CN102994407B - Flavobacterium strain and incision alginate lyase coding gene, preparation and application - Google Patents

Abstract

The invention discloses a gen sequence of incision alginate lyase Alg2A, as well as a preparation method and application thereof. The incision alginate lyase Alg2A is from soil new separated strain Flavobacterium sp.S20. The invention also provides a preparation method of the novel alginate lyase, namely a technical method by utilizing genetic engineering. According to the method, the gene of the novel alginate lyase is cloned to an escherichia coli expression vector to obtain an escherichia coli recombined strain capable of heterologously expressing the alginate lyase, and the alginate lyase Alg2A prepared by heterologous expression of the strain has the function of preparing sodium alginateoligosaccharide by degrading sodium alginate. The alginate lyase Alg2A provided by the invention can be widely applied to the fields of chemical industry, agriculture, food, feed additive, medicine, algae genetic engineering and the like.

Description

Flavobacterium strain and endo-alginate lyase coding gene and its preparation and application

technical field

The invention relates to a gene sequence of an endo-alginate lyase Alg2A, a preparation method and application thereof. The invention also provides the recombinant plasmid and recombinant genetic engineering strain of the endo-alginate lyase. The endo-alginate lyase Alg2A of the present invention can be widely used in the fields of chemical industry, agriculture, food, feed addition, medicine, seaweed genetic engineering and the like.

Background technique

The vast ocean is rich in seaweed resources, which are mainly composed of four major categories: cyanobacteria, green algae, red algae and brown algae. Algin is a straight-chain acidic polysaccharide. In the natural state, the main forms of algin in the cell wall of brown algae are water-soluble sodium alginate (sodium alginate), potassium and other alkali metal salts and water-insoluble alginic acid. (alginic acid) and its alginates combined with 2 or more valent metal ions. Sodium alginate (trade name: sodium alginate) or other alginates currently on the market are mainly obtained from brown algae. Studies have found that the oligosaccharides obtained by degrading sodium alginate have various biological activities, such as immune regulation, growth promotion, induction of plant resistance and improvement of protein stability, etc., so they can be widely used in chemical industry, agriculture, food, feed additives and medicine and other fields (Dou Yong, Guangxi Light Industry, 2009, 10: 12-13). Sodium alginate can be degraded by various methods, including chemical degradation, physical degradation and enzymatic degradation. The chemical degradation method is mainly based on acid degradation, but the degradation conditions of this method are difficult to control, and the operation is complicated and time-consuming. Physical degradation methods include radiation method and ultrasonic method, and are generally used together with other degradation methods. The limit molecular mass of the degradation product is about 50ku, and it is not easy to produce oligosaccharides. The degradation of sodium alginate with alginate lyase has the advantages of mild degradation conditions and high yield, and because of the substrate specificity of the enzyme, it can provide information for subsequent research on the chemical structure of oligosaccharides, so alginate lyase has gradually become a priority for degradation. Sodium alginate method. In addition, alginate lyase can also be used in the treatment of pulmonary cystic fibrosis, seaweed feed processing and seaweed genetic engineering research (Wong TY et al. Annual Review of Microbiology, 2000, 54: 289-340).

Alginate lyase is derived from marine animals and plants and various microorganisms (including marine bacteria, soil bacteria and fungi). Sodium alginate lyase can be divided into two categories according to its substrate specificity: 1,4-β-D-mannuronic acid fragment lyase (Ec 4.2.2.3) and 1,4-a-L-gulose Aldehydic fragment lyase (EC 4.2.2.11). So far, the production of alginate lyase mostly relies on the original enzyme-producing animals, plants or microorganisms to obtain enzyme protein. Although this method can effectively obtain a certain amount of enzyme protein, the output is limited and the cost is high, which is difficult to meet the actual application requirements. . With the development of biotechnology, a technical method for efficiently producing alginate lyase by genetic engineering is provided. Dong Eun Kim et al. designed PCR primers based on the similar sequences of known related functional genes, cloned an alginate lyase gene from the Streptomyces sp. ALG-5 strain, and successfully expressed it in Escherichia coli BL21 (DE3) (Kim et al. al. Marine Biotechnology. 2009.11: 10-16). Using this strategy requires a certain understanding of related gene sequences to design PCR primers, and what is found is a new molecule in a certain class of proteins with similar structures or functions, and it is difficult to discover brand new genes.

