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K-carrageenase encoding gene and preparation and application thereof

A carrageenase and carrageenan technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of high production cost, low carrageenase enzyme activity, not meeting the requirements of actual production, etc., and achieve efficient degradation. Effect

Active Publication Date: 2019-06-25
中科绿帅生物科技(广州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, carrageenase has not been applied to actual production. The main reason is that the enzyme activity of carrageenase produced by wild bacteria is not high and the production cost is high, which does not meet the requirements of actual production.

Method used

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  • K-carrageenase encoding gene and preparation and application thereof
  • K-carrageenase encoding gene and preparation and application thereof
  • K-carrageenase encoding gene and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 κ-carrageenase CgκP; full-length gene cloning

[0059] Genomic DNA of Agrobacterium hainanensis Q-13 was extracted according to the operation steps of the genomic DNA purification kit (Thermo, LOT 00105781). After multiple sequence alignment analysis of the κ-carrageenase gene sequence in The National Center for Biotechnology Information (NCBI) database, degenerate primers CgκP-F: 5'-AGCATATGATGAGCAAATTTATGTTTGTG-3'; CgκP-R: 5'- GCCTCGAGGTTAATCACCACTTTTTCG-3', using the extracted genomic DNA of Agrobacterium hainanensis Q-13 as a template, amplified the gene sequence encoding the mature protein of κ-carrageenase (excluding the signal peptide gene). The PCR reaction conditions were: 94°C for 3 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 prokaryotic expression vector pE...

Embodiment 2

[0060] Example 2 Kappa-carrageenase gene sequence analysis

[0061] 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 VectorNTI was used to analyze sequence information.

[0062] The coding region of the obtained κ-carrageenase gene (named as CgκP) has a length of 1566 bp, and its nucleotide sequence is shown in SEQ ID NO 1. CgκP encodes 512 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 58.83kDa, and the predicted isoelectric point is 7.6. The amino acids encoded by CgκP include the κ-carrageenase domain in the GH16 family and the C-terminal sorting domain of the Pro secretion system. .

Embodiment 3

[0063] Embodiment 3 Recombinant expression and purification of CgκP gene in Escherichia coli

[0064] 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 CgκP 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 N...

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Abstract

The invention discloses a k-carrageenase gene derived from Pedrobacter hainanensis Q-13, a preparation method of its enzyme and an application thereof, a genetic engineering technical method is used to clone the gene of the k-carrageenase to an E. coli expression vector to obtain a recombinant strain of Escherichia coli which can heterologously express the enzyme. The k-carrageenase prepared by heterogenous expression of the strain can efficiently degrade k-carrageenan. The k-carrageenase provided by the invention can be widely used in the fields of agriculture, food, feed additives, medicine,cosmetics and carrageenan oligosaccharide preparation.

Description

technical field [0001] The invention relates to a gene sequence of κ-carrageenase, its preparation method and application. The invention provides the recombinant plasmid and recombinant genetic engineering strain of the κ-carrageenase and its application in polysaccharide degradation. The κ-carrageenase provided by the invention can be widely used in the fields of agriculture, food, feed addition, medicine, cosmetics, oligosaccharide preparation and the like. Background technique [0002] Carrageenan (Carrageenan), also known as carrageenan, unicorn gum. It is a polysaccharide containing D-galactose extracted from the cell wall of red algae. Carrageenan belongs to the family of hydrophilic linear sulfated galactose, which is a sulfated linear polysaccharide formed by alternating linkages of β-1,3-D-galactose and α-1,4-D-galactose as the basic skeleton. These glycans can be divided into six basic forms according to the connection mode of the monosaccharide and the position...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/38C12N15/70C12N1/21C12P19/14C12P19/04C12P19/00C12R1/19
Inventor 尹恒
Owner 中科绿帅生物科技(广州)有限公司
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