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A kind of alginate lyase, its preparation method and application

A technology of alginate lyase and lyase, which is applied in the field of preparation of alginate lyase and its coding gene, can solve the problems of low enzyme activity, low activity of alginate lyase, inability to recombine expression and molecular transformation, and achieve Effects of strong tolerance, broad substrate specificity

Active Publication Date: 2021-02-05
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the alginate lyases obtained are alginate lyases that specifically degrade polymannuronic acid, and a few are alginate lyases that have the activity of degrading guluronic acid. Lyases are very rare, only alginate lyase Aly-SJ02 (Li Jianwei, Marine Drugs, 2011, 21:1374-80) derived from Pseudoalteromonassp., and alginate lyase AlyIH derived from Isoptericola halotolerans in termite gut (Dou Wenfang etc., Carbohydrate Polymers, 2013,98:1476-82) etc. have broad substrate specificity, but the enzyme activity is low, and the stability is poor, wherein AlyIH has only obtained the purified enzyme, has not yet obtained its coding gene, and cannot be expressed recombinantly and molecular modification; and most alginate lyases are less active

Method used

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  • A kind of alginate lyase, its preparation method and application
  • A kind of alginate lyase, its preparation method and application
  • A kind of alginate lyase, its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Gene mining of alginate lyase

[0042] The results of analysis with RAST software showed that the genomic DNA of the marine bacteria strain carried the gene alg509 encoding alginate lyase, and the analysis with the biological software DNAMAN showed that the theoretical molecular weight of the protein encoded by the gene was about 61kD. The signal peptide online prediction software SignalP4.1Server was used to predict and analyze, and the amino acid sequence displayed in the result contained secreted signal peptide.

Embodiment 2

[0043] Example 2 Construction of Alginate Lyase Alg509 Heterologous Expression Engineering Strain

[0044] The marine bacillus genome was extracted using a bacterial genome extraction kit, and primers were designed to amplify the gene of alginate lyase alg509. The PCR conditions were: pre-denaturation at 95°C for 3 minutes, followed by 32 cycles of 95°C for 30s, 55°C for 30s, and 72°C for 2 minutes, and finally extension at 72°C for 10 minutes. Agarose gel electrophoresis showed a specific band at about 1.70kb, which was excised from the agarose gel and purified using a DNA gel recovery kit.

[0045] Upstream primer: 5'-AAGAAGGAGATATACATATGAAAATCAACAGGTTACTTCCTTTC-3'

[0046] Downstream primer: 5'-TGGTGGTGGTGGTGCTCGAGATCGTGGGTGTGCTCAAGGG-3'

[0047] The purified DNA fragment was connected to the cloning vector pet-21a, transformed into Escherichia coli DH5α competent cells, cultured in LB solid medium (containing ampicillin), and a single colony was picked for PCR verificati...

Embodiment 3

[0048] Example 3 Heterologous Expression and Purification of Alginate Lyase

[0049] The heterologous expression method of alginate lyase is as follows:

[0050] (a) Induce the expression of the correct plasmid transfection strain Escherichia coli BL21 in the sequencing analysis, spread the plate and culture it in a 37°C incubator;

[0051] (b) Pick a single colony, inoculate it into a 30 mL test tube containing 5 mL of fermentation medium, and incubate with shaking at 37°C for about 12 hours;

[0052] (c) Inoculate 0.5% of the inoculum into a 250mL Erlenmeyer flask containing 100mL of fermentation medium, and cultivate for 3-4h at 37°C and 200r / min;

[0053] (d) When the OD600 of the bacterial solution grows to 0.6-0.8, add IPTG (final concentration 0.5mmol / L) and induce at 16°C for about 20-24h.

[0054] (e) Collect the bacteria cultured in step (d), centrifuge at 4°C and 6000r / min for 30min, collect the bacteria, resuspend the bacteria with 2mL of pH 9 buffer (20mM glycin...

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Abstract

The invention discloses a alginate lyase (Alg509) derived from marine bacteria and its gene. At the same time, the method for recombinant expression and preparation of the alginate lyase is also disclosed, which is about to alg509 The gene is cloned into an Escherichia coli expression vector, and the vector is transformed into an Escherichia coli host bacterium to obtain a recombinant engineering strain capable of heterologously expressing the enzyme. The alginate lyase Alg509 disclosed by the present invention has high enzyme activity, the specific enzyme activity can reach more than 48000U / mg, the optimum reaction pH is 10, the optimum reaction temperature is 55°C, and the enzyme activity has no dependence on various metal ions . The enzyme is active on sodium alginate, polyguluronic acid (polyG) and polymannuronic acid (polyM), and can completely degrade sodium alginate to produce fucobiose, fucoidanose, and fucoidan Sugar and other fucoidan oligosaccharides. The enzyme exhibits strong basophilicity, has a certain tolerance to high pH, ​​has a certain potential for industrial application, and can be widely used in agriculture, food, feed additives, medicine and other fields.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a alginate lyase and its coding gene, its preparation method and application. Background technique [0002] my country is rich in seaweed resources. In recent years, with the continuous development of marine drugs, the research on seaweed polysaccharides has been paid more and more attention. Brown algae is one of the seaweeds with wide application value. Representative plants include kelp, staghorn and horsetail. It is also an economical seaweed plant that is abundant in the ocean. It contains various polysaccharides such as algin, fucoidan and laminaran. Sodium alginate (trade name: sodium alginate) or other alginates currently on the market are mainly obtained from brown algae. [0003] Alginate is a linear polysaccharide composed of α-L-guluronic acid (G) and β-D-mannuronic acid (Mannuronicacid, M) linked by glycosidic bonds. M segments, poly G segme...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N15/75C12N15/81
CPCC12N9/88C12N15/70C12N15/75C12N15/81C12Y402/02003C12Y402/02011
Inventor 陈朋闫军军朱玥明曾艳门燕孙媛霞
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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