An endolytic alginate lyase and use thereof

Abstract

The application discloses an endolytic alginate lyase AlgC2m and application thereof. The biological enzyme is derived from a Paenibacillus-like bacterium enriched in a seaside soil sample. The application also discloses a recombinant expression vector and a recombinant host containing the alginate lyase AlgC2m gene. The application clones a new alginate lyase gene AlgC2m, which is 1320 bp in size, encodes 439 amino acids, and is classified into the PL-7 family. The similarity of the alginate lyase to alginate lyases in the same family is only 38%. The alginate lyase AlgC2m provided by the application has the function of degrading sodium alginate to prepare alginate oligosaccharide, and the main degradation product is unsaturated alginate disaccharide and trisaccharide, and has certain industrial application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to an endolytic alginate lyase and application thereof. BACKGROUND

[0002] Marine polysaccharides are biological macromolecules with multiple physiological activities derived from the ocean. Due to their functions such as anti-tumor, antioxidant, antiviral and immunomodulatory, marine polysaccharides have been widely used in medicine, food, chemical industry, agriculture and environment. However, due to the poor solubility of polysaccharides, the bioavailability of polysaccharides is also low, which greatly limits the further application of polysaccharides. Marine functional oligosaccharides are degradation products of polysaccharides treated by physical, chemical or enzymatic methods. Marine functional oligosaccharides retain the multiple activities of polysaccharides, and the solubility and bioavailability of oligosaccharides are greatly improved. Therefore, marine functional oligosaccharides have become a research hotspot in the field of marine biological resource development (Liu H, Nat. Prod. Res. Dev., 2012, 24: 201-204).

[0003] Alginate lyases catalyze the degradation of alginate by beta-elimination of 1,4-O-glycosidic bonds from the non-reducing end, resulting in alginate oligosaccharides with a double bond between C-4 and C-5 of the non-reducing end. According to the carbohydrate-active enzymes (CAZy) database, alginate lyases belong to PL families 5, 6, 7, 14, 15, 17 and 18. According to the specificity of the substrate, alginate lyases can be further divided into two categories, one is polyG lyase (EC 4.2.2.11) which specifically degrades guluronate fragments, and the other is polyM lyase (EC 4.2.2.3) which specifically degrades mannuronate fragments. Due to their efficient, specific and mild degradation characteristics, alginate lyases have attracted widespread attention in the preparation of alginate oligosaccharides and other functional marine oligosaccharides. So far, hundreds of alginate lyases from marine microorganisms, brown seaweed and mollusks have been purified, cloned and characterized. SUMMARY

[0004] The present application provides a novel endolytic alginate lyase derived from Paenibacillus sp., gene and application thereof. The enzyme can be used to produce alginate oligosaccharides and can be applied in the fields of food, medicine, chemical industry, energy and the like.

[0005] The present application also aims to provide a genetically engineered bacterium containing the above alginate lyase and a construction method thereof.

[0006] The present application finally aims to provide the application of the above alginate lyase.

[0007] To solve the above technical problems, the present application provides the following technical solutions:

[0008] An endolytic alginate lyase AlgC2m, the encoded amino acid sequence of which is shown as SEQ ID NO. 1.

[0009] A gene encoding the above-mentioned endolytic alginate lyase AlgC2m. A specific example, the encoded nucleotide sequence of which is shown as SEQ ID NO. 2.

[0010] The present application also provides an expression vector of the above-mentioned endolytic alginate lyase, comprising a gene encoding the alginate lyase of the present application.

[0011] A specific example, the expression vector is pET-22b(+).

[0012] The present application also provides a preparation method of the alginate lyase of the present application, comprising transforming a host cell with an expression vector encoding the alginate lyase gene of the present application, culturing the transformant, and obtaining the recombinant alginate lyase from the culture. The alginate lyase AlgC2m gene can also be transfected into a suitable eukaryotic host, including yeast and mammalian cells, etc.

