Exo-alginate lyase VsAly7C as well as recombinant strain and application thereof

Abstract

The invention discloses exo-type alginate lyase VsAly7C as well as a recombinant strain and application of the exo-type alginate lyase VsAly7C. The amino acid sequence of the exo-alginate lyase is as shown in SEQ ID No.4, and the nucleotide sequence for coding the amino acid sequence is as shown in SEQ ID No.1. The suitable reaction temperature of the exo-alginate lyase is 24-28 DEG C, and the exo-alginate lyase has high stability at 0-20 DEG C, so that the exo-alginate lyase can effectively degrade a substrate at low temperature, and belongs to cold-adapted alginate lyase. The exo-type alginate lyase has the capability of degrading alginate, polyG and polyM, the degradation mode is exo-type, and a final degradation product is unsaturated alginate trisaccharide and has NaCl dependence. The invention further constructs a recombinant vector and a recombinant thallus containing the exo-alginate lyase VsAly7C coding gene, and a good foundation can be provided for industrial application and production of unsaturated alginate oligosaccharides.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and in particular relates to an exo-acting alginate lyase VsAly7C, a recombinant strain thereof and an application thereof. Background Art

[0002] Alginate is the primary polysaccharide in the cell walls of brown algae, comprising up to 40% of its dry weight. Alginate is a linear polysaccharide composed of β-D-mannuronic acid (M) and its C5 diastereomer, α-L-guluronic acid (G), linked together in poly-β-D-mannuronic acid (polyM), poly-α-L-guluronic acid (polyG), and heteropolymers (polyMG). Alginate and its oligosaccharides are highly safe and are widely used in food, medicine, and industrial applications.

[0003] Alginate lyases degrade alginate via a β-elimination reaction, creating an unsaturated double bond between C4 and C5 at the non-reducing end. Cold-adapted alginate lyases are suitable for many industrial processes because they can catalyze at low temperatures and selectively inactivate at slightly elevated temperatures, saving energy and reducing biofouling. Based on their mode of action, alginate lyases are classified as exo- and exo-acting.

[0004] In recent years, a variety of alginate lyases have been discovered and characterized, some of which can produce alginate oligosaccharides with a single degree of polymerization. This facilitates the preparation of unsaturated alginate oligosaccharides and promotes the study of the bioactivity of oligosaccharides derived from polyG, polyM, and alginate. Summary of the Invention

[0005] The present invention provides an exo-acting alginate lyase VsAly7C, a recombinant strain thereof, and its application. The exo-acting alginate lyase of the present invention has high activity, broad substrate adaptability, cold adaptability, and high degradation activity, and has good market application prospects.

[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions: The present invention provides an exo-acting alginate lyase VsAly7C, the amino acid sequence of which is shown in SEQ ID No.4.

[0007] Furthermore, the nucleotide sequence encoding the amino acid sequence of the exo-acting alginate lyase VsAly7C is shown as SEQ ID No.1.

[0008] Furthermore, the suitable reaction temperature of the exo-acting alginate lyase VsAly7C is 24-28°C.

[0009] Furthermore, the optimal reaction temperature of the exo-acting alginate lyase VsAly7C is 28°C.

[0010] Furthermore, the suitable reaction pH of the exo-acting alginate lyase VsAly7C is 7.3-9.6.

[0011] Furthermore, the optimal reaction pH of the exo-acting alginate lyase VsAly7C is 8.0.

[0012] Furthermore, the exo-acting alginate lyase VsAly7C has good stability at pH 7.3-9.6.

[0013] Furthermore, the exo-acting alginate lyase VsAly7C has good stability at 0-20°C.

[0014] Furthermore, the suitable reaction NaCl concentration for the exo-acting alginate lyase VsAly7C is 0.4-0.8 M.

[0015] Furthermore, the optimal reaction NaCl concentration of the exo-acting alginate lyase VsAly7C is 0.5 M.

[0016] Furthermore, the exo-acting alginate lyase VsAly7C has a wide range of substrate adaptability and can significantly degrade alginate, polyM and polyG.