Constructing the genome library of enzyme-producing strains can break through the limitations of PCR cloning genes, fully excavate and utilize the genetic resources of microorganisms, and combined with the method of functional screening, new genes can be discovered. Caswell et al. cloned the alginate lyase gene that specifically degrades polyguluronic acid fragments from the genome library of the Klebsiella pneumoniae PG1 strain. The molecular weight of the enzyme is 28kDa (Caswell et al. Gene, 1989, 75: 127- 134). Maki and Kraiwattanapong et al. cloned two alginate lyase genes from the genome library of Pseudomonas sp. 1365bp, encoding 398 amino acids, with a molecular weight of about 50kDa (Maki et al. Journal of General Microbiology, 1993, 139:987-993). The other is alyII, the coding region of the gene is 2141bp long, encoding 713 amino acids, and the molecular weight is about 79kDa (Kraiwattanapong et al. Bioscience Biotechnology and Biochemistry, 1997, 61(11): 1853-1857). So far, there are few studies on alginate lyase produced by Flavobacterium. Takeuchi et al. purified two alginate lyases from the crude enzyme solution produced by Flavobacterium multivolum K-11 by column chromatography, one is polyguluronic acid lyase with a molecular weight of 43kDa (EC 4.2.2.11 ) (Takeuchi et al.Food Science and Technology International.1997.3(1):22-26), the other is 32kDa molecular weight, for the two fragment substrates of polyguluronic acid fragment and polymannuronic acid fragment Alginate lyase with the same degradation rate (Takeuchi et al. Food Science and Technology International. 1997.3(4): 388-392). An et al. newly isolated a strain of Flavobacterium sp.LXA producing alginate lyase, they partially purified the alginate lyase from the fermentation broth of the strain by ammonium sulfate precipitation, and used the enzyme for algin oligosaccharides Sugar production and activity research (An et al.Journal of Applied Microbiology.2009.106:161-170). Alginate lyase gene derived from Flavobacterium has not been reported yet.

Contents of the invention

The first object of the present invention is to provide a new strain of the genus Flavobacterium producing alginate lyase, which is Flavobacterium S20, named after classification: Flavobacterium sp., preservation number: CGMCC NO.5026.

The second object of the present invention is to provide a novel high-efficiency endoalginate lyase Alg2A and its coding gene.

The third object of the present invention is to provide a method for preparing a novel high-efficiency endo-alginate lyase Alg2A.

The fourth object of the present invention is to provide a recombinant expression plasmid containing the endo-alginate lyase Alg2A gene and a recombinant genetic engineering strain.

Another object of the present invention is to provide the application of novel high-efficiency endo-alginate lyase Alg2A in the degradation of sodium alginate.

The endoalginate lyase Alg2A provided by the present invention is derived from the new strain Flavobacterium sp.S20 isolated and purified in the soil, and its amino acid sequence has one of the following characteristics:

1) The sequence of amino acid residues at positions 1-288 or 23-288 of SEQ ID NO.2 in the sequence listing starting from the amino terminal, wherein positions 1-22 are signal peptides, and positions 23-288 are active alginate cleavage Amino acids of the enzyme Alg2A.

2) The sequence of amino acid residues at positions 1-288 or 23-288 of SEQ ID NO.2 in the sequence listing from the amino terminal has the ability to degrade sodium alginate after amino acid residue substitution and/or deletion and/or addition active protein.

3) A protein whose homology with the amino acid sequence defined by SEQ ID NO.2 in the sequence listing reaches 80% or more and has the activity of degrading alginate.

The present invention also provides the coding gene of endoalginate lyase Alg2A (named alg2A), which has one of the following nucleotide sequence characteristics:

1) the deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in the sequence listing;

2) the deoxyribonucleic acid (DNA) sequence of the amino acid sequence of SEQ ID NO.2 in the coding sequence list;

3) The homology with the deoxyribonucleic acid (DNA) sequence defined by SEQ ID NO.1 reaches 80% or more, and can encode the deoxyribonucleic acid (DNA) sequence that degrades alginate protein.

