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Xylanase, gene and its application

A kind of technology of gene and application, applied in the field of referring to xylanase, xylanase, gene and its application

Active Publication Date: 2021-08-17
ZHENGZHOU UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention proposes a xylanase protein, gene and application thereof, which solves the problem of hydrolyzing substances containing β-1,4-xylosidic bonds

Method used

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  • Xylanase, gene and its application
  • Xylanase, gene and its application
  • Xylanase, gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Embodiment 1, the acquisition of β-mannanase Xyl1 and its coding gene

[0081] Acquisition and assembly of short sequences of microbial communities in paper mill wastewater obtained by Illumina high-throughput sequencing technology

[0082] Get 2ml paper mill waste water, get the precipitate after centrifugation and wash with 2ml PBS buffer solution (137mmol l -1 NaCl, 2.7mmoll - 1 KCl, 1.5mmol l -1 K H 2 PO 4 , 8.1mmol l -1 Na 2 HPO 4 , pH7.4) washed 3 times. DNA extraction was performed using DNeasy PowerSoilPro Kit (Qiagen, Germany). After that, high-throughput sequencing was performed on the extracted paper mill wastewater DNA using Illumina high-throughput technology, and a total of 53,886,441 short metagenomic sequences were obtained, with an average length of 148.14bp. The basic analysis process of metagenomic sequencing data analysis includes: raw data quality assessment, quality control, removal of host sequences (definite host information is required)...

Embodiment 2

[0083] Example 2, expression of xyl1 in Escherichia coli

[0084] 1. Construction of recombinant expression vector in host Escherichia coli

[0085] The ORF coding gene (SEQ ID NO.1, encoding the amino acid shown in SEQ ID No. 2) of mannanase gene xyl1 was cloned by PCR using paper mill wastewater DNA as a template, and the forward primer used was: 5' GCCTGGTGCCGCGCGGCAGCATGATTAAATTAGGATGTATCAAAGGTC3' (SEQ ID NO.5), its 5' end is added with the recombination sequence of pET28 vector: GCCTGGTGCCGCGCGGCAGC; the reverse primer is 5'TCATTTATTCATAAGGTCTATCACACTTTATTCATAAGGTCTATCACAC3' (SEQ ID NO.6), its 5' end is added with the recombination sequence of pET28 vector: TCATTTATTCATAAGGTCTATCACAC.

[0086] The purified DNA fragment of the PCR product and the vector pET28 recovered after double digestion with Cla I and Xba I were obtained by using the one-step homologous recombination enzyme Hieff CloneTM Plus Multi One StepCloning kit from Shanghai Yisheng Company. Expression vector ...

Embodiment 3

[0094] Example 3, Analysis of the Enzymatic Properties of Recombinant Xyl1 Protein

[0095] The determination of the enzymatic activity of xylanase adopts DNS method, and concrete operation is as follows:

[0096] (1) DNS configuration

[0097] Weigh 10 grams of NaOH and dissolve in approximately 400ml ddH 2 O, then weigh 10g dinitrosalicylic acid, 2g phenol, 0.5g anhydrous sodium sulfite, 200g potassium sodium tartrate tetrahydrate, dissolve them in about 300ml ddH 2 O, mix the two solutions, dilute to 1 liter, and store in the dark.

[0098] (2) Standard curve preparation

[0099] Take 9 thin-walled centrifuge tubes and add the solution according to Table 3.

[0100] table 3

[0101] Standard No. 1 2 3 4 Total xylose (μg) 0 10 20 30 Xylose volume (µL) 0 1 2 3 Add pure water (µL) 100 99 98 97

[0102] The xylose concentration was 10 mg / ml. Add 100µL of DNS to each standard sample in the above table, develop color in a boilin...

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Abstract

The invention belongs to the field of biotechnology, and relates to a xylanase derived from a metagenome, in particular to a xylanase, a gene and an application thereof. The present invention clones a xylanase gene from a paper-making wastewater metagenomic library, and it is identified that the encoded protein can hydrolyze glycosidic bonds well in an endo-cutting manner, and can be highly expressed in Escherichia coli hosts. The xylanase of the invention has good stability and high activity under the conditions of high temperature and high pH, ​​so it can be applied in the papermaking industry.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a xylanase derived from a metagenome, in particular to a xylanase, a gene and an application thereof. Background technique [0002] Xylanase (endo-1,4-β-xylanases, EC3.2.1.8) is an important class of industrial enzymes, mainly including: endo-1,4-xylanase and β-xyloside Enzymes that can degrade xylan into oligosaccharides and xylose. Xylanase mainly hydrolyzes the β-1,4-glucosidic bonds in xylan or hemicellulose in an endo-cutting manner to generate xylooligosaccharides, xylose and a small amount of arabinose. Xylanase exists widely in nature, a variety of bacteria and fungi can produce xylanase, and xylanase has also been found in many microbial communities with cellulose substrates. Xylanases can be divided into different glycoside hydrolase families, and xylanases mainly exist in families 5, 7, 8, 10, 11 and 43. [0003] Xylanase is widely used in many industries, such as paper i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N15/76C12N15/74C12P19/14C12P19/02A23K20/189A23L29/00
CPCA23K20/189A23L29/06C12N9/2482C12N15/70C12N15/74C12N15/76C12P19/02C12P19/14C12Y302/01008
Inventor 买文宁郜继红魏勇军丁文洪张利兵冯小军王娜孟鑫晨代吉华唐启印刘兵梁家伟
Owner ZHENGZHOU UNIV
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