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A novel alginate lyase aly08 and its application

A technology of alginate lyase and molecular alginate, applied in the directions of lyase, carbon-oxygen lyase, application, etc., can solve the problems of poor thermal stability and thermal recovery, unable to achieve industrial application, easy inactivation, etc., and achieve high activity , Good industrial application prospects, simple purification method effect

Active Publication Date: 2019-05-21
QINGDAO UNIV
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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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  • A novel alginate lyase aly08 and its application
  • A novel alginate lyase aly08 and its application
  • A novel alginate lyase aly08 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Sequence analysis of alginate lyase Aly08

[0034] The enzyme-producing gene aly08 of alginate lyase Aly08 in the present invention is a fully synthetic sequence (synthesized by Huada Gene Company), which contains 897 base sequences and encodes 299 amino acid sequences. Using the conserved domain analysis of the National Center for Biotechnology Information (NCBI) to analyze the Conserved domain (CDD) and the multiple sequence alignment Basic Local Alignment Search Tool (Blast), it was found that the sequence contained a second superfamily of alginate lyase (Alginate lyase 2 superfamily) conserved region. Among the reported alginate lyases, the one with the highest amino acid sequence similarity to Aly08 is the alginate lyase AlyL2 (Genbank AJO61885) of the polysaccharide lyase family 7 (PL-7). The amino acid sequence similarity between the two ( Identity) is 78%.

[0035] The amino acid sequence of the alginate lyase Aly08 described in the present invention...

Embodiment 2

[0037] Example 2 Recombinant expression of alginate lyase Aly08

[0038] The fully synthesized algA1 gene sequence in Example 1 uses restriction enzymes Nco I and Xho I as restriction sites, and the recombinant primers are designed as follows (underlined restriction endonuclease sites, italics restriction endonucleases protected bases):

[0039] Forward primer: SEQ ID NO.3: Paly08F:

[0040] 5'-CATG CCATGG GTATGTTATTGAACAAAAT-3’ (Nco I)

[0041] Reverse primer: SEQ ID NO.4: Paly08R:

[0042] 5'-CCG CTCGAG GTAAGAGTAGTTGTC -3' (Xho I)

[0043] PCR amplification conditions were: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute, a total of 30 cycles; extension at 72°C for 5 minutes; stabilization at 4°C for 15 minutes. The DNA polymerase used in the PCR reaction was PrimerstarHS purchased from Dalian Bao Biological Company.

[0044] The PCR product was double-digested with restric...

Embodiment 3

[0046] Example 3 Fermentation and purification preparation method of alginate lyase Aly08

[0047] The Escherichia coli BL21(DE3) / pET22b-Aly08 constructed in Example 2 was transferred to LB liquid medium (50 μg / mL ampicillin), and 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 were The solution was inoculated into a 5 L fermenter according to the inoculum amount of 5%. Adjust the initial ventilation rate to 60L / h, the initial rotation speed to 300 rpm, the temperature at 37°C, and the dissolved oxygen at 10-60%; when the bacteria grow to OD 600 = 5.0, add the inducer isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.1 mM, and induce at 20°C for 60 h. Feed was carried out during this period. Feed medium (300 g glycerol dissolved in 100 mM pH7.6 phosphate bu...

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Abstract

The invention relates to endonuclease alginate lyase Aly08 with pH stability and thermal restoration characteristics and an application thereof. An amino acid sequence of the alginate lyase Aly08 is as shown by SEQ ID NO.1. The sequence of the alginate lyase provided by the invention is manually designed, and the identity between the amino acid sequence of the alginate lyase of provided by the invention and the alginate lyase with known functions is only 78 percent. The most appropriate reaction temperature of the alginate lyase of provided by the invention is 45 DEG C, the alginate lyase hasthermal restoration characteristics, after being boiled for 5 minutes, the alginate lyase is incubated for 40 minutes in icy water, and 78.3 percent of activity can be restored. The most appropriate reaction pH is 8.3, and the pH stability (t to 10.6) is wide. The enzyme acting way is an endonuclease way, a main product of the degradation of the endonuclease alginate lyase Aly08 is unsaturated disaccharide and trisaccharide. The alginate lyase of provided by the invention is high in yield, novel in performance and good in industrialized application potential.

Description

technical field [0001] The invention relates to an endo-type alginate lyase Aly08 with pH stability and heat recovery properties and application thereof, belonging to the field of biotechnology. Background technique [0002] Alginate is the main component of the cell wall of various brown algae, which can account for 20-40% of the dry weight of brown algae. my country is a big country in seaweed farming, and the production of alginate ranks first in the world. Alginate is composed of α-L-mannuronic acid (Mannuronicacid, M) and β-D-guluronic acid (G), which are epimers, linked by 1,4 glycosidic bonds linear polysaccharides. According to different arrangement and combination, alginate can be divided into polymannuronic acid fragments (PolyM), polyguluronic acid fragments, and alternating fragments of mannuronic acid and guluronic acid (PolyMG). Alginate is widely used in chemical, pharmaceutical, food and other industries. In recent years, more and more reports have confir...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12P19/00C12P19/12
CPCC12N9/88C12P19/00C12P19/12C12Y402/02003Y02P20/59
Inventor 李尚勇韩彦弢王亚男
Owner QINGDAO UNIV
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