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Endonuclease-type alginate lyase with warm restoration characteristics as well as application thereof

A technology of alginate lyase and heat recovery, applied in the direction of lyase, carbon-oxygen lyase, application, etc., can solve the problems of unreachable industrial application, easy inactivation, poor thermal stability and heat recovery, and achieve good industrial application Prospect, simple preparation method, effect of enhancing heat recovery properties

Inactive Publication Date: 2018-12-18
王存良
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the reported alginate lyases have poor thermal stability and thermal recovery, are easily inactivated, and cannot meet the requirements of industrial applications.

Method used

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  • Endonuclease-type alginate lyase with warm restoration characteristics as well as application thereof
  • Endonuclease-type alginate lyase with warm restoration characteristics as well as application thereof
  • Endonuclease-type alginate lyase with warm restoration characteristics as well as application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Sequence Analysis of Alginate Lyase AlyV11

[0032] Alginate lyase gene of the present invention alyV11 The artificially designed sequence contains a sequence of 1,014 bases, encoding a sequence of 338 amino acids, and retains the traditionally found conserved functional domain of alginate lyase (Pro 13 -His 338 ), a hinge region containing 12 amino acids (Met 1 -Thr 12), the hinge region can form an α-helical secondary domain, which affects the thermal stability and thermal recovery of the enzyme. The gene sequence is fully synthesized by BGI. Conserved domain analysis (CDD, https: / / www.ncbi.nlm.nih.gov / cdd) using the conserved domain in NationalCenter for Biotechnology Information (NCBI, https: / / www.ncbi.nlm.nih.gov / ) Compared with the Basic Local Alignment Search Tool (Blast, https: / / blast.ncbi.nlm.nih.gov / ) for multiple sequences, it was found that the sequence contained a conserved sequence of Alginatelyase 2 superfamily Area. Blast analysis found...

Embodiment 2

[0033] Example 2 Gene cloning and recombinant expression of alginate lyase AlyV11

[0034] Fully synthesized in Example 1 alyV11 restriction endonuclease Nco I and xho I (purchased from Dalian Bao Biological Co., Ltd.) is the enzyme cutting site and the protection base of the enzyme cutting site is designed, and the recombinant primers are designed as follows (the underline is the restriction endonuclease site, and the italic is the restriction endonuclease protection base) :

[0035] Forward primer: SEQ ID NO.3: PalyV11EF:

[0036] 5'- CATG CCATGG GTATGCTGCTGACCCCGTGCTCT-3’ ( Nco I)

[0037] Reverse primer: SEQ ID NO.4: PalyV11ER:

[0038] 5'- CCG CTCGAG GTGGTAGAAGGTAGCCTGAAC-3’ ( xho I)

[0039] The above-mentioned recombinant primers were used to PCR amplify the alginate lyase gene, and the high-fidelity DNA polymerase Primerstar HS used in PCR was purchased from Dalian Bao Biology Company. The specific PCR amplification conditions are: pre-denatur...

Embodiment 3

[0043] Example 3 Fermentation process and purification preparation method of alginate lyase AlyV11

[0044] The Escherichia coli BL21(DE3) / pET22b-alyV11 constructed and stored at -80°C in Example 2 was streaked on the LB solid plate, and after culturing at 37°C for 16 hours, single clones were picked; 50 μg / mL ampicillin in LB liquid medium (500 mL Erlenmeyer flask loaded with 50 mL liquid medium), cultured in a shaker at 37°C at 180 rpm to OD 600 =0.6. The 5 L fermenter was loaded with 60% (3 L) Terrific Broth (TB) medium, and sterilized in advance; 50 μg / mL ampicillin was added to the fermenter, and the cultured bacteria in the Erlenmeyer flask The solution was inoculated into a 5 L fermenter according to the inoculum amount of 2%. Adjust the initial ventilation rate to 50 L / h, the initial rotation speed to 350 rpm, the temperature at 37°C, and the dissolved oxygen at 15-40%; when the bacteria grow to OD 600 When =5.0, add the inducer isopropyl-β-D-thiogalactoside (IPTG) a...

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Abstract

The invention relates to an endonuclease-type alginate lyase with warm restoration characteristics as well as an application thereof. The alginate lyase is a novel alginate lyase AlyV11, and an aminoacid sequence is as shown by SEQ ID NO.1. An end N of the alginate lyase comprises a section of hinge area (Met1-Thr12), and the identity between the amino acid sequence of the endonuclease-type alginate lyase and the sequence of the alginate lyase with a known function is only 79 percent. The alginate lyase AlyV11 of provided bythe invention has the warm restoration characteristics, after being boiled for 5 minutes, the alginate lyase AlyV11 is incubated for 10 minutes at 30 DEG C, and about 61.5 percent of activity can be restored. The acting way of the lyase is an endonuclease way, a main product of the degradation of the endonuclease-type alginate is unsaturated alginate disaccharide and alginate trisaccharide. The alginate lyase ofprovided by the invention is high in yield, novel in performance, and high in industrialized application potential.

Description

technical field [0001] The invention relates to an endo-type alginate lyase with heat recovery properties and application thereof, belonging to the field of biotechnology. Background technique [0002] Alginate is an important component of brown algae cell wall. , A linear polysaccharide linked by 4 glycosidic bonds. Generally, alginate is processed from large brown algae such as kelp, sargassum, and macroalgae. my country is a big country in seaweed farming, and the production of alginate accounts for more than 70% of the world's total production. Alginate is widely used in chemical, pharmaceutical, food and other industries. Alginate oligosaccharides with different degrees of polymerization have different biological activities. The latest research shows that GV-971, a poly-M segment oligosaccharide drug prepared from alginate, can inhibit the aggregation and cytotoxicity of β-amyloid cells with multiple targets. Phase III clinical trials have been completed; poly-G oli...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12P19/00C12P19/12
CPCC12N9/88C12P19/00C12P19/12C12Y402/02003Y02P20/59
Inventor 王存良李尚勇
Owner 王存良
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