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A gene encoding endo-β-1,4-mannanase and its preparation and application

A technology of mannanase and gene, which is applied in the field of endo-β-1,4-mannanase coding gene and its preparation and application, and can solve the problems of weak degradation activity and unsuitability for large-scale application

Active Publication Date: 2021-07-06
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there have been many studies at home and abroad on the physiological activity and antibacterial activity of various strains of this genus, but there are very few reports on the mannanase gene, and the reported endo-β-1,4-mannan The degradation activity of carbohydrases on konjac polysaccharides is weak, which is not suitable for large-scale application

Method used

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  • A gene encoding endo-β-1,4-mannanase and its preparation and application
  • A gene encoding endo-β-1,4-mannanase and its preparation and application
  • A gene encoding endo-β-1,4-mannanase and its preparation and application

Examples

Experimental program
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Embodiment 1

[0055] Example 1 Endo-β-1,4-mannanase 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 performing multiple sequence alignment analysis on the endo-β-1,4-mannanase gene sequence in The National Center for Biotechnology Information (NCBI) database, a degenerate primer ppman-F: 5'-CGGACGCATATGGAATCARCTMMCTTGGTCAC-3' was designed; ppman-R: 5'-GACGAGCTCGAGCTCCGCTTYATTYTTGGABRAAGTA-3', using the extracted Paenibacillus polymyxa genomic DNA as a template to amplify the gene sequence encoding the mature protein of endo-β-1,4-mannanase (excluding the signal peptide gene). The PCR reaction conditions were: 94°C for 2 min, 1 cycle; 94°C for 30 s, 55°C for 30 s, 72°C for 2 min, 30 cycles; 72°C for 5 min, 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 pro...

Embodiment 2

[0057] Example 2 Endo-β-1,4-mannanase 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 endo-β-1,4-mannanase gene (named ppman) is 1896 bp long, and its nucleotide sequence is shown in SEQ ID NO 1. ppman encodes 631 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 70.11kDa, and the predicted isoelectric point is 4.97. The amino acid encoded by ppman contains two carbohydrate binding modules CBM (Carbohydrate Binding Module) 35 and a GH26 family domain, and its domain characteristics are more similar to those of GH26 family members, thus indicating that ppman is a member of the GH26 family.

Embodiment 3

[0060] Recombinant expression and purification of embodiment 3ppman 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 ppman 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 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 μg / mL ampicillin, and extract the plasmid; use endonucleases ...

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Abstract

The invention discloses an endo-β-1,4-mannanase gene derived from Paenibacillus polymyxa and the preparation method and application of the enzyme. Cloning the gene of cutting β‑1,4‑mannanase into the expression vector of Escherichia coli to obtain a recombinant strain of Escherichia coli that can express the enzyme heterologously Glycanase can efficiently degrade konjac polysaccharides (also known as konjac glucomannan). The endo-β-1,4-mannanase 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 invention relates to a gene sequence of endo-beta-1,4-mannanase, its preparation method and application. The invention provides the recombinant plasmid and recombinant genetic engineering strain of the endo-β-1,4-mannanase and its application in polysaccharide degradation. The endo-β-1,4-mannanase 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 va...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N9/42C12P19/14C12P19/02C12P19/00
CPCC12N9/2494C12P19/00C12P19/02C12P19/14C12Y302/01078
Inventor 尹恒李悝悝谭海东王文霞
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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