Exo-inulinase mutant MutDR121EH9 with improved low-temperature activity

A technology of mutdr121eh9 and exo-inulinase, which is applied in the field of exo-inulinase mutants, can solve the problems of easy exposure of protease cleavage sites and easy degradation of enzymes, and achieve the effect of reducing the amount of enzymes used and shortening the reaction time

Active Publication Date: 2021-05-25
YUNNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to control the catalytic reaction of enzymes conveniently and effectively, enzymes that are easy to heat denature are needed; at the same time, in order to make the use of enzymes safer, it is necessary for enzymes to be easily degraded after simple treatment, and heat denaturation makes enzymes easy to expose protease enzymes cleavage site, causing the enzyme to be easily degraded

Method used

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  • Exo-inulinase mutant MutDR121EH9 with improved low-temperature activity
  • Exo-inulinase mutant MutDR121EH9 with improved low-temperature activity
  • Exo-inulinase mutant MutDR121EH9 with improved low-temperature activity

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Construction and Transformation of Embodiment 1 Wild Enzyme InuAMN8 Expression Vector

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

[0035](2) According to the exo-inulinase nucleotide sequence JQ863111 (SEQ ID NO.4) recorded in GenBank, 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, and after 30 cycles, 72 ℃ for 5 minutes. The PCR results obtained the co...

Embodiment 2

[0038] Construction and transformation of embodiment 2 mutant enzyme MutDR121EH9 expression vector

[0039] (1) Design primers 5'-TGAAGAAGACCGAAAGACGGGCCTGCACCAGGCGCAGTCGCTCGCC-3'(SEQ ID NO.7) and 5'-TGGTGCAGGCCCGTCTTTCGGTCTTTCTTCACTGTAGGCACTG-3'(SEQ ID NO.8), use plasmid pEasy-E1-inuAMN8 as template for PCR amplification, PCR reaction The parameters are: denaturation at 95°C for 30 sec; then denaturation at 95°C for 15 sec, annealing at 70°C for 15 sec, extension at 72°C for 3 min and 30 sec, and after 30 cycles, incubation at 72°C for 5 min. As a result of PCR, a recombinant expression linearized plasmid pEasy-E1-mutDR121EH9 containing mutDR121EH9 (SEQ ID NO.2) was obtained. mutDR121EH9 and pEasy-E1-mutDR121EH9 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-mutDR121EH9, and digest at 37° C. for 1 hour.

[0041] (3) Using Mut II Fast Mutagenesis Kit, place the digested product in ...

Embodiment 3

[0043] Embodiment 3 Preparation of recombinant wild enzyme InuAMN8 and mutant enzyme MutDR121EH9

[0044] (1) The recombinant strains BL21(DE3) / inuAMN8 and BL21(DE3) / mutDR121EH9 were inoculated in LB (containing 100 μg mL -1 Amp) medium, shake rapidly at 37°C for 16h.

[0045] (2) Then the activated bacterial solution was inoculated into fresh LB (containing 100 μg mL -1 Amp) culture solution, after rapid shaking culture for about 2-3 hours (OD600 reaches 0.6-1.0), add IPTG with a final concentration of 0.7mM for induction, and continue shaking culture at 20°C for about 20 hours. Centrifuge at 12000rpm for 5min to collect the bacteria. After suspending the cells with an appropriate amount of McIlvainebuffer with a pH of 7.0, the cells were disrupted ultrasonically in a low-temperature water bath. The crude enzyme solution concentrated in the cells above was centrifuged at 13,000rpm for 10min, the supernatant was aspirated and the target protein was affinity and purified wit...

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Abstract

The invention discloses an exo-inulinase mutant MutDR121EH9 with improved low-temperature activity, which has an amino acid sequence as shown in SEQ ID NO.1; the thermal activity and the thermal stability of the mutant MutDR121EH9 are changed, so that the mutant MutDR121EH9 has higher activity at low temperature, the thermal stability is reduced and the low-temperature activity is increased, and the use amount of enzyme is reduced or the reaction time is shortened during a low-temperature reaction; meanwhile, the degraded thermal stability makes the enzyme reaction process to be controlled through thermal treatment, wherein optimal temperature of the wild enzyme InuAMN8 is 35 DEG C, and the optimal temperature of the mutant enzyme MutDR121EH9 is 20 DEG C; after treatment at 50 DEG C, the enzyme activity of the wild enzyme InuAMN8 keeps 81% or above, and the enzyme activity of the mutant enzyme MutDR121EH9 is reduced from 32% to 14%. The mutant MutDR121EH9 disclosed by the invention can be applied to the industries of food, wine brewing, washing and the like.

Description

technical field [0001] The invention relates to an exo-inulinase mutant, in particular to an exo-inulinase mutant MutDR121EH9 with improved low-temperature activity. Background technique [0002] Jerusalem artichoke can be planted in most areas of our country. It is a high-density non-grain energy crop, which is resistant to drought, barrenness, salt and alkali, and has high yield. In the dry matter of Jerusalem artichoke tubers, the inulin content can be as high as 70%, and these characteristics make the effective utilization of Jerusalem artichoke become the focus of attention. [0003] Inulin is a polysaccharide formed by the polymerization of fructose. After hydrolysis by exo-inulinase, fructose syrup with a sugar content of up to 95% can be obtained. Fructose is widely used in food, medicine, bio-energy and other industries. It can be used as a natural sweetener to replace sucrose. It can be eaten by diabetics and used as a raw material to produce bioethanol. Therefor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N1/21C12R1/19
CPCC12N9/2402C12N15/70C12Y302/01007Y02E50/10
Inventor 张蕊周峻沛黄遵锡岑潇龙许波韩楠玉唐湘华李俊俊吴倩高艳秀
Owner YUNNAN NORMAL UNIV
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