The amino acid sequence of the alginate lyase Alg2A and its nucleotide coding sequence of the present invention can also be artificially synthesized according to the predicted amino acid sequence of Alg2A and its nucleotide coding sequence.

The method for preparing the recombinant enzyme Alg2A is to clone the coding gene alg2A into a recombinant expression vector, introduce it into a host cell, and obtain a recombinantly expressed endoalginate lyase.

The expression vector of the recombinant expression endo-alginate lyase Alg2A can be Escherichia coli expression vector, yeast expression vector, Bacillus subtilis expression vector, lactic acid bacteria expression vector, Streptomyces expression vector, phage vector, filamentous fungus expression vector, plant Expression vectors, insect expression vectors, or mammalian cell expression vectors, etc.

Recombinant bacteria or transgenic cell lines for recombinantly expressing endoalginate lyase Alg2A can be Escherichia coli host cells (such as Escherichia coli BL21, Escherichia coli JM109, Escherichia coli DH5α, etc.), yeast host cells (such as Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyceslactis, etc.), Bacillus subtilis host cells (such as Bacillus subtilis R25, Bacillus subtilis 9920, etc.), lactic acid bacteria host cells (such as Lactic acid bacteria COCC101, etc.), actinomycetes host cells (such as Streptomyces spp., etc.), filamentous fungal hosts Cells (such as Trichoderma viride, Trichoderma reesei, Aspergillus niger, Aspergillus nidulans, etc.), insect cells (such as Bombyxmori, Antharaea eucalypti, etc.) or mammalian cells (such as Chinese hamster ovary cells CHO, young hamster kidney cells BHK, Chinese hamster lung cells CHL wait).

Alg2A of the present invention is derived from Flavobacterium sp.S20, a new strain of Flavobacterium sp.S20 isolated and purified in the soil. Through the activity screening of the Flavobacterium sp.S20 genomic DNA library, a positive clone capable of degrading sodium alginate was obtained, and further sequenced, homologous The full-length coding sequence of the endoalginate lyase Alg2A was obtained by the alignment method. The coding region of the gene is 867 bp long, encodes 288 amino acids, and has a molecular weight of about 33 kD. It belongs to the alginate lyase family 2. Alg2A obtained by recombinant expression in Escherichia coli has the highest enzyme activity at 45°C and pH 8.5 when sodium alginate is used as the substrate, and the specific activity reaches 300U/mg. Compared with the two substrates of sodium alginate and polymannuronic acid fragment (polyM), Alg2A has a higher degradation activity for polyguluronic acid fragment (polyG).

The endo-alginate lyase Alg2A of the present invention can be widely used in the fields of chemical industry, agriculture, food, feed addition, medicine, seaweed genetic engineering and the like.

Description of drawings

Figure 1: Protein three-dimensional structure model of endoalginate lyase Alg2A

Figure 2: Polyacrylamide gel electrophoresis (SDS-PAGE) of the expression and purification of the recombinant endo-alginate lyase Alg2A. The samples added to each lane are: M: protein marker (marker), the size of the band from top to bottom is 116kD, 66.2kD, 45kD, 35kD, 25kD, 18.4kD, 14.4kD; lane 1: after the recombinant bacteria are broken Supernatant, loading volume 10 μl, lane 2: the effluent from the supernatant purified by nickel column after the crushing of recombinant bacteria, loading volume 10 μl, lane 3: 100 mmol imidazole elution collection, loading volume 10 μl.

Fig. 3: The effect curve of pH value on the activity of endo-alginate lyase Alg2A.

Figure 4: Curve of the effect of temperature on the activity of endo-alginate lyase Alg2A.

Figure 5: Curves of the influence of temperature on the stability of endo-alginate lyase Alg2A.

Figure 6: The influence curve of pH value on the stability of endo-alginate lyase Alg2A.

Figure 7: Substrate preference curve of the endo-alginate lyase Alg2A.

Figure 8: Electrospray mass spectrometry (ESI-MS) analysis diagram of the product obtained from the degradation of sodium alginate by endo-alginate lyase Alg2A.

A strain of the genus Flavobacterium, which is Flavobacterium S20, classified and named: Flavobacterium sp., preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures, address: Chinese Academy of Sciences, No. 1, No. 1 Beichen West Road, Chaoyang District, Beijing Institute of Microbiology, deposit number: CGMCC NO.5026, date of deposit: July 05, 2011.

