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

Endonuclease mutant S35F07, as well as preparation method and application thereof

An endoxylanase, S35F07 technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve problems such as limited applications

Active Publication Date: 2020-09-25
YUNNAN NORMAL UNIV
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Insufficient adaptability of xylanase to high temperature and / or high salt limits its further application in the field of food and feed

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
  • Endonuclease mutant S35F07, as well as preparation method and application thereof
  • Endonuclease mutant S35F07, as well as preparation method and application thereof
  • Endonuclease mutant S35F07, as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Construction of embodiment 1 mutant library

[0032] The construction process of the mutant library is as follows:

[0033] (1) Extract the genomes of Arthrobacter sp. and Lechevalieria sp. according to the instructions of the GENE STAR Bacterial Genome Extraction Kit;

[0034](2) According to the Arthrobacter sp. endoxylanase nucleotide sequence JQ863105 (SEQ ID No.3) recorded in GenBank, primers 5'-GTGCAGCCGGAGGAAAAACG-3'(SEQ ID No.5) and 5'-GATGAAGGCAGGATCCGGGGT-3'(SEQ ID No.6), using the Arthrobacter sp. genome as a template for PCR amplification to obtain the endoxylanase gene xynAGN16L; Lechevalieria sp. endoxylanase nucleotide sequence JF745868 (SEQ ID No.4), designed primers 5'-GTCTCGGCCCCGCCGGACGT-3'(SEQ ID No.7) and 5'-GGCTCGCTTCGCCAGCGTGG-3' (SEQ IDNo.8), using the Lechevalieria sp. genome as a template for PCR amplification to obtain the endoxylanase gene xynAHJ3; the PCR reaction parameters are: denaturation at 94°C for 5min; then 94°C Denaturation for 3...

Embodiment 2

[0040] Screening of embodiment 2 mutants

[0041] The screening process of mutants is as follows:

[0042] (1) Take 2 μL of bacterial liquid from the 96-well cell culture plate of the preserved mutant library, and inoculate it into 200 μL / well liquid LB culture medium (containing 100 μg mL -1 Amp) in a 96-deep-well plate at 37°C with shaking at 200rpm until OD 600 >1.0 (about 20h), add 2mM IPTG and 100μg mL -1 200 μL liquid LB culture solution of Amp, induced overnight at 20°C, 160 rpm;

[0043] (2) Add 40 μL / well of PopCulture after induction TM Cell lysate, shake and lyse the cells at 25°C for 30 minutes;

[0044] (3) Take 50 μL of McIlvaine buffer (pH=7.0) containing 1.0% (w / v) beech xylan and 50 μL of cell lysate, and react in a 96 deep-well plate in a 70° C. incubator for 2 hours. After the reaction, add 150 μL of DNS reagent to terminate the reaction, incubate in a 140°C incubator for more than 20 minutes and cool to room temperature, and use a microplate reader to ...

Embodiment 3

[0048] Example 3 Enzyme Preparation of Mutant S35F07 and Wild Enzymes rXynAGN16L and rXynAHJ3

[0049] The recombinant strains containing mutant S35F07, wild enzymes rXynAGN16L and rXynAHJ3 were inoculated in LB (containing 100 μg mL -1 Amp) medium, shake rapidly at 37°C for 16h.

[0050] 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 (isopropylthiogalactopyranoside) at a final concentration of 0.1 mM for induction, and continue shaking culture at 20° C. for about 20 h. Centrifuge at 12000rpm for 5min to collect the bacteria. After suspending the bacteria with an appropriate amount of pH=7.0 Tris-HCl buffer solution, the bacteria were disrupted ultrasonically in a low-temperature water bath.

[0051] The crude enzyme solution concentrated in the cells above was centrifuged at 13,000rpm for 10min, the supernatant was aspirated, an...

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 endonuclease mutant S35F07, as well as a preparation method and application thereof. The mutant S35F07 has an amino acid sequence as shown in SEQ ID NO.1. Compared with wildenzyme, the mutant has the advantages that the activity of the mutant in CaCl2, MgSO4, ZnSO4, CoCl2, NiSO4, FeCl3, FeSO4, EDTA, Pb(CH3COO)2 and beta-Mercaptoethanol of 10.0mM and the activity of themutant in NaCl and Na2SO4 of 3.0-30.0 percent (w / v) can be improved. The endonuclease mutant S35F07 can be applied to the bio-technical fields of food processing, feed additives and the like at a high-temperature and / or high salinity environment.

Description

technical field [0001] The invention relates to an endo-xylanase mutant, in particular to an endo-xylanase mutant S35F07 and its preparation method and application. Background technique [0002] Hemicellulose is a heterogeneous polymer second only to cellulose in nature and widely exists in the cell walls of green plants. Among them, xylan has the highest content in hemicellulose, which can be as high as one-third of the dry weight of plant cells. The endoxylanase can randomly degrade the xylan backbone formed by the connection of xylose residues through β-1,4 glycosidic bonds, and produce xylooligosaccharides and / or xylose. Therefore, xylanase plays an important role in the fields of biotechnology involving hemicellulose conversion, such as food, feed, papermaking, environmental protection and energy, especially in the food and feed industry (Chakdar et al.3Biotech, 2016, 6:150.). [0003] Enzymes with good thermal activity and salt resistance can have better applicabili...

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): C12N9/24C12N15/56C12N15/70C12N1/21C12R1/19
CPCC12N9/2482C12N15/70C12Y302/01008
Inventor 张蕊周峻沛黄遵锡胡家乐张朴蕊沈骥冬吴倩慕跃林
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