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A kind of endoxylanase mutant s35f07 and its preparation method and application

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: 2022-03-08
YUNNAN NORMAL UNIV
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  • 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

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  • A kind of endoxylanase mutant s35f07 and its preparation method and application
  • A kind of endoxylanase mutant s35f07 and its preparation method and application
  • A kind of endoxylanase mutant s35f07 and its preparation method and application

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...

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Abstract

The invention discloses an endoxylanase mutant S35F07 and its preparation method and application. The mutant S35F07 has the amino acid sequence shown in SEQ ID NO.1. CaCl 2 , MgSO 4 , ZnSO 4 、CoCl 2 、NiSO 4 , FeCl 3 , FeSO 4 , EDTA, Pb(CH 3 COO) 2 , activity in β-Mercaptoethanol, NaCl and Na at 3.0-30.0% (w / v) 2 SO 4 Activity in 15.0~30.0% (w / v) NaNO 3 The activity in has been improved. The mutant endo-xylanase S35F07 of the present invention can be applied to biotechnology fields such as food processing and feed additives under high temperature and / or high salt 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

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Application Information

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Patent Type & Authority Patents(China)
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
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