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L-carrageenan enzyme with thermal stability and application thereof

A carrageenase and carrageenan technology, applied in the biological field, can solve the problems of inability to achieve industrial application, poor thermal stability and thermal recovery of ι-carrageenase, easy inactivation, etc., and achieve good industrial application prospects and excellent physical and chemical properties. , The effect of purification method is simple

Active Publication Date: 2019-05-10
QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And most of the reported ι-carrageenases have poor thermal stability and thermal recovery, are easily inactivated, and cannot meet the requirements of industrial applications

Method used

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  • L-carrageenan enzyme with thermal stability and application thereof
  • L-carrageenan enzyme with thermal stability and application thereof
  • L-carrageenan enzyme with thermal stability and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1 ι-carrageenase CgiV sequence analysis

[0032]The enzyme-producing gene CgiV of the iota-carrageenase CgiV of the present invention is derived from the marine bacterium Vibrio sp.SY01, and the strain is preserved in the China Center for Type Culture Collection with the preservation number CCTCC No: M2018769. Contains 1359 base sequences, encoding 452 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 AlignmentSearch Tool (Blast), it was found that the sequence contained a conserved region of the polysaccharide hydrolase (GH 82) family . Among the reported ι-carrageenases, the one with the highest amino acid sequence similarity to CgiV is the ι-carrageenase (Genbank APX55175) from the GH82 family of Flavobacterium sp., the amino acid sequence similarity between the two (Identity) was 88%.

Embodiment 2

[0033] Recombinant expression of embodiment 2-carrageenase CgiV

[0034] The iota-carrageenase CgiV gene sequence in Example 1 uses restriction endonucleases Nco I and Xho I as the enzyme cutting sites, and the recombinant primers are designed as follows (the underline is the restriction endonuclease site, and the italic is the restriction endonuclease site. Dicer protected bases):

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

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

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

[0038] 5'-CCG CTCGAG TTTACCTTGTTTACAGTTTTTAC-3' (Xho I)

[0039] 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 1.5 minutes, 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.

...

Embodiment 3

[0042] Fermentation and purification preparation method of embodiment 3-carrageenase CgiV

[0043] The Escherichia coli BL21(DE3) / pET22b-CgiV constructed in Example 2 was transferred to LB liquid medium (50 μg / mL ampicillin), and cultured in a shaker at 37°C at 180rpm to OD 600 = 0.6. Add the inducer isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.1 mM, and induce at 20°C for 24 hours. The activity of iota-carrageenan enzyme was measured by DNS method, the specific method was: 100 μl of enzyme solution was added with 900 μl of 0.3% iota-carrageenan substrate (20 mM phosphate buffer, pH=7.0), reacted at 40 ° C for 10 min, and added 750 μl of DNS The reagent terminates the reaction, boils in boiling water for 10min, and measures the value of A520 with a spectrophotometer. After the control group was added to the sample, 750 μL of DNS reagent was directly added and boiled in boiling water for 10 minutes before detection. Enzyme activity is defined as: ...

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Abstract

The invention relates to an l-carrageenan enzyme CgiV with thermal stability and application thereof. A provided l-carrageenan enzyme gene is derived from marine bacteria Vibrio sp.SY01, a strain is preserved in the China's typical culture collection center, and the preservation number of the strain is CCTCC No: M2018769. The amino acid sequence of the l-carrageenan enzyme CgiV is shown as SEQ IDNO.1. The similarity between the amino acid sequence of the l-carrageenan enzyme CgiV and the amino acid sequence of an l-carrageenan enzyme with known functions is only 88%. The l-carrageenan enzymeis obtained through purification of recombinant expression engineering strains and has thermal stability, the activity half-life of the l-carrageenan enzyme at 40 DEG C is 6.5 hours, and the activityhalf-life of the l-carrageenan enzyme at 30 DEG C is 42 hours. The l-carrageenan enzyme has industrial application potential.

Description

technical field [0001] The invention relates to a novel ι-carrageenase CgiV with thermal stability and its application, which belongs to the field of biotechnology. Background technique [0002] Carrageenan is a sulfated linear polysaccharide formed by alternately linking 1,4-α-D-galactopyranose and 1,3-β-D-galactopyranose as the basic skeleton. According to whether it contains 3,6-inner ether galactose structure and the content of sulfate groups, the carrageenan currently produced and used in industry is mainly ι-type, ι-type and λ-type, and the disaccharide units are respectively Contains 1, 2 and 3 sulfate groups. Carrageenan oligosaccharides, as a sulfated carbohydrate rich in sulfate groups, have a wide range of biological activities such as anti-virus, anti-tumor, hypolipidemic, immune regulation and anti-coagulation. [0003] Carrageenan degrading enzyme is a kind of glycohydrolase, which degrades macromolecular carrageenan into oligosaccharides by breaking the β-1,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/38C12N15/56C12P19/14C12P19/12C12P19/00
CPCC12N9/2468C12N15/70C12Y302/01157C12N1/205C12R2001/63
Inventor 于惠青李尚勇
Owner QINGDAO UNIV
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