure water, and the temperature of the Sugar-Pak column was 85°C. as attached figure 2 As shown, AcDFA-IIIase catalyzed 40g L -1After 12 hours of inulin, double fructose anhydride and a small amount of inulin were produced, and as time went on to 24 hours, the amount of double fructose anhydride and inulin incr...

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Abstract

The invention discloses a method for synthesizing inulobiose by taking synanthrin as a substrate, belonging to the technical field of genetic engineering. AcDFA-IIIase as difructose anhydride hydrolase is capable of catalyzing a substrate difructose anhydride into inulobiose, the optimal pH value of the catalytic reaction is 6.5, and the optimal temperature of the catalytic reaction is 55 DEG C. AcDFA-IIIase is also capable of catalyzing synanthrin under the same condition so as to generate difructose anhydride and inulobiose. Therefore, AcDFA-IIIase has the capability of continuously catalyzing synanthrin, namely that AcDFA-IIIase is capable of firstly converting synanthrin into difructose anhydride and further converting difructose anhydride into synanthrin. According to the method for synthesizing inulobiose by taking cheap synanthrin as the substrate, a great reference value is provided for the industrial production of inulobiose.

Description

technical field [0001] The invention relates to a method for synthesizing inulin with inulin as a substrate, and belongs to the technical field of genetic engineering. Background technique [0002] Inulin is a straight-chain polysaccharide formed by fructose connected by β-2,1-glycosidic bonds. There is a glucose group at the reducing end of the chain. The molecular formula of inulin is expressed as GF n . Among them, G represents the glucose group at the reducing end, F represents the fructose molecule, and n represents the number of fructose units. The degree of polymerization of inulin is 2-60, and the molecular weight is between 3500-5000 Da. Inulin can improve the drought tolerance of plants and also act as a vacuolar osmotic buffer and cryoprotectant. This is mainly due to the fact that plant inulin is mainly distributed in the cell vacuole, which plays a role in regulating osmotic pressure. Inulin, a fructan that has energy storage properties like glucans like sta...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/14C12P19/12C12N15/70C12N1/21C12R1/19
CPCC12N9/24C12N15/70C12P19/12C12P19/14
Inventor 沐万孟江波郁书怀张涛朱莺莺程园园
Owner JIANGNAN UNIV
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