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Novel alginate lyase, preparation method and application thereof

A technology of alginate lyase and expression vector, applied in the field of its preparation, alginate lyase and its coding gene, can solve the problems of low activity of alginate lyase, low enzyme activity, poor stability, etc., and achieve wide substrate specificity The effect of resistance and strong tolerance

Active Publication Date: 2019-02-01
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 and only come from Pseudoalteromonas sp. alginate lyase Aly-SJ02 (Li Jianwei, Marine Drugs, 2011, 21:1374-80), derived from termite gut Isoptericola halotolerans The alginate lyase AlyIH (Dou Wenfang et al., Carbohydrate Polymers, 2013, 98:1476-82) and the like have broad substrate specificity, but the enzyme activity is low and the stability is poor. AlyIH has only obtained purified enzymes and has not yet obtained Its coding gene cannot be recombinantly expressed and molecularly modified; and most alginate lyases have low activity

Method used

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  • Novel alginate lyase, preparation method and application thereof
  • Novel alginate lyase, preparation method and application thereof
  • Novel alginate lyase, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Gene mining of alginate lyase

[0043] The results analyzed by RAST software showed that the genomic DNA of the marine bacteria strain carried the coding gene of alginate lyase alg509 , analyzed with the biological software DNAMAN, showing that the theoretical molecular weight of the protein encoded by the gene is 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

[0044] Example 2 Construction of Alginate Lyase Alg509 Heterologous Expression Engineering Strain

[0045] Use the Bacterial Genome Extraction Kit to extract the genome of the marine bacilli, and design primers to amplify the alginate lyase alg509 Gene. The PCR conditions were: pre-denaturation at 95°C for 3 minutes, followed by 32 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 2 min, and finally extension at 72°C for 10 min. 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.

[0046] Upstream primer: 5'-AAGAAGGAGATATACATATGAAAATCAACAGG

[0047] TTACTTCCTTTC-3'

[0048] Downstream primer: 5'-TGGTGGTGGTGGTGCTCGAGATCGTGGGTGTG

[0049] CTCAAGGG-3'

[0050] 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...

Embodiment 3

[0051] Example 3 Heterologous expression and purification of alginate lyase

[0052] The heterologous expression method of alginate lyase is as follows:

[0053] (a) Sequence analysis of the correct plasmid transfection strain Escherichiacoli Induced expression in BL21, plated and cultured in a 37°C incubator;

[0054] (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;

[0055] (c) Inoculate 0.5% inoculum into a 250 mL Erlenmeyer flask containing 100 mL of fermentation medium, and incubate for 3-4 hours at 37°C and 200 r / min;

[0056] (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.

[0057] (e) Collect the cells cultured in step (d), centrifuge at 4°C and 6000 r / min for 30 min, collect the cells, and resuspend the cells with 2 mL of pH 9 buffer (20mM glycine-sodium hydroxide buf...

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Abstract

The invention discloses an alginate lyase (Alg509) derived from marine bacteria and a gene thereof, and also disclosed a method for recombinant expression and preparation of the alginate lyase. According to the method, the alg509 gene is cloned into an E. coli expression vector, and the vector is transformed into an E. coli host strain to obtain a recombinant engineering strain which can heterologously express the enzyme. The alginate lyase Alg509 disclosed in the invention has high enzyme activity, the specific enzyme activity can reach up to 48000 U / mg and above, the optimum reaction pH is 10, the optimum reaction temperature is 55 DEG C, and the enzyme activity has no dependence on various metal ions. The enzyme is active to sodium alginate, poly-guluronic acid (polyG) and ploymannuronic acid (polyM), and can completely degrade sodium alginate to produce alginate oligomers such as alginate disaccharide, alginate trisaccharide, alginate tetrasccharide, etc. The enzyme exhibits strongbasophilia, has certain tolerance to high pH, has certain potential of industrial applications, and can be widely applied in the fields of agriculture, food, feed additive, medicine and the like.

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 (Mannuronic acid, M) linked by glycosidic bonds. Poly-M segments, poly-G...

Claims

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Application Information

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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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