[0017] Furthermore, the degradation mode of the exo-acting alginate lyase VsAly7C is exo-acting, and its final degradation product is unsaturated trisaccharide.

[0018] The present invention also provides a recombinant expression vector comprising the gene encoding the highly active exo-acting alginate lyase VsAly7C.

[0019] Furthermore, the recombinant expression vector is a pET-24a (+) vector.

[0020] The present invention also provides a recombinant strain comprising the gene encoding the highly active exo-acting alginate lyase VsAly7C.

[0021] Furthermore, the recombinant strain is Escherichia coli BL21 (DE3).

[0022] Furthermore, the preparation method of the exo-acting alginate lyase VsAly7C is as follows: the recombinant strain is inoculated into a culture medium and cultured at 37°C until the OD 600When the pH value is 0.4-0.5, IPTG is added to a final concentration of 0.1 mM, and the mixture is induced at 18°C ​​for 48 h. The bacteria are centrifuged to remove the supernatant, the precipitate is resuspended and crushed, and the supernatant is collected by centrifugation to obtain a crude enzyme solution. The crude enzyme solution is separated and purified using a nickel ion affinity column to obtain the exo-acting alginate lyase VsAly7C.

[0023] The present invention also provides the use of the highly active exo-acting alginate lyase VsAly7C in the preparation of an enzyme preparation for degrading alginate, polyM and polyG.

[0024] Compared with the prior art, the advantages and technical effects of the present invention are: The exo-type alginate lyase VsAly7C of the present invention can be obtained by culturing, inducing and isolating and purifying the recombinant bacteria containing the VsAly7C encoding gene. The optimum reaction temperature of the enzyme is 28 ° C, and it has good stability at 0-20 ° C. Therefore, the enzyme belongs to a cold-adapted alginate lyase, which can effectively degrade the substrate at low temperatures, and the degradation mode of the enzyme is exo-type, and the final degradation product is an unsaturated trisaccharide. In addition, the optimum pH of the exo-type alginate lyase VsAly7C is 8.0, and it has good stability at pH 7.3-9.6. It also has a wide range of substrate degradability, can degrade alginate, polyG and polyM and has a high specific activity, and the exo-type alginate lyase VsAly7C has a high degradation rate for alginate and can quickly produce unsaturated trisaccharides. The present invention also constructs a recombinant vector and recombinant bacteria containing the exo-acting alginate lyase VsAly7C, and determines its enzymatic properties and degradation rate, which can provide a good basis for industrial application and production of unsaturated alginate trisaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Polyacrylamide gel electrophoresis of the expressed and purified exo-algin lyase VsAly7C (1, 100 mM imidazole elution fraction; 2, 200 mM imidazole elution fraction; 3, pure VsAly7C enzyme).

[0026] Figure 2 : Results of the effect of pH on the endo-algin lyase TsAly7C (A, effect of different reaction pH on enzyme activity; B, pH stability of the enzyme).

[0027] Figure 3 : Results of the effect of temperature on the endo-algin lyase TsAly7C (A, effect of different reaction temperatures on enzyme activity; B, temperature stability of the enzyme).

[0028] Figure 4: The substrate preference results of the exo-acting alginate lyase VsAly7C.

[0029] Figure 5 : The degradation results of the exo-acting alginate lyase VsAly7C.

[0030] Figure 6 : Results of the final degradation products of the exo-acting alginate lyase VsAly7C (A, thin layer chromatography results; B, gel filtration chromatography results).

[0031] Figure 7 : The NaCl dependence results of the exo-acting alginate lyase VsAly7C. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further explained in detail below with reference to the accompanying drawings and embodiments.