[0013] As a preferred mode of embodiment, the host cell is Escherichia coli.

[0014] As a preferred mode of embodiment, the recombinant alginate lyase is obtained by transforming the host cell Escherichia coli BL21 (DE3) with an expression vector encoding the alginate lyase gene of the present application, and inducing with IPTG to obtain soluble expression.

[0015] The present application also provides an application of the alginate lyase of the present application in preparing oligosaccharides, which are obtained by degrading marine polysaccharides with the alginate lyase.

[0016] Preferably, the marine polysaccharides are alginate, algin, agar, carrageenan, fucoidan, etc.

[0017] The oligosaccharides are alginate oligosaccharides, agar oligosaccharides, carrageenan oligosaccharides, fucoidan oligosaccharides, etc.

[0018] The substrate of the alginate lyase of the present application can be alginate, algin, agar, carrageenan, fucoidan, etc.

[0019] As a preferred mode of embodiment, with sodium alginate as the substrate, the optimal reaction temperature of the alginate lyase is 45℃, and the optimal reaction pH is 8.0.

[0020] The alginate lyase AlgC2m of the present application is stable at 40℃, and the activity obviously decreases after 40℃. The enzyme has good stability at pH 4.0-10.0. By studying the effects of different concentrations of NaCl, CaCl2, MgCl2, and KCl on the activity of the enzyme, it is found that the enzyme has good stability in the presence of 0.1-0.5 mol / L NaCl, 0.1-0.5 mol / L CaCl2, 0.1-0.5 mol / L MgCl2, and 0.1-0.5 mol / L KCl. +The results show that Cu 2+ , Ni 2+ and EDTA greatly inhibit the enzyme activity.

[0021] Advantages of the present application:

[0022] The endolytic alginate lyase AlgC2m described in the present application has wide substrate adaptability and can significantly degrade alginate, polyM and polyG. Compared with the alginate lyases reported in the same family, the alginate lyase of the present application has only 38% homology with them, and is a novel endolytic alginate lyase. Among more than 100 alginate lyases characterized, only a few enzymes exhibit high activity at low temperature. The alginate lyase of the present application retains more than 60% activity below 15℃, has high activity and stability at low temperature, and can be selectively inactivated by slightly increasing the temperature, thereby saving energy and reducing biological pollution, and is suitable for many industrial production purposes. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Figure 1 is a polyacrylamide gel electrophoresis (SDS-PAGE) of the recombinant alginate lyase AlgC2m expressed and purified. M is a standard protein Marker; 1 is the alginate lyase after purification.

[0024] Figure 2 Figure 2 is a graph showing the effects of temperature and pH on the activity and stability of the recombinant alginate lyase (2a: effect of temperature on the activity of the recombinant enzyme; 2b: effect of temperature on the stability of the recombinant enzyme; 2c: effect of pH on the activity of the recombinant enzyme; 2d: effect of pH on the stability of the recombinant enzyme).

[0025] Figure 3 Figure 3 is an analysis graph of the effect of metal ions on the activity of the alginate lyase.

[0026] Figure 4 Figure 4 is an analysis graph of the degradation of sodium alginate, polyM and polyG by the alginate lyase AlgC2m.

[0027] Figure 5 Figure 5 is a thin layer chromatogram (TLC) of the product of the degradation of sodium alginate by the alginate lyase. DP2 and DP3 are alginate disaccharide and trisaccharide standards, respectively.

[0028] Figure 6 Figure 6 is an analysis graph of the electrospray mass spectrum (ESI-MS) of the product of the degradation of sodium alginate by the alginate lyase. DETAILED DESCRIPTION

[0029] The application will be further described in connection with the following examples. The examples described are only used to illustrate the application and not to limit the application.

