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Beta-1,4-glucanase encoding gene and preparation method and application thereof

A glucanase and coding technology, applied in application, glycosylase, genetic engineering and other directions, can solve the problems of few hemicellulose substrates, unsuitable for large-scale application, weak degradation activity of konjac polysaccharide, etc., and achieve high efficiency. Degradation effect

Active Publication Date: 2018-10-02
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on glucanase is mostly focused on the degradation of cellulose substrates, while the research on hemicellulose substrates (such as konjac) is relatively less
Moreover, the reported dextranases have weak degradation activities on konjac polysaccharides, which are not suitable for large-scale application.

Method used

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  • Beta-1,4-glucanase encoding gene and preparation method and application thereof
  • Beta-1,4-glucanase encoding gene and preparation method and application thereof
  • Beta-1,4-glucanase encoding gene and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Endo-β-1,4-glucanase full-length gene cloning

[0057] Genomic DNA of Paenibacillus polymyxa was extracted according to the operation steps of Genomic DNA Purification Kit (Thermo, LOT 00105781). After performing multiple sequence alignment analysis on the endo-β-1,4-glucanase gene sequence in The National Center for Biotechnology Information (NCBI) database, a degenerate primer Ppglu-F: 5'-CGGACGCATATGGCCAGYGTGAAAGGATATTATC-3' was designed; Ppglu-R: 5'-GATGATCTCGAGTTCGGCKSYTGCTTYMRCTTGC-3', using the extracted Paenibacillus polymyxa genomic DNA as a template to amplify the gene sequence encoding the mature protein of endoβ-1,4-glucanase (excluding the signal peptide gene). The PCR reaction conditions were: 94°C for 3min, 1 cycle; 94°C for 30s, 68°C for 30s (0.5°C drop per cycle), 72°C for 1min30s, 30 cycles; 72°C for 5min, 1 cycle. PCR products were analyzed by agarose gel electrophoresis (see figure 1 ), the target gene was cleaned and recovered, connecte...

Embodiment 2

[0058] Example 2 Endo-β-1,4-Glucanase Gene Sequence Analysis

[0059] The sequencing results were analyzed using Basic Local Alignment Search Tool (BLAST) in the GenBank database, DNAMAN software was used for multiple sequence alignment, and Vector NTI was used to analyze sequence information.

[0060] The coding region of the obtained endo-β-1,4-glucanase gene (named Ppglu) is 1125 bp long, and its nucleotide sequence is shown in SEQ ID NO 1. Ppglu encodes 374 amino acids and a stop codon, its amino acid sequence is shown in SEQ ID NO 2, the theoretical molecular weight of the protein is 42.18kDa, and the predicted isoelectric point is 5.58. The amino acids encoded by Ppglu mainly contain a cellulase domain, and its domain characteristics are more similar to those of GH1 family members, so it is classified as GH1 family.

Embodiment 3

[0061] Recombinant expression and purification of embodiment 3Ppglu gene in Escherichia coli

[0062] In order to facilitate the recombinant expression of the gene, NdeI and XhoI restriction sites were respectively introduced into the designed upstream and downstream primers. The PCR cleaning product Ppglu and the expression vector pET21a were double-digested with NdeI and XhoI, respectively, and the digested products were cleaned and recovered, and T 4 DNA ligase connection (ligation system: (5μLT 4 DNA Ligase 0.5μL, 10×T 4 DNA LigaseBuffer 0.5 μL, pET21a 2 μL, PCR product 2 μL), ligation conditions: overnight at room temperature. ). Take 5 μL of the ligation product to transform E.coli TOP10 competent cells, spread on solid Luria-Bertani medium containing 100 μg / mL ampicillin, and culture at 37°C for 12-16h. Pick a single clone, use degenerate primers for colony PCR verification, insert the correctly amplified single clone into liquid Luria-Bertani medium containing 100 ...

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Abstract

The invention discloses a preparation method and application of internally tangent beta-1,4-glucanase gene derived from paenibacilluspolymyxa and its enzyme. The preparation method is a technique method utilizing genetic engineering. The internally tangent beta-1,4-glucanase gene is cloned to an escherichia coli expression vector to obtain an escherichia coli recombination strain which can heterologously express the enzyme, and the strain heterologously expresses the prepared internally tangent beta-1,4-glucanase and can efficiently degrade konjac glucomannan (also known as konjac glucomanna).The prepared internally tangent beta-1,4-glucanase can be widely applied the fields of preparation of agriculture, foods, feed additives, medicine, oligo-glucomannan and the like.

Description

technical field [0001] The invention relates to a gene sequence of an endo-beta-1,4-glucanase and its preparation method and application. The invention provides the recombinant plasmid and recombinant genetic engineering strain of the endo-β-1,4-glucanase and its application in polysaccharide degradation. The endo-β-1,4-glucanase provided by the invention can be widely used in the fields of agriculture, food, feed addition, medicine, oligosaccharide preparation and the like. Background technique [0002] Konjac (Amorphophalluskonjac), commonly known as konjac, is a perennial herb of the family Araceae Amorphophallus. The main ingredient in konjac is konjac polysaccharide (also known as konjac glucomannan), which is composed of glucose and mannose. Konjac glucomannan is formed by linking β-D-glucose and β-D-mannose at a molar ratio of 1:1.6 or 1:1.69 through β-1,4 pyranoside bonds, and every 19 sugar residues There is an acetyl group, and its special structure makes it hav...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12P19/14C12P19/02C12P19/04C12P19/12C12P19/00C12R1/01
CPCC12N9/2437C12P19/00C12P19/02C12P19/04C12P19/12C12P19/14C12Y302/01004
Inventor 尹恒李悝悝曹海龙贾晓晨
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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