Application of f52-2 protein and its coding gene and hydrolyzed xylan
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A protein and coding region technology, applied in the fields of application, hydrolase, genetic engineering, etc., can solve problems that limit the efficient development of the biofuel industry
Active Publication Date: 2017-03-22
PEKING UNIV
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Problems solved by technology
This limits the efficient development of the biofuel industry and is also an important factor restricting the reduction of production costs of biofuels
In order to achieve higher heat resistance and thermal stability of enzymes, many scholars screen new enzymes from thermophilic microorganisms, but they are rarely applicable in industrial conditions
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Embodiment 1
[0040] Cloning of embodiment 1, F52-2 gene
[0041] In the previous study, the metagenomic library of the dry fermented sludge system was constructed using the Fosmid vector. The ORF was predicted by sequencing and softberry, and the NCBI and Pfam databases were annotated. Get the F52-2 gene, the nucleotide sequence of this gene is the 11th-2170th nucleotide from the 5' end of sequence 1 in the sequence listing, the protein encoded by it is named F52-2, and its amino acid sequence is the sequence in the sequence listing 2 Amino acids 1-719 from the N' terminal. After comparison, the F52-2 protein has the highest similarity of 63% with the β-xylosidase from Clostridium stercorarium, and it is predicted to be a β-xylosidase.
Embodiment 2
[0042] Embodiment 2, the application of F52-2 as β-xylosidase
[0043] 1. Construction of recombinant vector
[0044] Using the artificially synthesized sequence 1 as a template, using F52-2F and F52-2R as primers, and using TAKARA's PrimeStar high-fidelity enzyme to perform PCR amplification, a 2179bp PCR product was obtained, and its nucleotide sequence was sequence 1.
[0045] F52-2F:5'-GGAATTC CATATG CTTTTCCAGCGAAGTGACCTGC-3' contains Nde I restriction site
[0046] F52-2R:5'-CCC AAGCTT TCAGCGGGGCAGCTCCAG-3' contains a Hind III restriction site
[0047] The above PCR amplification system is as follows:
[0048]
[0049] The PCR program is as follows:
[0050] Pre-denaturation at 94°C for 1 min
[0051] 98°C 10s
[0052] 68°C 120s
[0053] Back to 2, 24 cycles
[0054] 72℃ 5min
[0055] end
[0056] The above PCR product was double digested with Nde I and Hind III, and the resulting digested product was ligated with the pET-28a vector (Novagen pET-28a DNA Ca...
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Abstract
The invention discloses F52-2 protein and application of a coding gene of the F52-2 protein and hydrolyzed xylan. The protein disclosed by the invention is protein (1) or (2), wherein the protein (1) is formed by amino acid residues shown in a sequence 2 in a sequence table, and the protein (2) is formed through enabling a residue sequence of an amino acid sequence of the sequence 2 in the sequence table to be subjected to the substitution and / or deletion and / or addition of one or more amino acid residues and is protein which has the same functions and is derived from the protein (1). According to the F52-2 protein and the application of the coding gene of the F52-2 protein and the hydrolyzed xylan, proved by experiments, the optimal temperature for novel beta-xylosidase F52-2 is 60 DEG C under the conditions of taking p-nitrophenol xyloside (pNPX) as a substrate and being in the pH value of 5.0, and activity of 85% can still be maintained after the novel beta-xylosidase F52-2 is incubated for 2 hours at the temperature of 50 DEG C; over 60% residual activity can be maintained in the pH value of 3.5-5.5 after the novel beta-xylosidase F52-2 is incubated for 24 hours in buffer solutions with different pH values at the temperature of 4 DEG C.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to the application of F52-2 protein, its coding gene and hydrolyzed xylan. Background technique [0002] Xylan (Xylan) is an important component of hemicellulose in plant cell walls, accounting for about 35% of cell dry weight, and is an abundant renewable resource in nature, second only to cellulose. Xylanase (Xylananse) is a class of enzymes that can hydrolyze xylan into xylooligosaccharides and xylose. Many natural microorganisms contain xylanase. Because xylanase can be widely used in textile, paper, food and biofuel industries, it has attracted more and more research interests of scholars in recent years. However, during the hydrolysis process of xylanase, a large amount of xylobiose and oligosaccharides produced will strongly inhibit the activity of xylanase, resulting in a sharp drop in the hydrolysis efficiency of xylan and a reduction in the overall production efficiency. β-X...
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