Difructose anhydride hydrolase mutant E160F with improved thermal stability

A double fructose anhydrolase technology, applied in the field of enzyme engineering, can solve the problems of low activity of double fructose anhydrolase, poor thermal stability, and limited property research, and achieve the effect of broadening industrial applications and solving poor thermal stability

Active Publication Date: 2022-03-25
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no pure sample of inulin on the market, and there are few reports on the synthesis of inulin
The limited production of inulin may limit the study of its properties, mainly due to the low activity of difructoanhydrolase and poor thermal stability (half-life of 2 hours at 55 °C)

Method used

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  • Difructose anhydride hydrolase mutant E160F with improved thermal stability
  • Difructose anhydride hydrolase mutant E160F with improved thermal stability
  • Difructose anhydride hydrolase mutant E160F with improved thermal stability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Preparation and Enzyme Activity Determination of Wild Type Double Fructose Anhydrolase

[0031] The genome number of the microbial Arthrobacter chlorophenolicus A6 strain in GenBank is CP001341, which contains a double fructoanhydrolase gene (number Achl_2895), and the full length of the gene is 1338 nucleotides (SEQ ID NO.2). The protease number coded by the gene is ACL40859.1, with a total of 445 amino acids (SEQ ID NO.1).

[0032] 1) Construction of recombinant strains:

[0033] The gene encoding double fructoanhydrolase is molecularly cloned and expressed in Escherichia coli engineering bacteria, and the nucleotide sequence such as SEQ ID NO.2 double fructoanhydrolase gene is constructed to the Xho I of the plasmid vector pET-22b (+) Between the Nde I restriction site, a recombinant plasmid was constructed and named pET-22b(+)-AcDFA-IIIase.

[0034] Transform the plasmid pET-22b(+)-AcDFA-IIIase into E.coli BL21(DE3) competent cells in a medium containin...

Embodiment 2

[0041] Example 2: Preparation and expression purification of double fructoanhydrolase mutant E160F

[0042] (1) Preparation of mutant E160F

[0043] The nucleotide sequence such as SEQ ID NO.2 double fructoanhydrolase gene is used as a template for primer design, and the carrier pET-22b(+)-AcDFA- IIIase was used as template for site-directed mutagenesis to construct mutant plasmid pET-22b(+)-E160F.

[0044] Mutant Forward Primer: 5'-GCGGTCGCCATCCG TTC AATACCTA-3'

[0045] Mutant reverse primer: 5'-ATTGGCATAGGTATT GAA CGGATGGCGA-3'

[0046] The PCR reaction system is: 10×PCR Buffer 5 μL, dNTP (2 mmol / L) 4 μL, mutant forward primer (10 μM) 1 μL, mutant reverse primer (10 μM) 1 μL, template pET-22b(+)-AcDFA-IIIase 1 μL, Taq Plus DNA polymerase (5U / μL) 0.5 μL, use double distilled water to make up the volume of the reaction system to 50 μL.

[0047] PCR amplification conditions were: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 1 min, annealing at 56°C for 1 ...

Embodiment 3

[0056] Embodiment 3: Enzyme thermostability assay

[0057] (1) Detection of optimum temperature:

[0058] In a 1mL reaction system, include a final concentration of 10g L -1 Substrate bisfructose anhydride, pH 6.5 phosphate buffer at a final concentration of 50 mM and 100 nmol L -1 Pure enzyme solution was reacted at 30°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, and 80°C for 10 minutes, 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. 1U of enzyme activity is defined as the amount of enzyme required to produce 1 μmol of inulin per minute at pH 6.5 and 55°C.

[0059] The results showed that the optimal catalytic temperature of d...

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Abstract

The invention discloses a difructose anhydride hydrolase mutant E160F with improved thermal stability, and belongs to the technical field of enzyme engineering. According to the invention, difructose anhydride hydrolase (AcDFA-IIIase) derived from a microorganism Arthrobacter chloropherolicus A6 is used as a parent enzyme, the 160th glutamic acid Glu of the amino acid sequence of the difructose anhydride hydrolase is replaced by phenylalanine Phe by using a gene mutation technology to obtain a single-point mutant enzyme E160F, and the single-point mutant enzyme E160F can be used for catalyzing the difructose anhydride hydrolase under the optimal catalysis condition (pH 6.5, pH 7). The thermal stability of the mutant is improved from the half-life period of 2 hours at 55 DEG C to the enzyme activity basically unchanged at 55 DEG C, and the half-life period at 65 DEG C is improved from 30 minutes to 8 hours. The mutant E160F provides a favorable guarantee for further industrial application of the difructose anhydride hydrolase, and is especially suitable for long-time immobilized catalytic production.

Description

technical field [0001] The invention relates to a double fructoanhydrolase mutant E160F with improved thermal stability, belonging to the technical field of enzyme engineering. Background technique [0002] Inulin is a dietary fiber widely used in food. Inulin is a kind of fructan polymerized by fructosyl through β-(2,1) linkage, and its end contains a molecule of glucose. Inulin can be metabolized by various enzymes, including being degraded into dietary fiber fructo-oligosaccharide by inulinase, which is widely used in yogurt, beverages and other products. Or it can be converted into prebiotic difructose anhydride III (DFA-III) by inulin fructotransferase (IFTase). This metabolic pathway is a novel pathway, and the resulting double fructose anhydride is a new type of functional sweetener with prebiotic effects, which has been used in food systems in Japan. Difructoan is not digested and absorbed by the human body, but it is hydrolyzed into inulin by the difructoan hydro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N1/21A23L29/00C12R1/19
CPCC12N9/2402C12N15/70A23L29/06
Inventor 郁书怀赵伟徐寒冰李绮婷
Owner JIANGNAN UNIV
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