A class of xylanase mutants with improved stability and their coding genes and applications
A technology of xylanase mutation and encoding gene, which is applied in the field of genetic engineering, can solve the problems of easy inactivation, denaturation of enzyme preparations, increase the production cost of enzyme preparations, etc., and achieves good application potential, improved stability, thermal stability and The effect of improving gastric juice tolerance
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Embodiment 1
[0042] Example 1: Mutation Screening for Mutants of Xylanase XYNTH0
[0043] Referring to the amino acid sequence of XYNTH0 (SEQ ID NO: 1), the optimized gene sequence was obtained according to the codon preference of Pichia pastoris, and its nucleotide sequence was shown in SEQ ID NO: 2. The gene was synthesized and placed in the 5' An EcoR I restriction site was designed at the end, and a Not I restriction site was designed at the 3' end.
[0044] SEQ ID NO: 1:
[0045]
[0046] SEQ ID NO: 2:
[0047]
[0048] Use the GeneMorph II Random Mutation PCR Kit and use the xylanase XYNTH0 gene as a template to perform random mutations. The primer sequences used are as follows:
[0049] XYNTHF: 5'-A GAATTC GATACCACTATTAC-3';
[0050] XYNTHR: 5'-C GCGGCCGC TTAGTTGGCGGT-3';
[0051] The amplified random mutation PCR product was double-digested with EcoR I and Not I, purified and recovered, then connected to the pET-21a(+) vector, transformed into Escherichia coli BL21-DE...
Embodiment 2
[0065] Embodiment 2: fusion PCR integration xylanase forward mutation site
[0066] Using the gene sequence of XYNTH1 as a template, PCR amplifies the DNA fragment at the position of 1-513 bp in the gene, and the primers used are as follows:
[0067] XYNTHF: 5'-A GAATTC GATACCACTATTAC-3';
[0068] XYNTHR(513): 5'-TTGGTAATCATGAGATCCTAAG-3';
[0069] Using the gene sequence of XYNTH2 as a template, PCR amplifies the DNA fragment at the 491-902 bp position in the gene, and the primers used are as follows:
[0070] XYNTHF(491): 5'-CTTAGGATCTCATGATTACCAA-3';
[0071] XYNTHR: 5'-CGCGGCCGCTTAGTTGGCGGT-3'.
[0072] The two PCR products amplified above were gel-cut and recovered, fused by PCR, using the fusion product as a template, amplified with the above-mentioned XYNTHF / XYNTHR primers, and the obtained PCR products were sequenced and verified.
[0073] Sequencing results proved that the DNA sequence shown in SEQ ID NO: 8 has been obtained, indicating that the two mutation sit...
Embodiment 4
[0080] Example 4: Construction of xylanase mutant Pichia pastoris high expression strain
[0081] Xylanase mutant genes XYNTH1, XYNTH2 and XYNTH3 were connected to pPIC9K plasmid with EcoR I and Not I double restriction sites respectively and transformed into Escherichia coli DH5α to obtain the expression vector pPIC of each mutant in Pichia pastoris -XYNm, sequence verification; the expression vectors of each mutant were linearized with Sal I digestion and then electrotransformed into Pichia pastoris GS115, and the transformants of the mutant expression strains were obtained by MD plate screening. A transformant expressing xylanase XYNTH0 was obtained by the same method. The transformants of each mutant were washed with sterile water, diluted appropriately, and spread sequentially on YPD plates containing different concentrations of geneticin (1mg / mL, 2mg / mL, 4mg / mL, 8mg / mL) for screening. High-copy transformants: Streak the high-copy transformants on the YPD plate for activ...
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