Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

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
View PDF7 Cites 9 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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

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 MutDP121ET6 expression vector

[0039] (1) Design primers 5'-TGAAGAAGACCGAAAGACGGGCCGGCAGGCGCAGTCG-3'(SEQ ID NO.7) and 5'-CCGTCTTTCGGTCTTTTTCACTGTAGGCACTG-3'(SEQ ID NO.8), use plasmid pEasy-E1-inuAMN8 as template for PCR amplification, PCR The reaction parameters were: 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, the recombinant expression linearized plasmid pEasy-E1-mutDP121ET6 containing mutDP121ET6 (SEQ ID NO.2) was obtained. mutDP121ET6 and pEasy-E1-mutDP121ET6 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-mutDP121ET6, and digest it at 37° C. for 1 hour.

[0041] (3) Using Mut II Fast Mutagenesis Kit, place the digested product in step (2) at 37...

Embodiment 3

[0043] Embodiment 3 Preparation of recombinant wild enzyme InuAMN8 and mutant enzyme MutDP121ET6

[0044] (1) The recombinant strains BL21(DE3) / inuAMN8 and BL21(DE3) / mutDP121ET6 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 medium, rapid shaking culture for about 2–3h (OD 600 After reaching 0.6-1.0), add IPTG with a final concentration of 0.7mM for induction, and continue shaking culture at 20°C for about 20h. 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 with Nickel-...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com