A low-temperature exo-inulinase mutant mutp126r stable at mesophilic temperatures

An exo-inulinase and mutant technology, applied in the direction of enzymes, hydrolases, glycosylases, etc., can solve the problems of poor heat resistance, low-temperature enzymes are prone to heat denaturation, etc., and achieve good thermal stability

Active Publication Date: 2022-06-24
YUNNAN NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, low-temperature enzymes generally have poor heat resistance. During the production, storage, and transportation of enzymes, low-temperature enzymes are prone to thermal denaturation.

Method used

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  • A low-temperature exo-inulinase mutant mutp126r stable at mesophilic temperatures
  • A low-temperature exo-inulinase mutant mutp126r stable at mesophilic temperatures
  • A low-temperature exo-inulinase mutant mutp126r stable at mesophilic temperatures

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Construction and transformation of wild enzyme InuAMN8 expression vector

[0034] (1) Extraction of Arthrobacter genomic DNA: Centrifuge the bacterial liquid cultured for 2 days to take the bacterial cells, add 1 mL of lysozyme, treat at 37°C for 60 min, and then follow the bacterial genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) Arthrobacterial genomic DNA was extracted according to the instructions and placed at -20°C for later use.

[0035] (2) Design primers 5'-ATGAATTCATTGACGACGGC-3' (SEQ ID NO. 5) and 5'-TCAACGGCCGACGACGTCGA-3' ( SEQ ID NO.6), using Arthrobacter genomic DNA as a template for PCR amplification, the PCR reaction parameters are: denaturation at 95°C for 5min; then denaturation at 95°C for 30sec, annealing at 58°C for 30sec, extension at 72°C for 1min 30sec, after 30 cycles of 72 ℃ for 5 min. PCR results obtained the gene inuAMN8 encoding wild exoinulinase InuAMN8. According to the exoinulinase nucleotide ...

Embodiment 2

[0038] Example 2 Construction and transformation of mutant enzyme MutP126R expression vector

[0039] (1) Design primers 5'-CCGCTTCGTGGCCGGCAGGCGCAGTCGCTCGC-3' (SEQ ID NO. 7) and 5'-TGCCGGCCACGAAGCGGCGCGGCGTCACTGTA-3' (SEQ ID NO. 8), use plasmid pEasy-E1-inuAMN8 as a template for PCR amplification, PCR The reaction parameters were: denaturation at 95°C for 30sec; then denaturation at 95°C for 15sec, annealing at 70°C for 15sec, extension at 72°C for 3min 30sec, followed by 30 cycles of incubation at 72°C for 5min. As a result of PCR, a recombinant expression linearized plasmid pEasy-E1-mutP126R containing mut P126R (SEQ ID NO. 2) was obtained. mut P126R and pEasy-E1-mutP126R can also be obtained by gene synthesis.

[0040] (2) Add 1 μL of DpnI enzyme to 50 μL of the PCR product of the linearized plasmid pEasy-E1-mutP126R, and digest at 37° C. for 1 h.

[0041] (3) Utilize Mut II Fast Mutagenesis Kit, the digestion product in step (2) was ligated at 37°C for 30min.

[0042...

Embodiment 3

[0043] Example 3 Preparation of recombinant wild enzyme InuAMN8 and mutant enzyme MutP126R

[0044] (1) The recombinant strains BL21(DE3) / inuAMN8 and BL21(DE3) / mutP126R were inoculated in LB (containing 100 μg mL of 0.1% inoculum) respectively -1 Amp) medium, 37°C for rapid shaking for 16h.

[0045] (2) The activated bacterial solution was then inoculated into fresh LB (containing 100 μg mL of 1% inoculum) -1 Amp) medium, shake quickly for about 2-3 hours (OD600 reaches 0.6-1.0), add IPTG with a final concentration of 0.7 mM for induction, and continue shaking at 20 °C for about 20 hours. The cells were collected by centrifugation at 12000 rpm for 5 min. After suspending the cells with an appropriate amount of pH=7.0 McIlvainebuffer, the cells were disrupted by ultrasonic waves in a low temperature water bath. After the above intracellular concentrated crude enzyme solution was centrifuged at 13,000 rpm for 10 min, the supernatant was aspirated and the target protein was af...

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Abstract

The invention discloses a low-temperature exo-inulinase mutant MutP126R stable at medium temperature. The mutant MutP126R has the amino acid sequence shown in SEQ ID NO.1, and its thermal activity and thermal stability have changed. Elevated temperature and better thermal stability are beneficial to the production, storage and transportation of enzymes. The optimal temperature of the purified wild enzyme InuAMN8 was 35°C, and that of the mutant enzyme MutP126R was 40°C; after treatment at 55°C for 10–60 min, the activity of the wild enzyme InuAMN8 decreased from 70% to 17%, and that of the mutant enzyme MutP126R Enzyme activity decreased from 70% to 26%. The low-temperature exo-inulinase mutant MutP126R of the present invention can be applied to industries such as food, brewing, and bioenergy.

Description

technical field [0001] The present invention relates to a low-temperature exo-inulinase mutant, in particular to a low-temperature exo-inulinase mutant MutP126R that is stable at medium temperature. Background technique [0002] Jerusalem artichoke has very strong disease resistance, strong regeneration, easy proliferation, high yield, cold resistance, barren resistance and drought resistance. Inulin is the main component that makes up Jerusalem artichoke tubers and can account for 19% of its wet weight or 70% of its dry weight. [0003] Inulin is a fructan with a glucose residue at the end. Exoinulinase can degrade inulin to fructose and a small amount of glucose to produce fructose syrup with a content of up to 95%. Fructose can promote the absorption of iron in children, promote the absorption of calcium in postmenopausal women, prevent obesity, and promote the formation of beneficial intestinal flora to prevent colon cancer. Fructose can also be used to produce bioetha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N1/21C12R1/19
CPCC12N9/2402C12N15/70C12Y302/01007
Inventor 周峻沛张蕊黄遵锡岑潇龙唐湘华许波李俊俊韩楠玉吴倩高艳秀
Owner YUNNAN NORMAL UNIV
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