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Incision alginate lyase Alg2B and coding gene, preparation and application thereof

A kind of algin lyase, endo-algin technology, applied in lyase, application, genetic engineering and other directions

Inactive Publication Date: 2016-08-03
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The Flavobacterium genus is a typical aerobic heterotrophic bacteria. It has been reported that Flavobacterium has a good ability to degrade sodium alginate ( BERNARDETetal, Prokaryotes, 2006, 7:481–531; AnQDetal, Canadian Journal of Microbiology, 2008, 54:314-320), there are few reports on the alginate lyase gene produced by the strains of this genus, so the Flavobacterium strain Flavobacterium sp.S20 As the starting bacteria, mining its alginate lyase gene resources

Method used

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  • Incision alginate lyase Alg2B and coding gene, preparation and application thereof
  • Incision alginate lyase Alg2B and coding gene, preparation and application thereof
  • Incision alginate lyase Alg2B and coding gene, preparation and application thereof

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Embodiment 1

[0029] Example 1 Cloning of the full-length gene of alginate lyase

[0030] Genomic DNA of Flavobacterium strain S20 was extracted according to the operation steps of the bacterial genomic DNA extraction kit (TaKaRa Company). After performing multiple sequence alignment analysis on the alginate lyase gene sequence of the lyase 17 family in the CarbohydrateActiveEnzyme (CAZy) database, degenerate primers PL17-F: 5'-TTYTGGCARTGYYTAAAYGA-3'; PL17-R: 5'-GTATTRTGNGCWATNGTTTGTTTKGCCCA were designed -3'. PCR amplification was performed using the genomic DNA of strain S20 as a template. The PCR reaction conditions were: 94°C for 5 min, 1 cycle; 94°C for 30 s, 45°C for 30 s, 72°C for 1 min 30 s, 30 cycles; 72°C for 10 min, 1 cycle. After the PCR product was analyzed by agarose gel electrophoresis, the target fragment was recovered by cutting the gel, connected to the pMD18-T vector and then sequenced. Nested specific primers were designed according to the obtained enzyme gene nucleo...

Embodiment 2

[0031] Example 2 Alginate Lyase Gene Sequence Analysis

[0032] The results of the sequencing were analyzed using the BasicLocalAlignmentSearchTool (BLAST) in the GenBank database, and the software VectorNTISuite8.0 was used for multiple sequence alignments to analyze their homology. The domains of the sequences were analyzed using the SimpleModular Architecture Research Tool (SMART) online tool.

[0033] The coding region of the obtained alginate lyase gene (named alg2B) is 2265 bp long, and its nucleotide sequence is shown in SEQ ID NO1. The nucleotide sequence of alg2B has the highest identity (70%) with the gene of heparanase II / III family protein (accession number CP002453.1) derived from Cellulophaga algicola DSM14237 strain. alg2B encodes 754 amino acids and a stop codon, its amino acid sequence is shown in SEQ ID NO2, the theoretical molecular weight of the protein is 84.48kDa, and the predicted isoelectric point is 8.26. SMART analysis showed that in the amino acid ...

Embodiment 3

[0034] Recombinant expression of embodiment 3 alg2B gene in escherichia coli

[0035] Using the genomic DNA of strain S20 as a template, the designed upstream primer (NX-F: 5'-CCCCCATATGCAAGGGCATCCAAGACTAATCATG-3') and downstream primer (NX-R: 5'-CCCCTCGAGTTTAATTAATGTATTATAATGTAC-3') were used to amplify the coding alginate lyase The gene sequence of the mature protein (not including the signal peptide gene). The upstream and downstream primers contain NdeI and XhoI restriction sites respectively. The PCR amplified product and the expression vector pET21a (Novagen, USA) were double-digested with NdeI and XhoI. After the digested products were separated by electrophoresis and recovered from the gel, the double-digested PCR product was combined with the same double-digested pET- The 21a vector was ligated with T4 DNA ligase. The ligation product was transformed into Escherichia coli TOP10 competent cells, spread on the solid plate of Luria-Bertani medium containing 100 μg / mL a...

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Abstract

The invention discloses a gene sequence of Incision alginate lyase Alg2B from Flavobacterium strain (Flavobacterium sp. S20). The invention also provides a method for preparing novel alginate lyase, which is characterized in that by using a gene engineering technical method, the gene of the alginate lyase is cloned to an escherichia coli expression vector, the escherichia coli recombinant strain capable of realizing heterogenous expression of enzyme can be obtained, the recombinant strain is used for heterogenous expression of the alginate lyase Alg2B, and thereby monosaccharide and oligosaccharides produced by sodium alginate can be degraded. The provided alginate lyase Alg2B can be widely used in the fields of agriculture, food, feed addictives, medicine and alga genetic engineering.

Description

technical field [0001] The present invention relates to a gene sequence of endo-alginate lyase Alg2B derived from Flavobacterium sp. S20, preservation number: CGMCC NO.5026, a preparation method and application thereof. The invention also provides the recombinant plasmid and recombinant genetic engineering strain of the endo-alginate lyase. The endo-alginate lyase Alg2B of the present invention can be widely used in the fields of agriculture, food, feed addition, medicine, seaweed genetic engineering and the like. Background technique [0002] Brown algae is an economical seaweed plant abundant in the ocean. It contains various polysaccharides such as algin, fucoidan and laminaran. In algae, alginate mainly exists in the form of alginic acid (alginic acid), alkali metal salts (sodium salt, potassium salt) and divalent salts (calcium salt, magnesium salt) (TusiSKetal., Biomaterials, 2011, 32 : 5438-5458; Qin Guokui et al., China Biotechnology Journal, 2004, 24: 26-33). Sod...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N15/63C12N1/21C12P19/02C12P19/00C12N5/10C12R1/19C12R1/125C12R1/01C12R1/645C12R1/66
Inventor 尹恒黄李淑馨曹海龙李曙光王文霞
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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