[0030] Example 1: Culture and identification of Paenibacillus lautus strain JCM 9073 The strain used in the present application was isolated from a soil sample collected from different seashores. The screening medium used in the present application was as follows: 10.0 g sodium alginate, 1.0 g peptone, 5.0 g (NH4)2SO4, 2.0 g K2HPO4, 10.0 g NaCl, 2.0 g MgSO4·7H2O and 0.01 g FeSO4·7H2O, and the pH value was 7.0. The solid medium was added with 2% agar.

[0031] The collected soil sample was vortexed and allowed to stand with sterile water. 100 μl of the sample was inoculated into 3 ml of rich medium, and the mixture was cultured at 37°C for 24 h at 180 rpm. The bacterial genomic DNA was extracted using TIANGEN DNA KIT. The extracted bacterial genome was used as a template, and universal primers 27F and 1492R were used as primers to perform PCR amplification to obtain the full-length sequence of 16S rDNA gene. The PCR conditions were as follows: 96°C for 3 min, 96°C for 30 s, 58°C for 30 s, 72°C for 1 min, 35 cycles, and finally 72°C for 10 min. After the PCR reaction, 1% agarose was used for identification, and the Axygen gel recovery kit was used to recover the desired PCR product fragment. The 16S bacterial identification was performed by General Biotechnology (Anhui) Co., Ltd., and the bacterial genomic nucleotide sequence is shown in SEQ ID NO. 3.

[0032] Example 2: Cloning and identification of alginate lyase AlgC2m encoding gene

[0033] According to the possible alginate lyase gene in the genome of Paenibacillus lautus strain JCM 9073, the following amplification primers were designed: upstream primer (5'-GTGAAACGAAGGCATGCTTTTTC-3', SEQ ID NO. 4) and downstream primer (5'-TTAAGGGTGCAGGTGCGTTAGAG-3', SEQ ID NO. 5). The extracted strain genome was used as a template to amplify the alginate lyase AlgC2m gene. The PCR conditions were as follows: 95°C for 3 min, 95°C for 15 s, 58°C for 15 s, 72°C for 2 min, 30 cycles, and finally 72°C for 10 min.

[0034] The purified DNA fragment was ligated to cloning vector pMD18-T Vector, and transformed into E. coli DH5a competent cells. After culture in LB solid medium, white colonies were picked and subjected to colony PCR verification using amplification primers. The PCR conditions were as follows: 94°C pre-denaturation for 3 min, followed by 95°C pre-denaturation for 3 min, followed by 95°C for 15 s, 58°C for 15 s, and 72°C for 2 min for 30 cycles, and finally 72°C extension for 10 min. The PCR product was subjected to sequencing analysis. The results showed that the full-length nucleotide sequence of the alginate lyase gene was 1320 bp, the nucleotide sequence was shown as SEQ ID NO. 2; it encoded 439 amino acids, the amino acid sequence was shown as SEQ ID NO. 1, and the theoretical molecular weight of the protein was 43.96 kDa.

[0035] Example 3: Construction of recombinant expression vector of alginate lyase AlgC2m.

[0036] According to the full sequence of the alginate lyase gene obtained by sequencing, an upstream primer (5'-GGAATTCCATATGGCTGGAGAGAGCAAGTACTCGG-3', SEQ ID NO. 6) and a downstream primer (5'-CCCAAGCTTAGGGTGCAGGTGCGTTAGAG-3', SEQ ID NO. 7) were designed for PCR amplification. The PCR conditions were as follows: 95°C pre-denaturation for 3 min, followed by 95°C for 15 s, 55°C for 15 s, and 72°C for 2 min for 30 cycles, and finally 72°C extension for 10 min. After purification by a gel recovery kit (Novozyme), the PCR product was ligated with the expression vector pET22b(+) after enzyme digestion with Nde I and Hind III; the recombinant plasmid pET222b-AlgC2m was transformed into E. coli BL21(DE3), and the transformants with ampicillin resistance were screened, and the gene sequence was confirmed by sequencing.