Detailed ways

Embodiment 1 Flavobacterium new strain Flavobacterium sp.S20 cultivation and production of alginate lyase

The strain used is Flavobacterium sp. S20, and the single clone of Flavobacterium sp.S20 strain (CGMCC NO.5026) was picked and inoculated into 50ml liquid medium, and then placed in a shaking table with a temperature of 30°C and a rotation speed of 220rpmin. After 48 hours, the culture fluid was centrifuged to collect Flavobacterium sp.S20 cells and the supernatant culture medium was retained. The bacteria were resuspended with 5 ml of phosphate buffer (20 mM, pH 7.0) and then ultrasonically disrupted.

The liquid medium formula used is (g/L): beef extract 5g, glucose 15g, yeast extract powder 1.0g, NaCl 5.0g, MgSO ₄ 7H ₂ O 0.5g, CaCl ₂ 0.2g, KH ₂ PO ₄ 1.0g , FeSO ₄ ·7H ₂ O 0.02g, the pH value is 7.0.

Enzyme activity unit (U) definition: the amount of enzyme required to catalyze sodium alginate to produce 1 μmol reducing sugar per minute. According to the method of measuring alginate lyase activity with dinitrosalicylic acid (Qing-Da An et al. Process Biochemistry, 2008, 43: 842-847), the enzyme activity in the liquid medium of Flavobacterium sp.S20 strain was measured is 0.37U/ml, and the enzyme activity of the cell lysate is 1.34U/ml, so the total alginate lyase activity of the culture solution obtained by culturing the Flavobacterium sp.S20 strain with 50ml liquid medium for 48h is 1.71U/ml.

Example 2 Extraction of Flavobacterium sp.S20 bacterial strain genomic DNA

Take 2ml of fresh Flavobacterium S20 cultured overnight, and centrifuge (12,000rmp, 3min) to collect the bacteria. Wash the bacteria three times with phosphate buffer (20mM, pH7.0), add 650μl DNA extraction buffer (100mM Tris-HCl; 100mM Na ₂ EDTA; 100mM Na ₃ PO ₄ ; 1.5M NaCl; 1% (w/ v) Hexadecyltrimethylammonium bromide; pH 8.0), after mixing, freeze at -80°C, then place in a water bath at 65°C to thaw, repeat the freeze-thaw process three times. After cooling, add 4 μl lysozyme (100 mg/L) and shake horizontally (37 ° C, 225 rpm) for 30 minutes in a shaker, then add 3 μl proteinase K (20 mg/mL) and continue shaking for 30 minutes, and finally add 50 μl 20% (w/v) Sodium lauryl sulfate, after mixing, keep warm at 65°C for 2h (mix by inverting the centrifuge tube up and down every 20min). Centrifuge at room temperature for 10 min at 12,000 rpm, collect the supernatant, add 500 μl of saturated phenol:chloroform:isoamyl alcohol (volume ratio 25:24:1) to extract twice, then add 250 μl chloroform:isoamyl alcohol (volume ratio 24 : 1) After extraction once, 0.6 times the volume of isopropanol was added, left at room temperature for 20 minutes, and then centrifuged at 12,000 rpm for 15 minutes. Rinse the precipitate with 70% ethanol, dry it and dissolve it with 60 μl TE buffer (10 mM Tris-HCl, 1 mM Na ₂ EDTA, pH 8.0), add 1 μl ribonuclease (to remove RNA) and store at -20 °C .

Example 3 Construction of total genomic DNA library of Flavobacterium sp.S20 strain and screening of clones expressing alginate lyase activity from the library

