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Sucrose isomerase mutant and application thereof

A sucrose isomerase and mutant technology, applied in the field of enzyme engineering, can solve the problems of increased difficulty in downstream process development, high preparation cost, inability to large-scale application, etc., and achieves immobilized enzyme activity and stability improvement, and final products. The effect of high concentration and improved thermal stability

Active Publication Date: 2021-10-08
HUNAN FLAG BIOTECHNOLOGY CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, neither the wild-type sucrose isomerase nor the reported modified enzymes can take into account good stability and high conversion efficiency.
Poor stability will make it difficult for sucrose isomerase to carry out continuous enzyme conversion and enzyme storage and transportation in industry. The low conversion rate not only increases the difficulty in the development of downstream processes, but also leads to waste of raw materials, both of which lead to isomaltulose The cost of enzymatic preparation remains high and cannot be applied on a large scale

Method used

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  • Sucrose isomerase mutant and application thereof
  • Sucrose isomerase mutant and application thereof
  • Sucrose isomerase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Construction of a prokaryotic expression strain of sucrose isomerase derived from Pantoea disperse

[0040] Download the amino acid sequence of sucrose isomerase (SIM) from Pantoea dispersed in GenBank (SEQ ID NO.1 in this article, corresponding to GenBank accession number: AAP57083.1), remove the 1-21 signal peptide (MFLNGFKTVIALTMASSFYLA) and provide it to Beijing Qing Kebio Technology Co., Ltd. carried out the whole gene synthesis of the coding nucleic acid (using E. coli preferred codons). The C-terminal of the synthetic gene has a His tag (the pET30a(+) vector comes with a His tag), and it is constructed into the prokaryotic expression vector pET30a(+). The prokaryotic expression vector restriction site: 5' end Nde I, 3' end Xho I. Pass the constructed plasmid pET30a(+)-SIM through CaCl 2 Transformed into Escherichia coli expression strain BL21(DE3) by heat shock transformation method, spread on LB solid medium plate containing 50 μg / ml Kanamycin, and ...

Embodiment 2

[0042] Example 2: Purification and immobilization of sucrose isomerase from dispersed Pantoea

[0043]Using the His tag carried in the SIM recombinant protein, the fermentation broth obtained in Example 1 was treated with activated IDA resin (purchased from Anolon (Beijing) Biotechnology Co., Ltd., specific model: His.Bind Resin, Ni-charged). Carry out protein purification, the specific method is as follows: 4 ℃, 10000r / min, centrifuge the fermentation broth for 10min, discard the supernatant, collect the bacterial cells, the bacterial cells are repeatedly washed twice with phosphate buffer (pH 7.5, 0.1mol / L), centrifuged Afterwards, the cells were concentrated 5 times and resuspended in 20ml of phosphate buffer (pH 7.5, 0.1mol / L). The above-mentioned treated bacterial liquid was placed in ice water for ultrasonic crushing until clarification, the ultrasonic crushing conditions were: working for 2s, interval of 5s, ultrasonic power 500W. The crushed lysate was centrifuged in ...

Embodiment 3

[0047] Example 3: Construction of SIM prokaryotic expression strain E.coli BL21(DE3) / pET30a(+)-SIM error-prone mutation library

[0048] The pET30a(+)-SIM recombinant plasmid was used as a PCR template, and conventional T7F / R was used as a universal primer (primer sequence: T7F: 5'-TAATACGACTCACTATAGGG-3'T7R: GCTAGTTATTGCTCAGCGG See SEQ ID NO.3 and 4) for the SIM gene Error-prone PCR amplification, adjust the Mg in the PCR amplification reaction system 2+ , Mn 2+ , dCTP and dTTP oligonucleotide concentrations, so that the base mismatch rate of the mutant library is only 2 / 1000, that is, it is guaranteed that only 1 to 2 amino acids are mutated in a mutant.

[0049] Error-prone PCR reaction system:

[0050]

[0051] Error-prone PCR reaction conditions: pre-denaturation at 95°C for 5 minutes; then denaturation at 94°C for 30 seconds, annealing at 56°C for 1 minute, and extension at 72°C for 1.5 minutes, a total of 25 cycles; finally, extension at 72°C for 10 minutes.

[00...

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Abstract

The invention belongs to the technical field of enzyme engineering, and relates to a sucrose isomerase mutant and application thereof. The mutant carries out mutation on R434P and E273N in wild type sucrose isomerase with an amino acid sequence as shown in SEQ ID NO. 1. Compared with wild type sucrose isomerase, the mutant has the advantage that thermal stability and fermentation activity are obviously improved. The immobilized enzyme prepared from the mutant is higher in activity, has remarkably improved stability, an improved sucrose conversion rate and isomaltulose generation rate, has a lower impurity content and is more suitable for industrial application.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering, and relates to a sucrose isomerase mutant and application thereof. Background technique [0002] Sucrose isomerase (EC 5.4.99.11) is an enzyme capable of converting sucrose to isomaltulose or trehalulose. Compared with sucrose, isomaltulose (Palatinose) not only has similar physical and chemical properties and taste as sucrose, but also has the advantages of good acid stability, low hygroscopicity, and high safety, and has broad market application prospects in food. At the same time, as a new type of sweetener, isomaltulose has the advantages of low sweetness (about half of the sweetness of sucrose), no dental caries, low calorie, etc., and is especially suitable for patients with diabetes and obesity. In addition, as a reducing sugar, it can be used as a precursor for the production of new functional edible sugar alcohols. [0003] At present, there are mainly four methods for the r...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N11/089C12N15/70C12P19/24C12P19/12
CPCC12N9/90C12N11/089C12N15/70C12P19/24C12P19/12C12Y504/99011
Inventor 刘洋郭川志周晶辉赵强赵士敏许岗
Owner HUNAN FLAG BIOTECHNOLOGY CO LTD
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