[0037] Example 4: Expression and purification of alginate lyase AlgC2m gene using recombinant expression strain.

[0038] E. coli strain BL21(DE3) (purchased from Novagen, USA) containing the recombinant expression plasmid pET222b-AlgC2m was inoculated into LB medium containing ampicillin resistance and cultured at 16°C for 20 h to induce expression of alginate lyase AlgC2m. The crude enzyme solution was purified by nickel affinity chromatography, and the column was equilibrated with equilibration buffer A (100 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole). Then, contaminating proteins were eluted with 5% and 10% elution buffer B (100 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole), and the target protein was eluted with 35% elution buffer B. The purification status of alginate lyase AlgC2m was detected by polyacrylamide gel electrophoresis, and the results are shown below. Figure 1 As shown, the purified AlgC2m appears as a single band on the electrophoresis gel, and its position matches the predicted molecular weight.

[0039] Example 5: Detection of alginate lyase activity

[0040] The activity of alginate lyase was determined by measuring the amount of reducing sugar released using the 3,5-dinitrosalicylic acid (DNS) method. 1 mL of enzyme solution was added to 1 mL of 0.6% (w / v) sodium alginate aqueous solution, and the reaction was carried out at 35 °C for 30 min. Then, 3 mL of DNS reagent was added, and the reaction was terminated by boiling for 5 min. After cooling to room temperature, the OD value was measured. 540nm The absorbance was measured at the test site. One unit of alginate lyase activity is defined as the amount of enzyme required to release 1 μmol / min of reducing sugar under the test conditions.

[0041] Example 6: Enzymatic properties of alginate lyase AlgC2m.

[0042] 1. Effect of temperature on recombinant alginate lyase.

[0043] The optimal reaction temperature for alginate lyase was determined within the range of 20℃–60℃. Using the system described above, reactions were carried out at 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, and 60℃ for 30 min, respectively. OD was measured using the DNS method. 540nm The absorbance value is taken as 100% of the highest enzyme activity. For example... Figure 2 As shown in Figure a, the optimal reaction temperature for the recombinant alginate lyase AlgC2m is 45℃. The enzyme's thermostability was analyzed by reacting at 4℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, and 55℃ for 30 min. The residual enzyme activity was determined using the DNS method at the optimal reaction temperature (45℃). The enzyme activity of the untreated enzyme solution was defined as 100% relative activity.Figure 2 As shown in Fig. b, the recombinant alginate lyase AlgC2m can remain relatively stable below 40°C, and the activity obviously decreases after 40°C, indicating that the recombinant enzyme AlgC2m belongs to cold-adapted enzyme.

[0044] 2. Effect of pH on the recombinant alginate lyase

[0045] The optimum pH of the alginate lyase was determined in the range of 4.0-10.0. The substrate buffer solution was prepared by using citric acid-sodium citrate buffer (pH 4.0-6.0), PB buffer (pH 6.0-8.0), Tris-HCl buffer (pH 8.0-9.0), and glycine-NaOH buffer (pH 9.0-10.0), respectively. The 0.6% (W / V) sodium alginate substrate solution was prepared by using different pH buffer solutions. The enzyme activity was determined by using DNS method, and the highest enzyme activity was defined as 100%. As shown in Fig. c, the optimum reaction pH of the recombinant alginate lyase AlgC2m is pH 8.0. The pH stability of the alginate lyase was determined in the range of 4.0-12.0. The enzyme solution was diluted with the above-mentioned buffer solution of different pH, and then incubated at 0°C for 2 h. The enzyme activity was determined by using DNS method under the optimum temperature (45°C) and optimum pH (pH 8.0) conditions, and the enzyme activity of the enzyme solution without pH treatment was defined as 100% relative activity. As shown in Fig. d, the recombinant alginate lyase AlgC2m has good stability at pH 7.0-9.0. Figure 2 Figure 2

[0046] Example 7: Effect of metal ions and chelating agents on the activity of AlgC2m

[0047] In the above reaction system, 1 mM of metal ions or chelating agents were added, respectively, and then mixed and incubated at the optimum temperature (45°C) for 30 min to determine the enzyme activity. The control group was the activity of AlgC2m without any metal ions (defined as 100%). As shown in Fig. e, Cu Figure 3 2+ , Ni 2+ and EDTA greatly inhibit the enzyme activity.

