A kind of xylanase mutant and its application
A technology of xylanase mutation and xylanase, which is applied in the field of xylanase mutants, can solve problems such as difficulty in meeting industrial needs and poor thermal stability
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Embodiment 1
[0028] According to the spatial structure of the xylanase with strong heat resistance of the GH11 family, the C-terminus has an obvious β-fold, and through sequence comparison and analysis of the difference in the temperature factor of the C-terminus in the crystal structure, the fungus Neoflagellates The C-terminus of the xylanase is modified, and the sequence is shown in SEQ ID No: 1, so that the heat resistance of the xylanase is improved. Amino acids in the amino acid sequence SEQ ID No: 3 of the fungus Neoflagellate xylanase have the following changes: N207S, G208S, and A210S. The specific scheme is to use the fungus Neoflagellate xylanase as a template to mutate the fungus Neoflagellate xylanase gene, and obtain a new mutant of the xylanase gene, SEQ ID No: 2, In addition to constructing a recombinant plasmid with PPIC9K, the mutated gene can also construct a recombinant plasmid with expression vectors such as PPIC9, PPICZaA\B\C, PPICZA\B\C, PGAPZaA\B\C, and transform th...
Embodiment 2
[0030] Such as figure 1 Shown, the construction method of xylanase mutant comprises the following steps:
[0031] S101: The recombinant plasmid with the xylanase gene of the fungus Neoflagellates linked to the ppic9k vector was used as a template for mutation PCR amplification; the PCR system was prepared in 50 μL according to the kit instructions;
[0032] S102: Take 10 μl of the PCR product, and detect it by 1% agarose gel electrophoresis. After the band is correct, add 1 μl DMT enzyme to the PCR product, mix well, and incubate at 37°C for 1 hour;
[0033]S103: Then transform, add 5 μl of the mutated product to 50 μl of DMT competent cells (add the product when the competent cells are just thawed), lightly flick and mix, ice bath for 30 minutes; accurately heat shock at 42°C for 45 seconds, place immediately Place on ice for 10 minutes; add 500 μl LB medium, 200 rpm, and incubate at 37°C for 1 hour; centrifuge at 7000 rpm for 3 minutes, discard part of the supernatant, ke...
Embodiment 3
[0037] Enzyme activity is defined as: the amount of enzyme required to release 1 μmol of reducing sugar per minute from a xylan solution with a concentration of 5.0 mg / mL at a temperature of 50 ° C and pH = 6.0 is an enzyme activity unit, expressed in U (QB / T 4483-2013).
[0038] 1. Test materials and reagents
[0039] Strains and vectors: Escherichia coli DMT competent was purchased from Beijing Quanshijin Biotechnology Co., Ltd., expression vectors PPIC9K or PPIC9, PPICZaA\B\C, PPICZA\B\C, PGAPZaA\B\C, PPINK Hc\Lc etc. and Pichia pastoris GS115 or (X33, SMD1168, PICHIAPINK) (from INVITROGEN).
[0040] 2. Enzymes and other biochemical reagents
[0041] DNA polymerase, nuclease endonuclease and dNTP were purchased from TaKaRa Company; xylan was purchased from Sigma Company; FastMutagenesis System kit was purchased from TRANSGENBIOTECH Company, and the others were domestic reagents (all of which can be purchased from common biochemical reagent companies).
[0042] Medium: LB...
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