[0033] Example 1 1. Cloning and acquisition of the gene encoding the exo-acting alginate lyase VsAly7C The full-length amino acid sequence of the exo-acting alginate lyase VsAly7C provided by the present invention is shown in SEQ ID No. 4. The full-length sequence of the gene encoding the exo-acting alginate lyase VsAly7C (SEQ ID No. 1) was used as a template for the PCR reaction, and PCR amplification was performed using the following primers containing pET-24a homology arms: F: 5'- taagaaggagatatacatatgATGAAACATATTTACCTCAAGAGCTTG -3' (SEQ IDNo. ​​2); R: 5'-gtggtggtggtggtgctcgagCTAGCCTTGGTACTTACCAAAAGATTG-3' (SEQ ID No. 3).

[0034] The PCR reaction was performed under the following conditions: 28 cycles of pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 1 min, followed by extension at 72°C for 5 min. The PCR product was sequenced to obtain the nucleotide sequence shown in SEQ ID No. 1.

[0035] The sequence shown in SEQ ID No. 1 obtained by the above PCR contains part of the homology arm of pET-24a and the nucleotide sequence of the catalytic region of the exo-acting alginate lyase VsAly7C, wherein the nucleotide sequence of the exo-acting alginate lyase VsAly7C is 1566 bp, and the nucleotide sequence is shown in SEQ ID No. 1; the nucleotide sequence shown in SEQ ID No. 1 encodes a total of 521 amino acid residues, and its amino acid sequence is shown in SEQ ID No. 4, with a theoretical molecular weight of 57.3 kDa.

[0036] 2. Construction of recombinant expression vector of exo-acting alginate lyase VsAly7C The PCR product was purified using a gel extraction kit (Sikejie) and ligated into a pET-24a(+) vector (Novagen) double-digested with the restriction endonucleases NdeI and XhoI. The product was then transformed into competent E. coli DH5α cells, and positive clones were selected for DNA sequencing. The nucleotide sequence of the exo-algin lyase VsAly7C obtained by sequencing is shown in SEQ ID No. 5, and the amino acid sequence is shown in SEQ ID No. 4, confirming that the sequencing was correct and the target fragment had been ligated. The recombinant expression plasmid that had been sequenced correctly was extracted using a plasmid extraction kit (Sikejie). The recombinant expression plasmid was then transformed into the expression strain E. coli BL21(DE3).

[0037] 3. Induced fermentation of exo-algin lyase VsAly7C The recombinant Escherichia coli BL21 (DE3) containing the pET24a-VsAly7C vector was streaked, and a single clone was picked into 5 mL of liquid LB medium containing kanamycin (kana, 50 μg / mL) and cultured in a shaking incubator at 37°C overnight.

[0038] Then transfer the cells to a 500 mL conical flask containing 100 mL of liquid LB (50 μg / mL kana) and culture at 37 °C until the OD 600 When the concentration is between 0.4 and 0.6, add IPTG (isopropyl-β-D-galactopyraNoside) to a final concentration of 0.25 mM and induce at 18 °C for 48 h. Centrifuge the bacteria at 6500 rpm for 30 min, remove the supernatant, and resuspend the precipitate in solution A (10 mL, 20 mM PB, 500 mM NaCl, pH = 8.0). Use a high-pressure crusher to crush the bacteria, and then centrifuge the mixture at 12000 rpm at 4 °C for 30 min. Collect the supernatant to obtain the crude enzyme solution. Use A 235The enzyme activity of crude enzyme solution was determined by measuring the light absorbance of the reaction system at 235 nm, and the enzyme solution inactivated at 100°C for 10 min was used as a control; under this condition, an increase of 0.1 in the light absorbance per minute was defined as one enzyme activity unit (U).

[0039] 4. Isolation and purification of exo-acting alginate lyase VsAly7C Equilibrate a nickel affinity column (HisTrap HP, GE) with equilibration buffer A (20 mM PB, 500 mM NaCl, pH 8.0) for 5–10 volumes. Disrupt the treated cells and apply the crude enzyme supernatant. Elute unbound proteins with equilibration buffer A. Elute other contaminants with equilibration buffer A supplemented with 25 mM imidazole and 50 mM imidazole. Elute the target protein with equilibration buffer A supplemented with 200 mM imidazole, and assay enzyme activity. Elute other proteins with equilibration buffer A supplemented with 500 mM imidazole. Rinse the column with water and store in 20% ethanol.

