Alginate lyase and gene and application thereof

A alginate lyase and gene technology, applied in the field of bioengineering, can solve problems such as difficulty in obtaining solubility, small molecular weight, cumbersome process, etc., and achieve good application prospects and mild reaction conditions

Inactive Publication Date: 2019-05-21
青岛海大生物集团股份有限公司
View PDF0 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the preparation of soluble fucoidan oligosaccharides is still a difficult problem. In actual use, brown algae (kelp) is used directly as a substrate, and it is difficult to obtain fu

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
  • Alginate lyase and gene and application thereof
  • Alginate lyase and gene and application thereof
  • Alginate lyase and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Embodiment 1 Expression and purification of alginate lyase

[0014] Carry out whole gene synthesis of SEQ ID NO: 1 according to conventional methods, connect to the prokaryotic expression vector pET-28a(+), and add a BamHI restriction site at the upstream of the gene, and a SacI restriction site at the downstream;

[0015] Transform the synthetic recombinant plasmid into Escherichia coli BL21(DE3) to obtain the genetically engineered bacteria BL21 / Alg3 producing Alg3;

[0016] The above-mentioned recombinant bacteria were inoculated into LB medium, cultured at 37°C to the logarithmic growth phase, then added IPTG to a final concentration of 0.1 mmol / L, and continued to be cultured overnight at 16°C for induction. The cells were collected by centrifugation, washed twice with saline, and a certain amount of buffer (50 mM Na 2 HP0 4 , 0.3M NaCl, pH8.0) to make a suspension of alginate lyase wet bacteria. Then ultrasonically destroy the bacteria in an ice-water bath (pow...

Embodiment 2

[0017] Enzyme activity detection of alginate lyase described in embodiment 2

[0018] Take 1ml of enzyme solution, add 1ml of 0.5% sodium alginate solution and 5ml of phosphate buffer solution with pH7.2, and bathe in constant temperature water at 40°C for 30min. The reducing sugar was used DNS method, the absorption wavelength was 540nm, the inactivated enzyme was used as a control, and the relative enzyme activity was calculated;

[0019] The alginate oligosaccharides produced by the degradation of sodium alginate by alginate lyase are reducing sugars. Enzyme activity is defined as: 1 μg of reducing sugar is released per gram of immobilized enzyme catalytic substrate per minute, which is an enzyme activity unit (U);

[0020]

[0021] Wherein, X is the enzyme activity (U / g) of the sample, A is the absorbance value at a wavelength of 540nm, the reaction time is 30min, M is the quality (g) of the enzyme added, and D is the dilution factor; m and n are reducing sugars respec...

Embodiment 3

[0022] Application of alginate lyase described in embodiment 3 in kelp degradation

[0023] Rinse the kelp with clean water, crush it with a pulverizer, and pass through a 60-mesh sieve to obtain dry powder; put the kelp powder into the enzymatic hydrolysis tank, add water 12 times the weight of the kelp powder, stir until fully suspended, and add 2% fiber by weight of the kelp powder Vegetase and 10% alginase crude enzyme solution, the ratio of the two enzymes is 1:2, stir evenly; adjust the pH to 5, hydrolyze at 55°C for 4 hours to obtain kelp hydrolyzate; hydrolyzate at 5000rpm Centrifuge for 20 minutes to separate the residue to obtain a soluble oligosaccharide supernatant; spray dry to obtain a dry powder of fucoidan oligosaccharide.

[0024] The obtained kelp hydrolyzed oligosaccharide is light yellow after drying, has good water solubility, a yield of 6%, simple operation, no pollution, no addition of chemical reagents, and has potential application value.

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 an alginate lyase and a gene and application thereof. The nucleic acid sequence of the alginate lyase gene is shown as SEQ ID NO:1; the amino acid sequence of the alginate lyase Alg3 encoded according to a nucleotide sequence is shown as SEQ ID N0:2. The invention also discloses a technical method for transforming a gene engineering strain of the alginate lyase gene into Escherichia coli by means of genetic engineering. The alginate lyase is prepared by connecting the alginate lyase gene with a vector, constructing tecombinant expression plasmid and transforming the Escherichia coli. The alginate lyase can specifically degrade sodium alginate to produce alginate oligosaccharide and can be used together with cellulase for hydrolyzing kelp to produce a water-soluble oligosaccharide product. Therefore, the alginate lyase can be used for preparation of alginate oligosaccharide; moreover, reaction conditions are mild, consumption of energy is low, no pollutants are produced, and the alginate lyase has an important industrial application value.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a alginate lyase, its gene and application. Background technique [0002] Fucoidan oligosaccharides refer to sugar chains composed of 2-20 monosaccharides with a degree of polymerization. They have the characteristics of good hydrophilicity, stability, high viscosity, and non-toxicity, and have unique biological activities, such as promoting plant growth and improving stress resistance. It has broad application potential in medical and fertilizer fields. Alginate oligosaccharides are degraded from alginate. Traditional chemical methods use strong acid and alkali or strong oxidants, and the products are mainly saturated oligosaccharides. The enzymatic hydrolysis method has a mild effect, and mainly breaks the chemical bonds between uronic acids to obtain unsaturated oligosaccharides, which have better physiological activity; and the enzymatic hydrolysis method is simple in p...

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): C12N15/60C12N9/88C12N1/21C12P19/00C12P19/12C12P19/04C12R1/19
Inventor 宋海妹单俊伟张术聪
Owner 青岛海大生物集团股份有限公司
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