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A kind of endocellulase coding gene and its preparation and application

An endo-cellulase and gene technology, which can be used in applications, genetic engineering, plant genetic improvement, etc., and can solve the problems of weak konjac polysaccharide degradation activity, few hemicellulose substrates, and unsuitable for large-scale application.

Active Publication Date: 2021-05-11
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 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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  • A kind of endocellulase coding gene and its preparation and application
  • A kind of endocellulase coding gene and its preparation and application
  • A kind of endocellulase coding gene and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055]Example 1 Full length gene cloning of cellulase

[0056]The genomic DNA of the Multiponded Bacillus is extracted with reference to the genomic DNA purification kit (Thermo, Lot 00105781). Gene sequence of the endocellulase The National Center for BiotechnologyInformation (NCBI) database for the analysis of multiple sequence alignments, degenerate primers Ppcell-F: 5'-CGGACGCATATGGAATCARCTMMCTTGGTCAC-3 '; Ppcell-R: 5'-GACGAGCTCGAGCTCCGCTTYATTYTTGGABRAAGTA -3 'The genomic DNA of the extracted multipondrobacillus is a template, and the gene sequence (not including the signal peptide gene) encoding the endocellulose mature protein is amplified. The PCR reaction conditions were: 94 ° C 3 min, 1 cycle; 94 ° C 30s, 55 ° C 30s, 72 ° C for 1 min 30s, 30 cycles; 72 ° C for 5 min, 1 cycle. After the PCR product is analyzed by agarose gel electrophoresis (seefigure 1 ), The target gene is subjected to rubber recovery, and the binding method is connected to the prokaryotic expression vector P...

Embodiment 2

[0057]Example 2 Analysis of Endulase Gene Gene Sequence

[0058]The sequencing results are analyzed by the Basic Local Alignment Search Tool (Blast) in the GenBank database, and the DNAMan software performs multi-sequence comparison, Vector NTI analysis sequence information.

[0059]The obtained endozyme gene (named PPCell) coding region is 1662 bp, and the nucleotide sequence is shown in SEQID NO 1. PPCell encodes 553 amino acids and a termination codon, and the amino acid sequence is shown in SEQ ID NO2, the protein theoretical molecular weight is 61.45 kDa, and the predicted isometure is 6.55. PPCell encoded amino acids comprise a GH1 family domain and a carbohydrate binding module CBM (Carbohydrate Binding Module) X2 domain, and its GH1 domain is in combination with the cellulase structure of the GH6 family, thereby indicating that PPCell is a kind of Double domain function enzyme.

Embodiment 3

[0060]Example 3 PPCell gene recombinant expression and purification in E. coli

[0061]In order to facilitate the recombinant expression of the gene, NDEI and XHOI enzyme sink were introduced into the designed upper and lower proders. The PCR cleaning product PPCell and the expression vector PET21a were used for bisase with NDEI and XHOI, and the enzyme-cut product was cleaned and used.4DNA connection enzyme connection (connection system: (5μlt)4DNA Ligase 0.5μL, 10 × T4DNA LigaseBuffer 0.5 μL, PET21A 2 μL, PCR product 2 μl), attachment conditions: room temperature overnight. ). 5 μl of the linked product was taking E.Coli Top10 sensitive state cells, coated on solid Luria-Bertani medium containing 100 μg / ml ampicillin, and incubated 12-16 h at 37 ° C. Picking the monoclonal, using a simple primer for colony PCR verification, the correct monoclonation of the amplified monoclonation was cultured in liquid Luria-Bertani medium containing 100 μg / ml ampicillin; the plasmid was extracte...

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Abstract

The invention discloses an endo-cellulase gene derived from Paenibacillus polymyxa and the preparation method and application of the enzyme, that is, the gene of the endo-cellulase is cloned by using the technical method of genetic engineering On the expression vector of Escherichia coli, a recombinant strain of Escherichia coli capable of heterologously expressing the enzyme is obtained, and the endocellulase prepared by the heterologous expression of the strain can efficiently degrade konjac polysaccharide (also known as konjac glucomannan). The endo-cellulase provided by the invention can be widely used in the fields of agriculture, food, feed additive, medicine, glucomannan oligosaccharide preparation and the like.

Description

Technical field[0001]The present invention relates to a gene sequence and a preparation method thereof of an endocellulose. The present invention provides the use of recombinant plasmids and recombinant genetic strains of the endocellulose and its application in polysaccharide degradation. The endocellulosic enzyme provided by the present invention can be widely used in agriculture, food, feed addition, pharmaceutical and oligose preparation.Background technique[0002]蒟蒻 (Amorphophalluskonjac), commonly known as konjac, Heavenly Star, Tag, is a perennial herb. The main ingredients in the konjac are konjac polysaccharides (also known as konjac glucosan), composed of glucose with mannose. Konjac gluconan is connected to the molar ratio of β-D-glucose and β-D-mannose by β-1,4-pyrose glycosidin, and is connected to the molar ratio of 1: 1.6 or 1: 1.69, each 19 sugar residues There is a acetyl group exists, and its special structure makes it a variety of biological functions. However, it ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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