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Alginate lyase encoding gene

A technology of alginate lyase and gene, which is applied in the field of genes encoding alginate lyase, can solve the problems of few types of enzyme preparations, restrict the development of enzyme applications, and high prices, and achieve stable properties, important industrial application value and scientific research Effect of high value, high product specificity

Active Publication Date: 2019-09-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Alginate lyase has a wide range of sources, mainly from seaweed plants, marine bacteria, fungi and molluscs, etc. However, there are few types of enzyme preparations and high prices, which limit the application and development of this enzyme

Method used

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  • Alginate lyase encoding gene
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Examples

Experimental program
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Embodiment 1

[0020] The production method of embodiment 1 alginate lyase Aly01

[0021] A Screening Method for Vibrio natriegens

[0022] (1) The sea mud was sampled near the kelp breeding factory in Rongcheng City, Shandong Province, and 1g of the sample was taken and dispersed evenly in 50mL of sterile water.

[0023] (2) Inoculate 1 mL of supernatant into 50 mL of screening liquid medium, culture at 28°C and 200 rpm for 2 days, and dilute 10 -6 And spread it on the screening plate medium, culture at 28°C for 2 days, pick single colonies of different shapes and streak the plates several times to obtain pure cultures.

[0024] (3) Pick single colonies of different forms, inoculate them in liquid medium, and culture them at 28°C and 200rpm for 2 days, take the supernatant to measure the enzyme activity of the strain, select a strain with higher enzyme activity, and entrust the China Center for Typical Culture Collection to preserve it. The morphological characteristics, physiological and...

Embodiment 2

[0039] Example 2 Sequence Alignment

[0040] After performing amino acid sequencing on the enzyme, primers were designed to amplify the gene encoding alginate lyase from the genome of Vibrio natriegens SK42.001, and the nucleotide sequence is shown in SEQ ID NO:2. DNA sequence BLAST results: The alginate lyase provided by the present invention has the closest DNA sequence homology to the alginate lyase derived from Vibrio alginolyticus FDAARGOS, but the similarity is only 85%, with 231 bases different and 8 gaps.

[0041] Amino acid sequence BLAST results: the alginate lyase provided by the present invention has the closest amino acid sequence homology with an alginate lyase derived from the genus Vibrio in the NCBI database, with a similarity of 93%, 39 amino acid differences, and 0 gap.

Embodiment 3

[0042] Embodiment 3 enzymatic properties

[0043] Enzyme activity definition: the amount of enzyme required to produce 1 μmol of reducing sugar per minute.

[0044] Enzyme activity detection method: 1mL enzyme reaction solution (50mM, pH7.0 PB buffer solution) contains: 5mg sodium alginate, 300mM NaCl, 0.84μg alginate lyase, react at 35°C for 30min, take the supernatant and use the DNS method to detect the enzyme live.

[0045] (1) Effect of temperature on enzyme activity: 1mL enzyme reaction solution (50mM, PB buffer solution with pH7.0) contains: 5mg sodium alginate, 300mM NaCl, 0.84μg alginate lyase; place the enzyme reaction solution in 4 °C, 20 °C, 30 °C, 35 °C, 40 °C, 50 °C, 60 °C and 70 °C in a water bath for 30 min, and measure the activity of alginate lyase at each temperature. Such as figure 2 As shown, the optimum reaction temperature is 35°C.

[0046] (2) Effect of pH on enzyme activity: 1mL enzyme reaction solution contains: 5mg sodium alginate, 300mM NaCl, 0...

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Abstract

The invention discloses an alginate lyase encoding gene, and belongs to the technical field of biology. Alginate lyase is high in degrading activity and stable in property, enzyme activity reaches 65U / mg, 98% or more of initial enzyme activity is still kept after the alginate lyase is stored for 18 months at the temperature of 4 DEG C, the alginate lyase has high product specificity, and brown algae oligosaccharide trisaccharide can be specifically produced. The alginate lyase has important industrial application values and scientific research values.

Description

technical field [0001] The invention relates to a gene encoding alginate lyase, which belongs to the field of biotechnology. Background technique [0002] Alginate is composed of β-D-mannuronic acid (β-D-mannuronate) and α-L-guluronate (α-L-guluronate) two sugar units polymerized by 1,4 glycosidic bonds my country is the largest producer of alginate in the world, with production exceeding 70% of the total production. Alginate Oligosaccharides (AOS) is a degradation product of alginate, containing 2-20 sugar units, with a small molecular weight, which can overcome the limitation that alginate macromolecules cannot pass through various biological barriers of the body, and has a wider range of applications. It shows more excellent biological activities than alginate polysaccharides, such as anticoagulation, lowering blood sugar and blood lipids, anti-inflammation, anti-oxidation, anti-tumor, immune regulation, etc., and can also promote the growth of beneficial microorganisms s...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N15/74C12N1/21C12P19/00C12R1/63
CPCC12N9/88C12Y402/02003C12Y402/02011C12N15/74C12P19/00Y02E50/10
Inventor 江波张涛孟青陈静静
Owner JIANGNAN UNIV
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