The extracted Flavobacterium S20 strain genomic DNA was randomly digested with Sau3A I enzyme, and the digested product was separated by agarose gel electrophoresis to obtain a 3-10kb DNA fragment. These fragments were ligated with the pGEM11z vector that had been digested with BamHI and the phosphate group at the 5' end was removed with alkaline phosphatase, the ligated product was transformed into E. 3-indole-β-D-galactoside, 40μg/ml), IPTG (isopropyl-β-D-thiogalactopyranoside, 40μg/ml), Amp (ampicillin, 50μg/ml) On a solid plate of Luria-Bertani medium, culture at 37°C for 12-16h. Pick the white Escherichia coli transformants that grew out into the wells of a 96-well culture plate (200 μl LB medium/well), and after culturing at 37°C for 12 to 16 hours, add 150 μl of 50% glycerol to each well of the culture plate, and then Stored at -80°C, all white E. coli transformants stored in the wells of the 96-well culture plate are genomic DNA library clones of the Flavobacterium S20 strain. Sau3A I enzyme, BamHI enzyme and alkaline phosphatase were all purchased from Bao Biological Company, and the reaction system, temperature and time of enzyme and substrate were operated according to the product instructions provided by the company.

The genomic library clone of the Flavobacterium sp.S20 strain was inoculated in a 96-well culture plate, and each well was filled with 200 μl of Luria-Bertani medium (containing 50 μg/ml ampicillin), cultured at 37°C for 12 hours, and collected by centrifugation. After adding 20 μl of phosphate buffer solution (20 mM, pH 7.0) to each well, the cells were frozen at -80°C, then thawed at 37°C, and the freeze-thaw was repeated three times to rupture the cells. Use the dinitrosalicylic acid method described above to detect whether the lysates of all library clones have enzyme activity. The results show that the library clone numbered VII 2H expresses alginate lyase activity, and the recombinant protein contained in the library clone The plasmid was named pGEM11z-VII 2H.

Example 4 Sequencing and sequence analysis of the gene expressing alginate lyase activity on the recombinant plasmid pGEM11z-VII 2H

The recombinant plasmid pGEM11z-VII 2H expressing alginate lyase activity was sent to Yingwei Jieji Company for sequencing. The sequencing results were analyzed with the software on NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/). The NCBI analysis software used is Open Reading Frame Finder (ORF Finder, http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and BasicLocal Alignment Search Tool (BLAST, http://blast.ncbi .nlm.nih.gov/Blast.cgi).

NCBI analysis results showed that the recombinant plasmid pGEM11z-VII 2H carried an alginate lyase gene (named alg2A), the gene coding region was 867bp long, and its nucleotide sequence was shown in SEQ ID NO 1. The 520 nucleotides in the whole genome sequence of alg2A and Lacinutrix sp.5H-3-7-4 (NCBI accession number: CP002825.1) share 71% homology.

The alginate lyase Alg2A encoded by alg2A consists of 288 amino acids, its amino acid sequence is shown in SEQ ID NO 2, and the theoretical molecular weight of the protein is about 33kD. Using the Simple Modular Architecture Research Tool (SMART, http://smart.embl-heidelberg.de/) to analyze the structural information of the alginate lyase Alg2A, the results show that the amino acid from the N-terminus to the 22nd amino acid is the signal peptide sequence, and the 23rd Amino acid at position -288 belongs to alginate lyase family 2. SWISS-MODEL homology modeling server (http://swissmodel.expasy.org) was used to carry out homology modeling on the protein three-dimensional structure of alginate lyase Alg2A, and the finally obtained three-dimensional structure model of Alg2A protein is shown in Figure 1.

