Low-temperature excision inulase mutant MutP126R stable at medium temperature

An exo-inulinase and mutant technology, which is applied in the direction of enzymes, hydrolases, glycosylases, etc., can solve the problems of poor heat resistance and low-temperature enzyme thermal denaturation

Active Publication Date: 2021-04-30
YUNNAN NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, low-temperature enzymes generally have poor heat resistance. During the production, st

Method used

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  • Low-temperature excision inulase mutant MutP126R stable at medium temperature
  • Low-temperature excision inulase mutant MutP126R stable at medium temperature
  • Low-temperature excision inulase mutant MutP126R stable at medium temperature

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033]Example 1 Construction and conversion of wild enzyme INUAMN8 expression vector

[0034](1) Extraction Pylori genomic DNA: Take the liquid culture 2D bacteria, add 1 mL of lysozyme, 37 ° C for 60 min, then according to the bacterial genomic DNA extraction kit (Tiangen Endochem Technology (Beijing) Co., Ltd.) The specification is extracted in genomic DNA, placed at -20 ° C.

[0035](2) According to GenBank records, the extracted laboase nucleotide sequence JQ863111 (SEQ ID No.4), design primer 5'-atgaattcatgacgacgcc-3 '(SEQ ID No.5) and 5'-Tcaacggcgcgacgtcga-3' ( SEQ IDNO.6), PCR amplification is performed in Pikacilus genomic DNA, and the PCR reaction parameters are: 95 ° C degeneration of 5 min; then 95 ° C degeneration 30SEC, 58 ° C for 30 SEC, 72 ° C for 1 min 30sec, 30 cycles 72 Holly in ° C for 5 min. The PCR results were obtained from the coding gene INUAMN8 of wild exotutozyme INUAMN8. According to the external troplayer nucleotide sequence JQ863111, INUAMN8 can also be obtain...

Embodiment 2

[0038]Example 2 Construction and transformation of the expression vector of mutant enzyme MUTP126R

[0039](1) Design primers 5'-ccgcttcgtggccgggcaggcgccgctcgccgggcaggcgcagtcgctcgcc-3 '(SEQ ID No.7) and 5'-TGCGGCCCCGAAGCGCGCGCGCGTCCCCGTA-3' (SEQ ID No.8), with plasmid PEASY-E1-INUAMN8 for PCR amplification, PCR The reaction parameters were: 95 ° C denaturation 30SEC; then 95 ° C degenerate 15 SEC, 70 ° C annealing 15SEC, 72 ° C for 3 min 30SEC, 30 cycles 72 ° C after 5 min after 72 ° C. The PCR results were obtained from recombinant expression of MUT P126R (SEQ ID No. 2) linearized plasmid PEASY-E1-MUTP126R. MUT P126R and PEASY-E1-MUTP126R can also be obtained by gene synthesis.

[0040](2) In the PCR product of 50 μl of linearized plasmid PEASIY-E1-MUTP126R, 1 μl of DPNI enzyme was added to 37 ° C for 1 h.

[0041](3) Using MUTThe II Fast Mutagenesis Kit kit, the digestive product in step (2) was placed at 37 ° C for 30 min.

[0042](4) Transform the ligation product in step (3) by a heat exci...

Embodiment 3

[0043]Example 3 Preparation of recombinant wild enzyme INUAMN8 and mutant enzyme MUTP126R

[0044](1) Recombinant strain BL21 (DE3) / INUAMN8 and BL21 (DE3) / MUTP126R were seeded in LB (including 100 μg ml) in LB (including 100 μg ml) at 0.1% inoculation-1AMP) In the culture solution, the rapid oscillation of 16 h at 37 ° C.

[0045](2) The activated bacterial liquid is then inoculated into fresh LB in a 1% inoculation amount, respectively, and 100 μg ml.-1AMP) In the culture solution, rapid oscillation culture was obtained from about 2 to 3 hours (OD600 reached 0.6-1.0), and IPTG of the final concentration of 0.7 mm was induced, and the oscillating culture was continued at 20 ° C for about 20 h. 12000 rpm is centrifuged for 5 min, and the bacteria were collected. Ultrasonic cracked bacteria was ultrasonic in a low temperature water bath with an appropriate amount of pH = 7.0 mcilvainebuffer suspension. After 11,000 rude concentrated crude enzyme solutions were centrifuged for 10 min, th...

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Abstract

The invention discloses a low-temperature exoinulinase mutant MutP126R stable at medium temperature, the mutant MutP126R has an amino acid sequence as shown in SEQ ID NO.1, the thermal activity and thermal stability of the mutant MutP126R are changed, the optimal temperature is increased, the thermal stability is better, and the mutant MutP126R is beneficial to production, storage, transportation and the like of enzymes. The optimum temperature of the purified wild enzyme InuAMN8 is 35 DEG C, and the optimum temperature of the mutant enzyme MutP126R is 40 DEG C; after the wild enzyme InuAMN8 is treated at 55 DEG C for 10-60 minutes, the enzyme activity of the wild enzyme InuAMN8 is reduced from 70% to 17%, and the enzyme activity of the mutant enzyme MutP126R is reduced from 70% to 26%. The low-temperature excision inulase mutant MutP126R disclosed by the invention can be applied to the industries of food, wine brewing, biological energy and the like.

Description

Technical field[0001]The present invention relates to a cryogenic extrapolase mutant, and more particularly to a medium temperature, stable low-temperature extractazaclopine enzyme mutant MUTP126R.Background technique[0002]The disease of Jerusalemooks is very strong, extremely strong, easy to proliferate, high yield, cold resistance, poor and drought, suitable for non-cultivated land of non-cultivated land in poor slope, arid saline, and do not compete with crops. The daisy powder is the main component constituting the tubers of Jerusalemooks, which can accounted for 19% or dry weight of the wet weight.[0003]The daisy powder is a fruit glycan containing a molecular glucose residue. Exogenous serotozymes can degrade synpring and a small amount of glucose with a small amount of fruit syrup with a content of up to 95%. Fruit sugar can promote the absorption of iron to iron, promote postmenopausal women's absorption of calcium, prevent obesity, promote the formation of intestinal benefi...

Claims

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Application Information

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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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