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A technology of xylanase and coding gene, which is applied in the field of xylanase coding gene, can solve problems such as container corrosion and achieve high-efficiency production
Active Publication Date: 2009-04-29
北京同力兴科农业科技有限公司
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Acidolysis is faster, but it is often accompanied by the generation of toxic substances, and it is easy to cause corrosion of containers
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Embodiment 1
[0024] Embodiment 1, the overlapping PCR method synthesis of xylanase coding gene
[0025] According to the reported amino acid sequence (GenBank Accession No. DQ168666) of Aspergillus thiochrome xylanase xynB and the codon preference of Pichia pastoris, 8 pairs of primers (see Table 1) were designed to amplify xylanase by overlapping PCR method. Carbohydrase coding gene sequence.
[0026] Table 1 Primer sequences for amplification of xylanase-encoding gene by overlapping PCR method
[0030] Embodiment 2, utilize the recombinant bacterium fermentative production xylanase that contains xylanase coding gene
[0031] 1. Construction of recombinant bacteria containing xylanase encoding gene
[0032] After the xylanase coding gene amplified by the above-mentioned Example 1 (i.e., the PCR product amplified with primers B-F-8 and B-R-8) was purified, the restriction endonucleases EcoRI and NotI were used to carry out double-enzyme Then, it was ligated with Pichia pastoris constitutive expression vector pGAPzαA (purchased from Invitrogen, USA) after the same enzyme digestion, and the ligated product was transformed into Escherichia coli Top10 competent cells, and positive clones were obtained after screening with the antibiotic Zeocin. The plasmid of the positive clone was extracted, and the obtained recombinant expression vector was named pGAP-xynB. After linearizing 10 μg of the recombinant expression vector pGAP-xynB with the restriction endonuclease BspHI (pur...
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Abstract
The invention discloses a coding gene of xylanase. The gene is any one of the following three genes: (1) a nucleotide sequence of a gene1 is sequence 1 in a sequence table; (2) under the strict condition, a gene2 can be hybridized with a DNA sequence restricted in the sequence 1 of the sequence table and encodes a DNA molecule of the xylanase; and (3) a gene3 has autoploidy with the gene1 by over 90 percent and encodes the DNA molecule of the xylanase. Through a gene engineering technology, the coding gene of the xylanase is synthesized and is transferred to pichia pastoris, thereby producing the xylanase in high efficiency. The research of shaking-flask fermentation shows that the output of the xylanase produced through the method reaches 20000U / mL; and the optimum catalytic temperature of the xylanase is 50 DEG C; the optimum catalytic PH is 4.4; and the xylanase is suitable for use in a feed additive of pigs, chickens and other monogastric animals.
Description
technical field [0001] The invention relates to a xylanase coding gene and its application. Background technique [0002] Xylan is a linear xylose polymer connected by β-1,4-xylosidic bonds, and a variety of different substituents are attached to the side chain. In nature, xylan is the second most abundant hemicellulose polysaccharide. In angiosperms, xylan accounts for 15% to 30% of the dry weight of the plant, and in gymnosperms, it accounts for 7% to 7% of the dry weight of the plant. 12%. Xylan cannot be digested and absorbed in the digestive tract of animals, and it will affect the absorption and utilization of other nutrients. wheat, etc.) applications. [0003] There are two main methods for degrading xylan in industry, namely acid hydrolysis and enzymatic hydrolysis. Acidolysis is faster, but it is often accompanied by the generation of toxic substances, and it is easy to cause corrosion of containers. The enzymes that degrade xylan are mainly endo-β-1,4-xylanas...
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