sequence listing

SEQ ID NO.1

ATGAGCATACAATTTTCAAAAATCTTATTACTAACGGTTCTA

GCAACTGCTACAATTAGTAATGCACAGGATAAAAAATCAAA

AAGCAAAACTGCTAAAATTGATTGGTCTCATTGGACGGTTAC

TGTGCCTGAGGAGAATCCAGATAAACCAGGTAAGCCGTACT

CTTTAGGGTATCCTGAAATACTAAATTATGCTGAGGATAAAA

TCGCATCCAAGTACATGTACGATGACCCAAAAGACAAGTCT

GTTGTTTTTTATGCCTTTCCTTCGGGAGTGACCACGGCTAATA

CGCATTATTCTCGTTCTGAGCTAAGAGAGACAATGGAAACTG

GTAGCAATAAGGTCAACTGGACATTTGCAAAAGGCGGTAAA

ATGAGAGGTACGTATGCTATTGACGACATTTCAAAAAGAGCC

AGATGGCAAATACAGCCGCGTTATTATTGCGCAAATTCACGG

TGTATTAACGGATGAACAACGTGATTTAATTGGTCAAAAAG

ACAACAATGCACCGCCTATTTTGAAAGTGTATTGGGATAAAG

GAAAAATTCGTGTGAAAACAAAAGTACTTAAAGATTTGAAC

GCGCCTTATAAAGAAATGCTTTTAGAACATGCTTGGGGTGAT

GATGAAGGTCGAAATTTTAAAGAGAAAATCGATTTAAACAC

AAGGTTTACTCTAGAAGTGAAAGTTTCGGATGGGCGAATGG

AAGTGATTTTAAATGATACAGAATCATTGGTTTACGATGATA

TTCACATGAAAAAATGGGGGATATTCGAAAATTATTTTAAAG

CAGGGAATTATTTTCAATCTAAAACACCAGGTACCTTTGCAA

AGGTAAAAAATATATTTCTTTACAAGTTACTCATTAG

SEQ ID NO.2

MSIQFSKILLTVLATATISNAQDKKSKSKTAKIDWSHWTVTVP

EENPDKPGKPYSLGYPEILNYAEDKIASKYMYDDPKDKSVVFY

AFPSGVTTANTHYSRSELRETMETGSNKVNWTFAKGGKMRGT

YAIDDISKEPDGKYSRVIIAQIHGVLTDEQRDLIGQKDNNAPPIL

KVYWDKGKIRVKTKVLKDLNAPYKEMLLEHAWGDDEGRNFK

EKIDLNTRFTLEVKVSDGRMEVILNDTESLVYDDIHMKKWGIFE

NYFKAGNYFQSKTPGTFAKVKIYSLQVTH

Embodiment 5 Recombinant expression of alg2A gene in Escherichia coli

Using the above-mentioned recombinant plasmid pGEM11z-VII 2H as a template, PCR amplification was carried out with the following primers. Primer pairs are as follows: forward primer Alg2A-F:

( CATATG CAGGATAAA

AAATCAAAAAGCAAAACTG), reverse primer Alg2A-R:

( CTCGAG ATGAG

TAACTTGTAAAGAATAT), the forward primer underlined is the restriction endonuclease Ned I site, and the reverse primer underlined is the restriction endonuclease Xho I site. Taq DNA polymerase was purchased from Bao Biological Company, and the PCR reaction system was operated according to the product instructions provided by the company. PCR reaction conditions: 94°C pre-denaturation for 5 minutes, then 94°C denaturation for 30 seconds-50°C annealing for 30 seconds-72°C extension for 1 minute, 30 cycles, and finally 72°C extension for 10 minutes. The PCR product was digested with Ned I and Xho I, and the digested PCR product was recovered by agarose gel electrophoresis. The product pET-21a vector purchased from Novagen, USA was digested with Ned I and Xho I, and the large fragment of the digested vector was recovered by agarose gel electrophoresis. Both Ned I and Xho I were purchased from Treasure Biotechnology Co., Ltd., and the reaction system, temperature and time of the enzyme and the substrate were operated in accordance with the product instructions provided by the company.

The double-enzyme-digested PCR product was connected to the pET-21a vector that also underwent double-enzyme digestion, and the ligated product was transformed into Escherichia coli Top10 strain and spread on a Luria-Bertani medium solid plate containing 50 μg/ml ampicillin, and cultured at 37°C 14h, single clone was picked; the single clone was cultured in liquid Luria-Bertani medium containing 50 μg/ml ampicillin, and the plasmid was extracted; the plasmid was subjected to colony PCR with forward primer Alg2A-F and reverse primer Alg2A-R Verified, the result obtained the amplified product of the correct size, which preliminarily proved that the recombinant plasmid constructed was correct; then the recombinant plasmid was sent to Yingwei Jieji Company for sequencing, and the results showed that at the Ned I and Xho I restriction sites of pET-21a The alg2A gene shown in SEQ ID NO 1 was inserted between them, and the insertion direction was correct, so it was further proved that the constructed recombinant plasmid was correct, and the recombinant plasmid was named pET21a-Alg2A.

