Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Preparation method and application of alginatelyase OalC6

A technology of alginate lyase and construction method, which can be applied in the directions of lyase, carbon-oxygen lyase, and botanical equipment and methods, and can solve problems such as the existence of food demand pressure

Inactive Publication Date: 2018-03-23
QINGDAO UNIV
View PDF2 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the raw materials for bioenergy are terrestrial crops. my country has a large population, and the pressure on food demand has existed for a long time. It is impossible to use a large amount of food to produce biofuels.

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
  • Preparation method and application of alginatelyase OalC6
  • Preparation method and application of alginatelyase OalC6
  • Preparation method and application of alginatelyase OalC6

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Cloning and identification of the gene encoding oligofucoidan lyase OalC6

[0027] According to Cellulophaga The possible oligofucoidan lyase gene in the sp. SY116 genome was designed with the following amplification primers: upstream primer (5'-CAAAAACATACATTATAC -3') and downstream primer (5'- CTAATTAATCCTAAATTTTT-3'). Using the extracted strain genome as a template, PCR amplification was performed to obtain the full-length sequence of the oligofucoidan lyase OalC6 gene. The PCR conditions were as follows: pre-denaturation at 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 54.8 °C for 30 s, and 72 °C for 1 min 30 s, and finally extended at 72 °C for 10 min.

[0028] After gene sequencing, the full-length 2,328 bp nucleotide sequence of the oligofucoidan lyase OalC6 gene was obtained, and the nucleotide sequence is shown in SEQ ID NO.1; encoding 775 amino acids, and the amino acid sequence is shown in SEQ ID NO.2 As shown, the theoretical molecu...

Embodiment 2

[0030] Example 2: Construction of oligofucoidan lyase OalC6 recombinant expression vector

[0031] The upstream primer (5'-CAAAAACATACATTATAC-3') and the downstream primer (5'-CTAATTAATCCTAAATTTTT-3') were designed according to the complete sequence of the oligofucoidan lyase gene. PCR amplification was carried out to obtain the full-length sequence of alginate lyase OalC6 gene. PCR conditions were: 94°C pre-denaturation for 5 min, followed by 30 cycles of 98°C for 30 s, 58°C for 30 s, 68°C for 2 min, and finally extension at 68°C for 10 min. PCR product and Escherichia coli expression vector pET28a(+) were digested, connected and transformed into E. coli For DH5α competent cells, single clones were picked and cultured in LB liquid containing resistance, plasmids were extracted and digested to identify positive clones. This recombinant plasmid was named pET28a-oalC6. Transform positive plasmids into expression hosts E. coli BL21 competent cells.

Embodiment 3

[0032] Example 3: Recombinant expression and purification of oligofucoidan lyase OalC6 gene in Escherichia coli

[0033] Transform the recombinant expression plasmid pET28a-oalC6 into the expression host E. coli BL21 competent cells. will reorganize E. coli Pick a single clone of BL21 bacteria into the LB liquid with the same resistance, and culture it with shaking at 37 °C until OD 600 At about 0.6, IPTG was added for induction (final concentration: 0.5 mM), and expression was induced at 25°C for 20 h. The target protein was separated and purified by Ni affinity chromatography. The purification of oligofucoidan lyase OalC6 was detected by polyacrylamide gel electrophoresis, and the results were as follows figure 2 As shown, the purified OalC6 presents a single band on the electrophoresis gel, and the position is consistent with the predicted molecular weight.

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 discloses exo-alginatelyase, a coding gene thereof and an application of the exo-alginatelyase. A novel alginatelyase gene is cloned from marine bacterium Cellulophaga sp. SY116, has thesize of 2328 bp, encodes 775 amino acids and belongs to the family of polysaccharide lyase PL-6. The gene can be expressed and purified in Escherichia coli to obtain recombinant OalC6, and the molecular weight of the gene is about 85.9 kDa. The exo-alginatelyase is active to sodium alginate and alginate oligosaccharide and preferential to guluronic acid fragment (polyG). The exo-alginatelyase haspotential application values in terms of production of bioethanol and development of new energy.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a preparation method and application of oligofucoidan lyase OalC6. Specifically, the present invention relates to a method for preparing oligofucoidan lyase OalC6 by using recombinant Escherichia coli and its application. Background technique [0002] my country's production of alginate ranks first in the world. Alginate is the cell wall component of various seaweeds. Its basic unit is monosaccharide, which is composed of mannuronic acid (M) and guluronic acid (G), which are epimers, through 1,4 glycosidic bonds. Linked linear polysaccharides. According to its structural characteristics, alginate polysaccharides can be divided into three types of fragments, the PolyM fragment composed of polymannuronic acid, the PolyG fragment composed of polyguluronic acid, and the alternation of mannuronic acid and guluronic acid. Arranged PolyMG fragments. [0003] Alginate is an important mari...

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): C12N15/60C12N9/88C12N1/21C12N15/70C12P19/02C12R1/19
CPCC12N9/88C12N15/70C12P19/02C12Y402/02
Inventor 陈雪红李尚勇韩彦弢
Owner QINGDAO UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com