A kind of β-agarase and its gene and application

Abstract

The present invention relates to a beta-agarase, and a gene and an application thereof. An amino acid sequence of the beta-agarase is shown in SEQ ID NO:1. A gene for encoding the beta-agarase is derived from a marine sediment microorganism metagenome sequencing data set. The beta-agarase is subjected to heterologous expression and purification. Through detection, the beta-agarase can catalyze degradation of agar into neoagarotetraose and neoagarohexaose, has a wide temperature application range and pH function range, can still maintain relatively strong hydrolysis performance at a condition of 50 DEG C for heat preservation for more than 20 h or in a buffer solution of pH 5.0-10.0 for warm bath for 60 h, and has relatively higher stability. The provided beta-agarase has good practicability, excellent gene resources and enzyme resources for industrial production of agar hydrolysis and neoagarooligosaccharode are provided, and the beta-agarase has wide application prospects.

Description

technical field

[0001] The invention relates to the field of biotechnology, in particular to a β-agarase and its gene and application. Background technique

[0002] Agar is a polysaccharide derivative extracted from marine red algae. In a certain way, macromolecular agar polysaccharides can be degraded into small molecular agar oligosaccharides, and agar oligosaccharides have many important biological activities including anti-inflammatory, anti-cancer, whitening, anti-oxidation, etc. , pharmaceuticals and cosmetics fields have important application value.

[0003] Agarase is a general term for a class of polysaccharide degrading enzymes that can catalyze the hydrolysis of agar polysaccharides to form agar oligosaccharides with a degree of polymerization of 2-10. In the natural environment, agarase is widely distributed, and many microorganisms and some marine molluscs can produce agarase. Most of the agarases currently reported come from marine bacteria, including Vibrio...

Examples

Embodiment 1

[0031] The acquisition of embodiment 1β-agarase gene agaM2 gene

[0032] The β-agarase gene in this example is obtained by analyzing the marine sediment microbial metagenomic sequencing data set, named agaM2 gene, and its nucleotide sequence is the sequence shown in SEQ ID NO:2. The whole sequence of the agaM2 gene is synthesized to obtain the β-agarase gene agaM2 gene.

Embodiment 2

[0033] Cloning expression of embodiment 2 agaM2 gene

[0034] The agaM2 gene synthesized in Example 1 was linked to pEASy @ -Blunt E1 vector (purchased from full gold), the connection method was carried out according to the instructions of the vector to obtain the pEASy-agaM2 recombinant vector; the obtained recombinant vector was transformed into Escherichia coli E.coli BL21 (DE3) competent cells, and coated with Spread on LB solid culture plates containing 100 μg / mL ampicillin, culture overnight at 37°C; inoculate positive clones into 2×YT liquid medium containing 100 μg / mL ampicillin and 0.7% glucose, and culture at 37°C until bacterial liquid OD 600 When the concentration is 0.6, add isopropylthio-β-D-galactoside (IPTG) at a final concentration of 1 mM, and induce the expression of the target protein rAgaM2 after 10 h at 25°C.

Embodiment 3

[0035] The separation and purification of embodiment 3rAgaM2 protein

[0036] The bacterial cells obtained by inducing expression in Example 2 were collected by centrifugation, resuspended in lysis buffer (0.5mol / L NaCl, 20mmol / L Tris-HCl, pH 8.0) for lysis, and the lysed suspension was lysed at 4°C Centrifuge at 4000 rpm for 20 min, and collect the supernatant. The supernatant was previously mixed with NiSO 4 Combined R10-Flammable (purchased from GE Healthcare) was mixed and eluted with imidazole at a concentration of 200mM to obtain the recombinant β-agarase rAgaM2 protein, whose amino acid sequence is shown in SEQ ID NO.1, and carried out by SDS-PAGE Electrophoresis analysis, electrophoresis test results see figure 1 , the third lane is the purified rAgaM2 protein, and its size is consistent with the predicted molecular weight (83.5kD).

technical field

[0001] The invention relates to the field of biotechnology, in particular to a β-agarase and its gene and application. Background technique

[0002] Agar is a polysaccharide derivative extracted from marine red algae. In a certain way, macromolecular agar polysaccharides can be degraded into small molecular agar oligosaccharides, and agar oligosaccharides have many important biological activities including anti-inflammatory, anti-cancer, whitening, anti-oxidation, etc. , pharmaceuticals and cosmetics fields have important application value.

[0003] Agarase is a general term for a class of polysaccharide degrading enzymes that can catalyze the hydrolysis of agar polysaccharides to form agar oligosaccharides with a degree of polymerization of 2-10. In the natural environment, agarase is widely distributed, and many microorganisms and some marine molluscs can produce agarase. Most of the agarases currently reported come from marine bacteria, including Vibrio...

Examples

Embodiment 1

[0031] The acquisition of embodiment 1β-agarase gene agaM2 gene

[0032] The β-agarase gene in this example is obtained by analyzing the marine sediment microbial metagenomic sequencing data set, named agaM2 gene, and its nucleotide sequence is the sequence shown in SEQ ID NO:2. The whole sequence of the agaM2 gene is synthesized to obtain the β-agarase gene agaM2 gene.

Embodiment 2

[0033] Cloning expression of embodiment 2 agaM2 gene

[0034] The agaM2 gene synthesized in Example 1 was linked to pEASy @ -Blunt E1 vector (purchased from full gold), the connection method was carried out according to the instructions of the vector to obtain the pEASy-agaM2 recombinant vector; the obtained recombinant vector was transformed into Escherichia coli E.coli BL21 (DE3) competent cells, and coated with Spread on LB solid culture plates containing 100 μg / mL ampicillin, culture overnight at 37°C; inoculate positive clones into 2×YT liquid medium containing 100 μg / mL ampicillin and 0.7% glucose, and culture at 37°C until bacterial liquid OD 600 When the concentration is 0.6, add isopropylthio-β-D-galactoside (IPTG) at a final concentration of 1 mM, and induce the expression of the target protein rAgaM2 after 10 h at 25°C.

Embodiment 3

[0035] The separation and purification of embodiment 3rAgaM2 protein

[0036] The bacterial cells obtained by inducing expression in Example 2 were collected by centrifugation, resuspended in lysis buffer (0.5mol / L NaCl, 20mmol / L Tris-HCl, pH 8.0) for lysis, and the lysed suspension was lysed at 4°C Centrifuge at 4000 rpm for 20 min, and collect the supernatant. The supernatant was previously mixed with NiSO 4 Combined R10-Flammable (purchased from GE Healthcare) was mixed and eluted with imidazole at a concentration of 200mM to obtain the recombinant β-agarase rAgaM2 protein, whose amino acid sequence is shown in SEQ ID NO.1, and carried out by SDS-PAGE Electrophoresis analysis, electrophoresis test results see figure 1 , the third lane is the purified rAgaM2 protein, and its size is consistent with the predicted molecular weight (83.5kD).

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