pET21a-Alg2A was transformed into Escherichia coli strain BL21(DE3) (purchased from Novagen, USA), and then alginate lyase Alg2A was induced, expressed and purified according to the operation steps provided by the company. Polyacrylamide gel electrophoresis was used to detect the purification of alginate lyase Alg2A. The results are shown in Figure 2. The purified alginate lyase Alg2A showed a single band on the electrophoresis gel, and its position matched the predicted molecular weight. Example 6 Enzymatic property analysis of alginate lyase Alg2A

(1) Effect of pH and temperature on enzyme activity

After mixing the mass concentration of 1% sodium alginate substrate, purified Alg2A enzyme solution and 20mM Tris-HCl buffer solution (pH range of 6.0-10.0) with different pH values in a ratio of 8:2:10 (volume ratio) , reacted at 40°C for 10 minutes, and measured the enzyme activity according to the aforementioned dinitrosalicylic acid method. The results show that Alg2A reaches the maximum activity at pH 8.5, indicating that the optimum reaction pH of Alg2A is 8.5 (as shown in Figure 3)

At the optimum pH, mix the mass concentration of 1% sodium alginate substrate, purified Alg2A enzyme solution and 20mM Tris-HCl buffer solution (pH8.5) in a ratio of 8:2:10 (volume ratio), respectively React at different temperatures (20°C-70°C) for 10 minutes, and measure the enzyme activity according to the aforementioned dinitrosalicylic acid method. The results showed that Alg2A reached the maximum activity at 45°C, indicating that the optimum reaction temperature of Alg2A was 45°C (as shown in Figure 4).

After mixing the mass concentration of 1% sodium alginate substrate, purified Alg2A enzyme solution and 50mM Tris-HCl buffer solution in the ratio of 8:2:10 (volume ratio), react at the optimum temperature and optimum pH for 10 Minutes, measure the enzyme activity according to the aforementioned dinitrosalicylic acid method, then measure the protein content of the Alg2A enzyme solution with the protein quantification kit purchased from the company, the results show that the specific activity of the recombinant Alg2A to sodium alginate is 300U/mg .

(2) Effect of pH and temperature on enzyme stability

Mix the Alg2A enzyme solution after heat treatment for 1 h at different temperatures (20°C-50°C) with the substrate solution of 1% sodium alginate at a mass concentration of 2:8 (volume ratio), and then mix it at the optimum temperature and the optimum temperature. The remaining enzyme activity was measured at an appropriate pH, and the activity of the enzyme solution without heat treatment was defined as 100% relative activity (relativie activity). The results showed that Alg2A had better thermal stability at a temperature lower than 40°C (as shown in Figure 5 ).

Mix the Alg2A enzyme solution pre-incubated at 30°C with different pH (pH4-10) for 24h and the substrate solution with a mass concentration of 1% sodium alginate in a ratio of 2:8 (volume ratio), and then at the optimum temperature And the remaining enzyme activity was measured at the optimum pH, and the activity of the enzyme solution without pH treatment was defined as 100% relative activity (relativie activity). The results showed that in the range of pH 5-10, the Alg2A enzyme activity still maintained more than 60%. It shows that Alg2A has a wide range of pH tolerance (as shown in Figure 6).

(3) Substrate preference of Alg2A

The purified Alg2A was mixed with three different substrates of sodium alginate, polymannuronic acid fragment (polyM) and polyguluronic acid fragment (polyG) with a mass concentration of 1% respectively in a 2:8 (volume ratio) Proportionally mixed, and then reacted at 30°C and pH 8.5. The products at different reaction time points were taken to measure the 235nm ultraviolet absorption value, and it was found that the 235nm absorption value gradually increased with the prolongation of the enzymatic hydrolysis time. According to the principle of measuring alginate lyase by ultraviolet method (Qing-Da An et al. , 43:842-847), confirming that Alg2A is a lyase. In addition, as shown in Figure 7, the degradation rate of Alg2A on polyguluronic acid fragments is faster than that on sodium alginate and polymannuronic acid fragments, indicating that Alg2A prefers to degrade polyguluronic acid fragments.

