Endo-cellulase coding gene as well as preparation and application thereof

A technology of endo-cellulase and cellulose, which is applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of less hemicellulose substrates, weak konjac polysaccharide degradation activity, and unsuitable for large-scale application, achieving The effect of efficient degradation

Active Publication Date: 2018-06-29
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • Endo-cellulase coding gene as well as preparation and application thereof
  • Endo-cellulase coding gene as well as preparation and application thereof
  • Endo-cellulase coding gene as well as preparation and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0055] Embodiment 1 endocellulase full-length gene cloning

[0056] Genomic DNA of Paenibacillus polymyxa was extracted according to the operation steps of Genomic DNA Purification Kit (Thermo, LOT 00105781). After multiple sequence alignment analysis of endocellulase gene sequences in The National Center for Biotechnology Information (NCBI) database, degenerate primers were designed Ppcell-F: 5'-CGGACGCATATGGAATCARCTMMCTTGGTCAC-3'; Ppcell-R: 5'-GACGAGCTCGAGCTCCGCTTYATTYTTGGABRAAGTA -3', using the extracted Paenibacillus polymyxa genomic DNA as a template to amplify the gene sequence encoding the endocellulase mature protein (excluding the signal peptide gene). The PCR reaction conditions were as follows: 94°C for 3min, 1 cycle; 94°C for 30s, 55°C for 30s, 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 recovered by cutting the gel, connected to the prokaryotic expression vect...

Embodiment 2

[0057] Embodiment 2 endocellulase gene sequence analysis

[0058] 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.

[0059] The coding region of the obtained endocellulase gene (named Ppcell) is 1662 bp long, and its nucleotide sequence is shown in SEQ ID NO 1. Ppcell encodes 553 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 61.45kDa, and the predicted isoelectric point is 6.55. The amino acid encoded by Ppcell contains a GH1 family domain and a carbohydrate binding module CBM (Carbohydrate Binding Module) X2 domain, and its GH1 domain overlaps with the cellulase domain of the GH6 family, thus indicating that Ppcell is a Dual-domain functional enzyme.

Embodiment 3

[0060] Recombinant expression and purification of embodiment 3 Ppcell gene in Escherichia coli

[0061] 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 Ppcell and the expression vector pET21a were double-digested with NdeI and XhoI respectively. 4 DNA ligase connection (ligation system: (5μLT 4 DNA Ligase 0.5μL, 10×T 4DNA 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 μg / mL ampicillin, and extract the plasmid; use endonuclease...

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Abstract

The invention discloses a gene of endo-cellulase derived from Paenibacilluspolymyxa as well as preparation and an application of the endo-cellulase. The gene of the endo-cellulase is cloned on an escherichia coli expression vector according to the technical method of genetic engineering to obtain an escherichia coli recombination strain capable of heterologously expressing the endo-cellulase, andthe endo-cellulase prepared through the heterologous expression of the strain effectively degrades konjac glucomannan (also known as konjac glucomannan). The provided endo-cellulase can be widely applied to the fields of preparation of feed additives, medicines and oligo-glucomannan in agriculture and foods.

Description

technical field [0001] The invention relates to a gene sequence of an endocellulase, a preparation method and application thereof. The invention provides the recombinant plasmid and recombinant genetic engineering strain of the endocellulase and its application in polysaccharide degradation. The endo-cellulase 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 have a variety of biolo...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/42C12P19/14C12P19/02C12P19/12C12P19/00
CPCC12N9/2437C12P19/00C12P19/02C12P19/12C12P19/14C12Y302/01004
Inventor 尹恒李悝悝王文霞谭海东
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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