A kind of xylosidase xyl_s and its coding gene and application

A technology of xylosidase and encoding gene, applied in the field of bioengineering, can solve the problem of not obtaining xylosidase reports and the like, and achieve the effect of improving the utilization rate

Active Publication Date: 2016-07-06
ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there is no report of xylosidase

Method used

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  • A kind of xylosidase xyl_s and its coding gene and application
  • A kind of xylosidase xyl_s and its coding gene and application
  • A kind of xylosidase xyl_s and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: the purification of glycosidase and the separation of active subunit thereof

[0026] 1. Cultivation of Cellulosimrobiumcellulans strain F16

[0027] Pick about 1 cm from the cultivated strain slant (xylan medium, containing 1.5% agar) 2 Square bacterial lawn, inoculated into 100ml aseptic wheat bran liquid medium (xylan medium composition: xylan 2%, yeast extract 0.2%, peptone 0.2%, K 2 HPO 4 0.1%), 30°C, 160rpm shake flask culture for 2 days.

[0028] 2. Separation and purification of xylosidase Xyl_S

[0029]10000g / min, after centrifugation for 2min, the supernatant collected is the crude enzyme solution. Proteins with β-xylosidase activity were tracked using p-nitrophenyl-β-D-xyloside (pNP-Xyl) as a specific chromogenic substrate. One enzyme unit is defined as the amount of enzyme required to catalyze the production of 1 μmol of p-nitrophenol within 1 hour at 30°C, pH 7.5, with pNP-Xyl as the substrate. After ammonium sulfate precipitation in tur...

Embodiment 2

[0030] Embodiment 2: Cloning of xylosidase Xyl_S coding gene

[0031] With fibrosis fiber microbacteria (Cellulosimerbiumcellulans) bacterial strain F16 genome DNA as template, the coding gene 4929bp of PCR amplification xylosidase Xyl_S (see Figure 4 ), cloned into the pMD-T vector, and the sequencing verification results showed that it was consistent with the sequence shown in SEQ ID NO.1. The PCR reaction system and reaction conditions are shown in the table below:

[0032]

Embodiment 3

[0033] Embodiment 3: Recombinant expression of xylosidase Xyl_S in Escherichia coli

[0034] Using the vector obtained in Example 2 as a template, PCR amplified the 4929bp CDS region of the target gene, added NdeI / BamHI restriction sites on both sides of the fragment, added 6*His tags before the stop codon TGA, cloned into the pColdIV expression vector, and selected 2 The plasmids of positive clones were sequenced and verified, and the results showed that the sequences were correct. The corresponding recombinant vector is obtained. The PCR reaction system, reaction conditions and primers used are shown in the table below:

[0035]

[0036]

[0037] Take 1 μl of the above-mentioned recombinant vector and transfer it into Escherichia coli BL21 competent cells, use LB / antibiotic Amp (100 μg / ml) plate, coat with 50ul transformation solution, and culture at 37°C O / N. ControlpColdIV does the same.

[0038] Pick a single colony into 50ml LB / Amp (100μg / ml) medium, culture O / N...

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Abstract

A xylosidase Xyl_S and its coding gene and application. The amino acid sequence of the xylosidase is shown in SEQ ID NO.2, and its nucleotide coding sequence is shown in SEQ ID NO.1. The enzyme can be prepared by genetic engineering or artificial synthesis. It has dual activities of β-xyloside hydrolase and β-glucosidase, and can specifically hydrolyze the corresponding xylosidic and glucosidic bonds. It can be widely used in chemical industry, Food, bio-energy, pharmaceutical industry and other application fields.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a xylosidase Xyl_S and its coding gene and application. Background technique [0002] β-Xylosidase is an exonuclease, which hydrolyzes xylobiose and xylooligosaccharides above xylobiose from the non-reducing end in an exocutive way, and the hydrolyzed product is xylose. It is one of the key enzymes for xylan degradation and has important industrial application value. In the energy industry, xylan in industrial and agricultural waste can be converted into xylose by xylanase system, and xylose can be converted into valuable fuels such as alcohol by bacteria and fungi; in the pharmaceutical industry, xylan The hydrolysis of specific substrates by glycanases can produce intermediate conversion products that have important application value in the pharmaceutical industry. For example, Patel et al. used Moraxella.spβ-xylosidase to hydrolyze the 7-position xylose residue of 10...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/19C12N1/21C12P19/14C12R1/19C12R1/645
CPCC12P19/14C12N9/2434C12P19/02C12Y302/01037
Inventor 杨凌窦同意栾宏伟刘兴宝李世阳
Owner ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI
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