The influence of embodiment 7 metal ions on Alg2A activity

Mix the mass concentration of 1% sodium alginate substrate, purified Alg2A enzyme solution and 50mM Tris-HCl buffer (pH8.5) in a ratio of 8:2:10 (volume ratio), and then add different The metal ions were added with a final concentration of 5mM or 10mM, and then reacted at 45°C for 10 minutes, and the enzyme activity was measured by the aforementioned dinitrosalicylic acid method. The control group is the activity of Alg2A without adding any metal ions (set as 100%), and the results are shown in the table below. The experimental results show that K ⁺ , Na ⁺ can increase the activity of Alg2A, Li ⁺ , Mg ²⁺ have basically no effect on the activity of Alg2A, and other ions such as Ca ²⁺ , Fe ²⁺ can inhibit the enzyme activity.

Table 1: Effect of metal ions on the activity of Alg2A

Example 8 Electrospray mass spectrometry (ESI-MS) analysis of the product obtained from the degradation of sodium alginate by Alg2A

After mixing the mass concentration of 1% sodium alginate substrate, purified Alg2A enzyme solution and 50mM Tris-HCl buffer solution in a ratio of 8:2:10 (volume ratio), react at pH 8.5, 45°C, The products with different enzymatic hydrolysis time (0.5, 1, 1.5, 2 hours) were selected for electrospray mass spectrometry analysis. Electrospray mass spectrometry conditions are positive ion mode, ion source voltage: 4.5kV; sheath gas flow: 30arb; capillary temperature: 275-300°C; tube mirror voltage: 250V; scan range: 150-2000.

The electrospray mass spectrogram of the enzymatic hydrolysis for 2 hours is shown in Figure 8. The mass spectrogram shows that Alg2A degrades sodium alginate for 2 hours with a series of sodium alginate oligosaccharides with different degrees of polymerization, which fully proves that Alg2A is an endo-alginate lyase. Therefore, the endo-alginate lyase Alg2A can be used in the preparation of sodium alginate oligosaccharides and the fields related to the degradation of sodium alginate, including chemical industry, agriculture, food, feed additive, medicine and seaweed genetic engineering and so on.

Claims (7)

1. the bacterial strain of Flavobacterium, it is Flavobacterium S20, Classification And Nomenclature: Flavobacterium Flavobacterium sp., be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, deposit number: CGMCC NO.5026, preservation date on 07 05th, 2011.

2. an inscribe alginate lyase gene Alg2A who derives from bacterial strain (Flavobacterium sp.S20) described in claim 1, its nucleotide sequence has one of following feature:

1) be thymus nucleic acid (DNA) sequence of SEQ ID NO.1 in sequence table;

2) thymus nucleic acid (DNA) sequence of coding SEQ ID NO.2 aminoacid sequence.

3. according to the inscribe algin catenase of inscribe alginate lyase gene coding claimed in claim 2, the aminoacid sequence of its coding is 1-288 or the 23-288 amino acids residue sequence that in sequence table, SEQ ID NO.2 starts from aminoterminal.

4. prepare the method for inscribe algin catenase as claimed in claim 3 for one kind, it is characterized in that: be that inscribe alginate lyase gene claimed in claim 2 is cloned into recombinant expression vector, import host cell, obtain recombinant expressed inscribe algin catenase;

The expression vector of described recombinant expressed inscribe algin catenase, refers to coli expression carrier, Yeast expression carrier, Bacillus subtilus expression vector, lactic acid bacteria expression vectors, streptomyces expression vector, phage vector, filamentous fungus expression vector, plant expression vector, insect expression vector or mammalian cell expression vector.

5. in accordance with the method for claim 4, it is characterized in that: for recombinant bacterium or the transgenic cell line of recombinant expressed inscribe algin catenase, refer to e. coli host cell, yeast host cell, Bacillus subtilus host cell, milk-acid bacteria host cell, actinomycetes host cell, filamentous fungal host cell, insect cell, the one in mammalian cell.

6. the application of inscribe algin catenase as claimed in claim 3 in alginate degraded, is characterized in that: have one of following purposes or more than two kinds;

1) for the glycosidic link of rupture alginate or algal polysaccharide, obtain alginate oligosaccharides;

2) for the alginate component of degrade red algae or brown alga cell walls, extracting protoplastis, protoplastis is for food and algae research.

7. application as claimed in claim 6, is characterized in that: after described inscribe algin catenase Alg2A mixes with other algin degrading enzymes, for the application of collaborative fracture alginate glycosidic link aspect.