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Kappa-carrageenase, gene encoding same and application of kappa-Carrageenase

A technology of carrageenanase and gene, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as poor temperature stability and large loss of enzyme activity

Active Publication Date: 2018-01-19
GREENFRESH FUJIAN FOODSTRUFF
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the temperature stability of the reported recombinant carrageenase is poor, and most of the carrageenase can maintain high activity at 0-30°C, and the enzyme activity will be greatly lost when the temperature is higher than 40°C, and the pH is greater than At 6.0, the enzyme activity begins to decline. Therefore, obtaining carrageenase with better heat resistance from nature and expressing it in E. coli is of great significance for the large-scale preparation of carrageenase and the industrial production of carrageenan oligosaccharides

Method used

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  • Kappa-carrageenase, gene encoding same and application of kappa-Carrageenase
  • Kappa-carrageenase, gene encoding same and application of kappa-Carrageenase
  • Kappa-carrageenase, gene encoding same and application of kappa-Carrageenase

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Experimental program
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Embodiment 1

[0033] Example 1: Kappa-Carrageenase Nucleotide and Amino Acid Sequences

[0034] strains using whole genome sequencing Pseudoalteromonas carrageenovora ASY5 was sequenced, its whole genome sequence was obtained, and related bioinformatics analysis was carried out on the gene. First, the genome was assembled using the Newbler version 2.8 method, and the sequencing data with the adapter sequence removed was de novo assembled to construct a scaffold; then the unique gene family of the bacteria was found and classified with the help of OrthoMC software; the strain was classified using bioinformatics software. P. carrageenovora GO functional enrichment analysis of ASY5 gene and biochemical reaction process analysis of memory genes. Finally, a complete genome is assembled: including 1 circular bacterial chromosome and 1 plasmid DNA. From the splicing results, it can be known that the strain P. carrageenovora The genome length of ASY5 is 4,489,108 bp, and the G+C content is 39...

Embodiment 2

[0037] Example 2: Construction of recombinant κ-carrageenase LJ 30

[0038] Carrageenan pseudoalteromonas ( P. carrageenovora ASY5, deposited in the China Industrial Microbiology Collection Center (CICC), with the preservation number 23819) Genomic DNA was used as a template, using P. carrageenovora ASY5 κ-carrageenase gene

[0039] The forward primer (SEQ ID NO.3) is: 5ʹ-CCG GAATTC CAAGAGTTTAAAAAACTA-3 (underlined as EcoR I recognition sequence),

[0040] The reverse primer (SEQ ID NO.4) is: 5ʹ-CGC GGATCCAAAACTGCTGTGGGGAATA-3ʹ (underlined as Bam HI recognition sequence),

[0041] Amplify the 16S rDNA sequence of the strain. Taq DNA polymerase was used to amplify in a PCR instrument, and the amplification temperature parameters were: 95°C, 5 min; (94°C, 60 s; 50°C, 45 s; 72°C, 60 s) 30 cycles; 4°C, hold. The amplified samples were detected by agarose gel electrophoresis at a concentration of 1%. figure 1 It can be seen from the figure that a band of about 750 ...

Embodiment 3

[0044] Example 3: Expression of recombinant κ-carrageenase in Escherichia coli

[0045] Take 500 μL of the verified bacterial solution, inoculate it in 50 mL LB medium containing 50 μg / mL kanamycin, and culture it at 37°C with shaking at 200 r / min until OD 600 When it reaches 0.8, add the inducer isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.05 mmol / L, and induce culture at 10°C for 24 h. The cells were collected by refrigerated centrifugation, and the cells were resuspended with the wall-breaking buffer. The supernatant was obtained by refrigerating and centrifuging the bacterial solution after ultrasonic breaking.

[0046] Figure 4 It shows that after induction by IPTG, the expression of recombinant κ-carrageenase can be observed, and the effective expression of the target gene is verified by measuring the activity of κ-carrageenase in the enzyme solution. Since we are using affinity chromatography to purify the target protein, since the puri...

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Abstract

The invention discloses kappa-carrageenase, a gene encoding the same and an application of kappa-carrageenase. The cloned kappa-carrageenase precursor gene car 30 of P.carrageenovora ASY5 is transformed into E. coli, a recombinant bacterium producing recombinant kappa-carrageenase is obtained, and the suitable temperature and pH of the recombinant kappa-carrageenase are 45 DEG C and 6.5; after heat preservation at 40 DEG C for 30 min, the remnant enzyme activity is 80.0% or higher, the recombinant kappa-carrageenase is treated for 60 min in a buffer solution with pH being 6.5-7.5, and the enzyme activity can still remain 50.0% or higher. The recombinant vector containing the kappa-carrageenase gene car 30 is constructed, heterologous expression is realized, and a good foundation is provided for industrialized production and application of the enzyme. The expressed kappa-carrageenase has a better degradation effect on kappa-carrageenan, kappa-carrageenan is degraded into disaccharide, tetrasccharide and the like, and the kappa-carrageenase can be applied to preparation of antibacterial agents, antiviral agents, immunomodulators, antioxidants and the like.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a method derived from pseudoalteromonas carrageenans ( P. Carrage Enovora Cloning of κ-carrageenase gene car 30 of ASY5) and construction and application of recombinant κ-carrageenase. Background technique [0002] Carrageenan is a structural polysaccharide present in the cell wall of marine red algae, which is composed of D-galactose and 3,6-anhydrogalactose units connected alternately by β-1,3 and α-1,4-glycosidic bonds . A large number of studies have confirmed that carrageenan oligosaccharides, the degradation products of carrageenan, and their derivatives have various physiological activities such as anti-virus, anti-tumor, anti-HIV, and immune regulation, and have high pharmaceutical development value. [0003] At present, the preparation methods of carrageenan oligosaccharides mainly include reductive hydrolysis, free radical depolymerization, chemical degradation an...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/38C12N15/70C12N1/19C12P19/14C12P19/12C12P19/00C12R1/19
Inventor 洪清林肖安风李佳佳嵇海峰钟晓婷陈垂烨肖琼倪辉蔡慧农姜泽东
Owner GREENFRESH FUJIAN FOODSTRUFF
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