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Recombinant alginate lyase AlyL7 and application thereof

A technology of alginate lyase and alginate oligosaccharide, applied in the direction of lyase, carbon-oxygen lyase, recombinant DNA technology, etc., can solve the problems of alginate oligosaccharide difficulty, low enzyme production of wild-type strains, etc., to overcome the catalytic Low efficiency, good application prospects, and the effect of improving plant stress resistance

Pending Publication Date: 2022-03-25
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The wild-type strain producing alginate lyase contains a variety of alginate lyases, which are essentially isozymes, but the alginate lyases produced by different coding genes have different substrate specificities, so wild bacteria can It is difficult to control the production of fucoidan oligosaccharides with uniform structure, and the wild-type strains have low enzyme production and cannot be applied industrially

Method used

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  • Recombinant alginate lyase AlyL7 and application thereof
  • Recombinant alginate lyase AlyL7 and application thereof
  • Recombinant alginate lyase AlyL7 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Source and sequence analysis of alginate lyase AlyL7

[0023] The Flavobacterium used in this example was provided by Fuzhou University.

[0024] Alginate lyase of the present invention alyl7 Flavobacterium Zobellia sp. .The genome is amplified by means of chromosome walking. This sequence contains 924 bases (as shown in SEQ ID NO.2), which encodes 307 amino acids (as shown in SEQ ID NO.1). Upload the base sequence Until NCBI obtained the sequence accession number MW561203.1, the homology comparison results showed that the similarity between it and the existing alginate lyase CaAly1 (ADV51457.1) nucleotide sequence was only 63.4%. According to the results of multiple sequence alignment, the amino acid sequence of recombinant alginate lyase AlyL7 includes the conserved sequence R(S / N)E(L / V)R, QIH, YFKAG(V / I)Y(N / P)O( figure 1 ), so the alginate lyase AlyL7 belongs to the 7th family of polysaccharide hydrolases (PL7).

Embodiment 2

[0025] Example 2: Construction of engineering bacteria for recombinant expression of alginate lyase AlyL7

[0026] With BamH Ⅰ and Xho Ⅰ as restriction sites, primers were designed so that the obtained alyl7 Restriction sites are placed at both ends of the gene sequence.

[0027] Forward primer AlyL7F (SEQ ID NO.3):

[0028] 5'-CG CGGATC CATGCAGTTTTTAAGCAACT-3' (the underline is the BamH Ⅰ restriction site)

[0029] Reverse primer AlyL7R (SEQ ID NO.4):

[0030] 5'-CC GCTCGA GGTGTATAACTTCTAAATCGT-3' (Xho Ⅰ restriction site is underlined)

[0031] The PCR reaction was carried out under the following conditions: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s; annealing at 55 °C for 20 s; extension at 72 °C for 2 min, for a total of 30 cycles, followed by extension at 72 °C for 10 min. The PCR product was gel-cut and recovered after 1% agarose nucleic acid electrophoresis to obtain the target gene with restriction site, and the target gene with restric...

Embodiment 3

[0032] Example 3: Induced fermentation of recombinant alginate lyase AlyL7

[0033] recombinant bacteria E.coil BL21(DE3)-pGEX-4T-1- alyl7 in the presence of 100 μg / mL Amp + Three-section line was performed on the LB solid medium plate, and a single colony was picked to 5 mL containing 100 μg / mL Amp + In LB liquid medium, cultivate overnight at 37°C in a shaker to obtain seed liquid. Inoculate the seed solution to 1 L containing 100 μg / mL Amp at an inoculum volume of 1 vol%. + LB liquid medium, cultured to OD 600When it is 0.6-0.8, add isopropylthiogalactopyranoside (IPTG) at a final concentration of 0.8 mM to the medium, and induce at 20 °C for 18 h. Centrifuge at 12,000 rpm at 4°C for 15 min to collect the bacteria, and use pre-cooled 40 mL buffer A (140 mM NaCl, 2.7 mM KCl, 10 mM NaCl 2 HPO 4 , 1.8 mM KH 2 PO 4 ; pH 7.4) for resuspension, ultrasonication for 30 min, and centrifugation at 12,000 rpm at 4°C for 15 min to collect the supernatant, which is the crude e...

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Abstract

The invention relates to a recombinant alginate lyase AlyL7 and application thereof. The amino acid sequence of the alginate lyase is shown as SEQ ID NO.1, and the nucleotide sequence of a coding gene of the alginate lyase is shown as SEQ ID NO.2. The recombinant alginate lyase belongs to bifunctional alginate lyase, the optimum reaction temperature of the recombinant alginate lyase is 40 DEG C, the optimum reaction pH of the recombinant alginate lyase is 9.0, the recombinant alginate lyase is stable under the conditions that the temperature is 0-25 DEG C and the pH is 5.0-10.0, and the recombinant alginate lyase has degradation activity on sodium alginate, polyguluronic acid and polymannuronic acid. The recombinant alginate lyase also belongs to an incision enzyme, and a final product obtained by enzymolysis of sodium alginate by the recombinant alginate lyase comprises unsaturated 1-5 sugars and mainly comprises unsaturated 2-3 sugars. The alginate lyase is high in reaction speed and high in efficiency, and has good industrial application potential.

Description

technical field [0001] The invention specifically relates to a recombinant alginate lyase AlyL7 and its application, belonging to the technical field of bioengineering. Background technique [0002] Alginate is a linear anionic polysaccharide polymer formed by linking β-D-mannuronic acid (M) and its C5 epimer α-L-guluronic acid (G) through 1,4-O glycosidic bonds . It has been used in food, biomaterials and pharmaceutical industries due to its high viscosity and gelling properties, but its low solubility limits the further development and application of alginate. Alginate can be degraded to obtain alginate oligosaccharides with low degree of polymerization and high solubility. Oligomeric alginate oligosaccharides have immune regulation, antibacterial, anti-tumor, anti-inflammatory, anti-coagulation, promote plant growth, improve plant stress resistance, As a fresh-keeping agent for fruits and vegetables, it has various physiological activities such as extending the shelf li...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12P19/12C12P19/04C12P19/02C12P19/00
CPCC12N9/88C12N15/70C12P19/12C12P19/04C12P19/02C12P19/00C12Y402/02003C12Y402/02011Y02A50/30
Inventor 严芬陈骏颖钟金福张少龙
Owner FUZHOU UNIV
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