Alginate lyase and application thereof

A technology of alginate lyase and algae oligosaccharide, which is applied in the directions of lyase, application, biochemical equipment and method, can solve the problems of high price, few types of enzyme preparations, restriction enzyme application and development, etc., and achieves high product specificity. , stable nature, important industrial application value and scientific research value effect

Inactive Publication Date: 2019-11-12
山东海之宝海洋科技有限公司
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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 the price is expensive, which limits the application and development of this enzyme.

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

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, 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 plate several times to obtain pure culture.

[0024] (3) Pick single colonies of different shapes, inoculate them in liquid medium, and culture them at 28°C and 200rpm for 2 days, take the supernatant to measure the activity of alginate lyase, select a strain with higher enzyme activity, and entrust the Chinese typical culture The morphological characteristics, physiological and biochemical characteristics and 16S rDNA se...

Embodiment 2

[0039] Example 2 Sequence Alignment

[0040] After amino acid sequencing of the enzyme, primers were designed to amplify the gene encoding alginate lyase from the genome of Vibrio natriegens SK42.001, the nucleotide sequence of which 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 base differences 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 (enzyme purified in Example 1), react at 35°C for 30min, The fermentation supernatant was taken to detect the enzyme activity by DNS method.

[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 ...

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Abstract

The invention discloses alginate lyase and an application thereof, and belongs to the technical field of biology. The alginate lyase provided by the invention is high in degradation activity, and theenzyme activity can reach 65U / mg; the alginate lyase is stable in nature, and after the alginate lyase is stored at 4 DEG C for 18 months, the enzyme activity still maintains 98% or above of initial enzyme activity; and the alginate lyase has very high product specificity, and can specifically produce alginate oligosaccharides and alginate tri-saccharide. The prepared alginate lyase has importantindustrial application value and scientific research value.

Description

technical field [0001] The invention relates to an alginate lyase and an application thereof, belonging 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 microo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N1/20C12P19/00C12R1/63
CPCC12N1/20C12N9/88C12P19/00
Inventor 江波刘晓勇张涛孟青陈静静王心怡
Owner 山东海之宝海洋科技有限公司
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