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Beta-xylosidase and encoding gene and application thereof

A technology for encoding genes and genes, which is applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of unreported literature, small expression of xylosidase, cumbersome purification steps, etc., and achieve environmental friendliness, simple purification process, The effect of high recovery rate

Inactive Publication Date: 2010-06-09
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there have been reports on the production of xylosidase by engineering bacteria at home and abroad, the problems of small expression of xylosidase and cumbersome purification steps generally exist in previous studies
This p

Method used

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  • Beta-xylosidase and encoding gene and application thereof
  • Beta-xylosidase and encoding gene and application thereof
  • Beta-xylosidase and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042]Embodiment 1, the discovery of β-xylosidase gene

[0043] 1. PCR amplification of the conserved region of the β-xylosidase genome

[0044] According to the amino acid sequence of filamentous fungal β-xylosidase in the GeneBank database, the conserved region was searched using the online software Block Maker (http: / / blocks.fhcrc.org / blocks / blockmkr / make blocks.html). According to the conserved amino acid sequence of xylosidase, the online design software CODHOP (http: / / blocks.fhcrc.org / codehop.html) was used to design degenerate primers xylDF and xylDR (see Table 2) to Paecilomyces thermophila ( The genomic DNA of Paecilomyces thermophila) J18 was used as a template, and the degenerate primers xylDF and xylDR in Table 2 were used for PCR amplification. The PCR amplification conditions were as follows: 95°C for 5min; ℃) 45min, 1min at 72℃, 10 cycles; 30s at 95℃, 45min at 55℃, 1min at 72℃, 20 cycles; 1min at 72℃. The conserved region fragment amplified by PCR (results see...

Embodiment 2

[0051] Embodiment 2, the efficient expression of β-xylosidase

[0052] 1. Acquisition of recombinant bacteria

[0053] 1. Acquisition of β-xylosidase gene

[0054] According to the genes shown in sequence 1 in the sequence listing, primers xylF and xylR at both ends were designed (see Table 2), and the cDNA of Paecilomyces thermophila (Paecilomyces thermophila) J18 was used as a template to carry out PCR amplification using primers xylF and xylR at both ends , the results of gel electrophoresis of the amplified product are shown in figure 2 .

[0055] The PCR amplified product was recovered by the gel kit and connected to the pMD-18T vector, transformed into Escherichia coli JM109 by heat shock method, and the positive recombinant plasmid was named pMD18T-xyl. Then sent for sequencing.

[0056] After sequencing, the amplified product has the 1-1014 nucleotides from the 5' end of sequence 1 in the sequence table, and no stop codon (1015-1017 from the 5' end of sequence 1),...

Embodiment 3

[0077] Embodiment 3, the application of β-xylosidase

[0078] 1. Preparation of corn cob steam explosion liquid

[0079] Weigh 100g corn cob (containing 32.4% xylan), add 800mL of distilled water, soak at room temperature for 12 hours, put it into a high-pressure tank, cover and seal it, heat it and start timing when the reaction temperature reaches 165°C, and open the ball valve immediately after keeping it warm for 30 minutes , Release the material (steam explosion liquid) into the collection tank. Suction filtration when the temperature of the collected material (steam explosion liquid) drops to 60-70°C, after the filtrate is cooled to room temperature, the thin layer chromatography (TLC) measurement result is as follows: Image 6 Shown, HPLC measures its xylooligosaccharide ratio (results are shown in Table 4). Adjust the pH of the steam explosion solution to 7.0 with 1M NaOH solution, and refrigerate at 4°C for later use.

[0080] Table 4. Effect of xylosidase enzymati...

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Abstract

The invention discloses a beta-xylosidase and an encoding gene and an application thereof. The beta-xylosidase is the following protein in 1 or 2: 1. the protein is formed from an amino acid sequence shown by a sequence 2 in a sequence table; and 2. the protein is formed from an amino acid residue sequence of the sequence 2 in the sequence table through the substitution and/or deletion and/or addition of one or more amino acid residues, has the activity of the beta-xylosidase and is derived from the protein in 1. A recombined colon bacillus formed by leading the encoding gene of the protein into a colon bacillus also belongs to the protection domain of the invention. An experiment proves that the beta-xylosidase produced by the recombined colon bacillus mainly is exoenzyme (the exoenzyme accounts for about 98 percent of the total enzymatic activity), and the enzyme amount of a fermented liquor reaches 103.9U/mL as the maximum when the fermented liquor is fermented for 48 hours. Because most of the combined xylosidase is exoprotein, the combined xylosidase is simply purified, and the recovery rate reaches 61.5 percent. The beta-xylosidase provided by the invention can hydrolyze a corncob steam-exploded fluid, has mild reaction conditions and is environmentally-friendly.

Description

technical field [0001] The invention relates to β-xylosidase, its coding gene and application. Background technique [0002] Xylan is the most important component of plant hemicellulose, and it is an abundant and available natural resource. It is composed of β-1,4 glycosidic bonds connecting β-xylopyranose units to form the main chain. Heterogeneous polysaccharides, the final product of complete degradation is xylose, an important industrial raw material. The entire hydrolysis process requires the synergy of xylanase systems including xylanase and xylosidase. β-xylosidase (β-1,4-D-xylan xylanohydrolase; EC.3.2.1.37) mainly acts on the non-reducing end xylosidic bond of xylobiose or xylooligosaccharide in exo mode and releases xylose, so In the whole coordinated hydrolysis process, it can reduce the hydrolysis product of xylan so that the inhibition of xylanase by the product can be relieved to a large extent, and it is the rate-limiting enzyme in the hydrolysis process of ...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N5/10C12N1/00C12N1/21C12P19/14C12R1/79C12R1/19
Inventor 江正强贾会勇滕超周鹏闫巧娟
Owner CHINA AGRI UNIV
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