[0048] Example 8: Substrate specificity of the recombinant alginate lyase AlgC2m

[0049] 0.1 mL of enzyme solution was added into 0.9 mL of 0.6% sodium alginate, polyM and polyG substrates, respectively, and then incubated at 45°C for 5 min. The activity of the alginate lyase was determined by analyzing the absorbance value at 235 nm. As shown in Fig. f, the recombinant alginate lyase AlgC2m has the highest activity on alginate, and the activity on polyM is lower than that on alginate, and the activity on polyG is the lowest. Figure 4 ​​​As shown, AlgC2m was able to degrade sodium alginate, polyM and polyG, indicating that AlgC2m has degradation activity on various structural types of substrates.

[0050] Example 9: Enzymatic product analysis of recombinant alginate lyase AlgC2m3 mL of alginate lyase was added to 2 mL of 0.6% sodium alginate substrate solution and the reaction was carried out at 30°C. Samples were taken at different time points by heating the mixture in boiling water for 5 min and then cooling on ice for 5 min to terminate the reaction. After centrifugation at 12,000 x g for 1 min at 4°C, the supernatant was collected as the hydrolysis product sample, 3 volumes of absolute ethanol was added and the mixture was precipitated in ethanol at -40°C overnight. The supernatant was concentrated by centrifugation and then analyzed by thin layer chromatography. The GF254 silica gel thin layer plate was first dried in an oven for 30 min, 1 μl of sample was spotted, the developing agent was (n-butanol:acetic acid:water = 2:1:2 (V / V / V)), and after spraying with 5% sulfuric acid color reagent, the plate was developed at 120°C for 5 min. The thin layer chromatography results are shown in Figure 6. Figure 5 As shown, the main degradation products were disaccharides and trisaccharides.

[0051] The enzymatic products were analyzed by electrospray mass spectrometry. The electrospray mass spectrometry conditions were in negative ion mode, and two characteristic peaks were identified, in which the disaccharide m / z was 351 ([ΔDP2-H] - ), and the trisaccharide m / z was 527 ([ΔDP3-H] - ). As shown in Figure 7. The mass spectrometry results Figure 4 Figure 6 showed that the sodium alginate degradation products of recombinant AlgC2m were further determined to be disaccharides and trisaccharides.

Claims

1. A alginate lyase, characterized in that: The amino acid sequence is shown as SEQ ID NO.

1.

2. A gene encoding the alginate lyase according to claim 1.

3. An expression vector comprising the gene encoding the alginate lyase according to claim 1.

4. The expression vector of claim 3, wherein: The expression vector is pET-22b(+).

5. A method for producing a alginate lyase, characterized by: The method comprises transforming host cells with the expression vector according to claim 3, culturing the transformants, and obtaining the recombinant alginate lyase from the culture.

6. The production method according to claim 5, characterized by: The host cells are Escherichia coli.

7. The production method according to claim 6, characterized by: The expression vector according to claim 2 encoding the gene of the alginate lyase according to claim 1 is transformed into host cells Escherichia coli BL21(DE3), and the recombinant alginate lyase is obtained by IPTG induction.

8. Use of the alginate lyase according to claim 1 in the preparation of oligosaccharides, which are obtained by degrading marine polysaccharides or polyM, polyG by the alginate lyase, and the marine polysaccharides are alginate.

9. Use according to claim 8, wherein: The oligosaccharides are alginate oligosaccharides.

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