Beta-agarase and coding gene and application thereof

A kind of agarase and gene technology, which is applied in application, genetic engineering, plant gene improvement, etc., can solve the problems of difficult large-scale production, instability, low activity, etc., and achieve easy purification, good enzyme activity, and good thermal stability Effect

Inactive Publication Date: 2017-03-29
XINXIANG MEDICAL UNIV
View PDF5 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although dozens of agarases have been studied, most of the existing agarases are mesophilic agarases with low activity, instability, and low yield, so i

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Beta-agarase and coding gene and application thereof
  • Beta-agarase and coding gene and application thereof
  • Beta-agarase and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Acquisition of β-agarase gene Aga0917

[0022] The entire genome analysis of the agarase-producing Pseudoalteromonas citrea (Pseudoalteromonas citrea) YTW1-15-1 strain was carried out, and the upstream and downstream primers were designed as follows:

[0023] Upstream primer: 5'-CGCGGATCCGCAGATTGGGACGCATATAG-3',

[0024] Downstream primer: 5'-CCGCTCGAGTTAGTTTGCTTTGTAAACACG-3'.

[0025] The Pseudoalteromonas citrea YTW1-15-1 bacterial strain was inoculated in the marine broth 2216 liquid medium, shaken and cultivated at 28°C and 120rpm for 24h, took 1.5ml of the culture solution, and followed the method in the instruction manual of the bacterial genomic DNA extraction kit (Takara), Genomic DNA of Pseudoalteromonas citrea YTW1-15-1 strain was extracted.

[0026] Using the genomic DNA of Pseudoalteromonas citrea YTW1-15-1 strain as a template, the PCR reaction was carried out under the action of the above-mentioned upstream and downstream primers. After the P...

Embodiment 2

[0031] Example 2: Construction of β-agarase gene cloning vector pMD19T-Aga0917

[0032] In order to increase the concentration of the target gene fragment (β-agarase gene Aga0917) when it is connected with the expression vector and ensure the complete digestion of the target gene fragment and reduce the false positive results of the recombinant vector, this example uses pMD19T vector and β-agar The enzyme gene Aga0917 was used to construct the cloning vector pMD19T-Aga0917.

[0033] The purified β-agarase gene Aga0917 was ligated with the pMD19T vector using a DNA ligation kit (DNA Ligation Kit) to obtain the ligation product—β-agarase gene cloning vector pMD19T-Aga0917.

[0034] The ligation reaction system is as follows (10 μl):

[0035] pMD19T vector (50ng / μl) 1.0μl,

[0036] β-agarase gene Aga0917 (165ng / μl) 4.0μl,

[0037] DNA ligation kit 5.0 μl.

[0038] Ligation reaction conditions: Incubate the reaction at 16°C for 16 hours.

Embodiment 3

[0039] Example 3: Construction of Escherichia coli recombinant strain DH5α-pMD19T-Aga0917

[0040] Thaw 100 μl on ice to a concentration of 5 x 10 7 cfu / ml Competent E. coli DH5α cells and 10 μl of the ligation product obtained in Example 2 were mixed, ice-bathed for 30 minutes, heat-shocked at 42°C for 90 seconds, quickly placed in ice for 3 minutes, and 890 μl of LB liquid medium was added. Recover on a shaker at 37°C and 170r / min for 1h, then centrifuge at 4°C and 4000r / min for 5min, suck out 890μl of supernatant, mix the remaining supernatant with bacteria, and mix the bacteria with 8μl of isopropyl - β-D-thiogalactopyranoside (IPTG), 40μl 5-bromo-4-chloro-3-indole-β-Dgalactoside (X-gal) was mixed and spread evenly in the solution containing 100μg / ml ampicillin On the LB solid medium of penicillin, place it upright for about 10 minutes, let the surface of the plate dry, then invert it, and incubate at 37°C for 12-16 hours.

[0041] White colonies were picked, and their p...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to beta-agarase and a coding gene and application thereof. The amino acid sequence of the beta-agarase is shown in the sequence table SEQ ID No:1. The nucleotide sequence of the coding gene of the beta-agarase is shown in the sequence table SEQ ID No:2. The beta-agarase has the good heat stability and enzymatic activity and can specifically degrade agarose, generate a single end-product of neoagarotetraose and provide a new enzyme source for preparation of agaro-oligosaccharide.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a β-agarase and its coding gene and application. Background technique [0002] The use of biotechnology to obtain medicines and functional foods from the ocean has attracted increasing attention. Macroalgae polysaccharide——agar (agar) is the main component of the cell wall of red algae. In recent years, with the in-depth study of glycobiology, it has been found that its degradation product agaro-oligosaccharide (AOS) has antibacterial, Anti-virus, value-added intestinal probiotics, anti-tumor, improving body immunity, anti-inflammation, anti-oxidation, scavenging hydroxyl free radicals, moisturizing and whitening and other special biological activities, making it widely used in medicine, food, cosmetics, etc. The industry has high application value. [0003] Traditional preparation methods of agar oligosaccharides include physical and chemical methods, wherein physical methods main...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N9/42C12N15/56C12N15/10C12N15/70C12N1/21C12P19/14C12P19/12C12P19/04C12R1/19
CPCC12N9/2468C12P19/04C12P19/12C12P19/14C12Y302/01081
Inventor 王燕张文博周晨妍朱新术解丽芹朱武凌郭帅张鹏陈超群
Owner XINXIANG MEDICAL UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap