Exo-inulinase mutant MutDP121ET6 with improved low-temperature activity

An exo-inulinase and mutant technology, applied in the direction of enzymes, hydrolases, glycosylases, etc., can solve the problems of easy exposure of protease cleavage sites and easy degradation of enzymes

Active Publication Date: 2021-05-18
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 safe

Method used

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  • Exo-inulinase mutant MutDP121ET6 with improved low-temperature activity
  • Exo-inulinase mutant MutDP121ET6 with improved low-temperature activity
  • Exo-inulinase mutant MutDP121ET6 with improved low-temperature activity

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[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...

Example Embodiment

[0038]Example 2 Construction and transformation of the expression vector of muto enzyme MUTDP121ET6

[0039](1) Design primers 5'-tgaagaagccgaAagcggccgggcgcgcagtcg-3 '(SEQ ID No.7) and 5'-ccgtctttcggtcttcttcactgtaggcactG-3' (SEQ ID No.8), 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 MUTDP121ET6 (SEQ IDNO.2) linearized plasmid PEASY-E1-MUTDP121ET6. MUTDP121ET6 and PEASIY-E1-MUTDP121ET6 can also be obtained by gene synthesis.

[0040](2) In 50 μl of linearized plasmid PEASY-E1-MUTDP121ET6, 1 μl of DPNI enzyme was added to give 1 h at 37 ° C.

[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) Transcend the connection product in step (3) by a heat excitation to E. coli BL21 (...

Example Embodiment

[0043]Example 3 Preparation of recombinant wild enzyme INUAMN8 and mutant enzyme MUTDP121ET6

[0044](1) Recombinant strain BL21 (DE3) / INUAMN / INUAMN / INUAMN / INUTDP121ET6 were inoculated in LB with 0.1% inoculation amount, respectively, inoculated in LB (including 100 μg ml)-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 at 1% inoculation, respectively, and 100 μg · ml.-1AMP) In the culture solution, rapid oscillation culture is about 2-3h (OD600After 0.6-1.0), an IPTG of the final concentration of 0.7 mm was added for induction, and the oscillation culture was continued at 20 ° C for about 20 h. 12000R PM 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...

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Abstract

The invention discloses an exo-inulinase mutant MutDP121ET6 with improved low-temperature activity. The mutant MutDP121ET6 has an amino acid sequence as shown in SEQ ID NO. 1. Compared with a wild enzyme InuAMN8, the thermal activity and the thermal stability of the mutant enzyme MutDP121ET6 are changed, the mutant enzyme MutDP121ET6 has higher activity at low temperature, reduction of the thermal stability and improvement of the low-temperature activity are beneficial to reduction of the use amount of the enzyme or shortening of the reaction time during low-temperature reaction, and deterioration of the thermal stability is beneficial to control of the reaction process of the enzyme through thermal treatment. After treatment at 55 DEG C, the enzyme activity of the wild enzyme InuAMN8 is reduced from 70% to 17%, and the enzyme activity of the mutant enzyme MutDP121ET6 is reduced from 74% to 8%. The low-temperature exo-inulinase mutant MutDP121ET6 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 MutDP121ET6 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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IPC IPC(8): C12N9/24C12N15/56C12N15/10C12N15/70C12N1/21C12R1/19
CPCC12N9/2402C12N15/1031C12N15/70C12Y302/01007C12Q2531/113Y02E50/10
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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