[0040] After collecting the target protein, place it on a dialysis membrane (molecular weight cutoff of 10 kDa) and dialyze it overnight at low temperature. The dialysis solution is 20 mM PB with 300 mM NaCl added, pH 8.0, to remove the imidazole in the sample. After dialysis, the target protein is stored at -20°C. Take 10 μL of the target protein sample and add it to an equal volume of 2× denaturing buffer (loading buffer). After boiling for 10 minutes, centrifuge it at 12,000 rpm at room temperature for 2 minutes. Take the supernatant and perform SDS-PAGE electrophoresis. The gel concentration is 10%, the voltage of the stacking gel is 80 V, and the voltage of the separation gel is 120 V. The electrophoresis gel is stained with Coomassie Brilliant Blue R-250 for 4 hours, destained with a destaining solution (acetic acid: ethanol: water = 2:1:17), photographed, and analyzed. Figure 1 As shown in the figure, the molecular weight of the exo-acting alginate lyase VsAly7C is approximately 57 kDa, which is consistent with the predicted molecular weight.

[0041] The protein concentration (unit: mg / mL) was determined using a protein concentration assay kit (New Cyme), and the specific activity (unit: U / mg) of the exo-algin lyase VsAly7C was calculated based on the enzyme activity (unit: U / mL) of the purified enzyme. Figure 1 As shown in lane 3, the purified alginate lyase shows a single electrophoretic band, confirming successful protein purification. Furthermore, using alginate as a substrate, the specific activity of the exo-acting alginate lyase VsAly7C was detected at 710.11 U / mg.

[0042] Example 2: Enzymatic properties of exo-acting alginate lyase VsAly7C 1. Effect of pH on exo-acting alginate lyase VsAly7C (1) Add 100 μL of appropriate concentration of alginate lyase VsAly7C to 900 μL of 0.3% alginate substrate (20 mM PB buffer, pH = 8.0) and react in buffers of different pH values ​​for 10 min. The A 235 The values ​​were calculated with the highest enzyme activity as 100%; the buffers used were 50 mM Na2HPO4-NaH2PO4 (pH 6.0-8.0), glycine-NaOH (pH 8.6-10.6), Na2HPO4-citric acid (pH 3.0-8.0), and Tris-HCl (pH 7.05-8.95), respectively. The relative enzyme activity of the exo-algin lyase VsAly7C under different pH conditions was calculated. The results are shown in the figure. Figure 2 As shown in A, the exo-acting alginate lyase VsAly7C is suitable for reaction at pH 7.3-9.6, and has the highest enzyme activity at pH 8.0.

[0043] (2) Store the pure enzyme at appropriate concentrations in the above-mentioned different pH buffers at 4°C for 12 h, take 100 μL and mix it with 900 μL substrate, react at 30°C for 10 min, and measure the A 235 The residual relative enzyme activity of exo-algin lyase VsAly7C at different pH values ​​was calculated with the untreated enzyme activity as 100%. Figure 2 As shown in Figure B, the exo-acting alginate lyase VsAly7C can still maintain a high activity after being placed in a buffer solution of pH 6.6-9.3 for 12 h, indicating that the enzyme has good pH stability at pH 7.3-9.6.

[0044] 2. Effect of temperature on exo-acting alginate lyase VsAly7C (1) 100 μL of pure alginate lyase VsAly7C of appropriate concentration was added to 900 μL of 0.3% alginate substrate (20 mM PB buffer, pH = 8.0) and reacted at different temperatures (10, 15, 20, 25, 30, 40, 50, 60 °C) for 10 min. The A 235 The relative enzyme activity of VsAly7C at different temperatures was calculated with the highest enzyme activity as 100%. Figure 3 As shown in A, the optimal reaction temperature of the exo-acting alginate lyase VsAly7C is 28°C, and it has high activity at 20-28°C, indicating that the exo-acting alginate lyase VsAly7C is a cold-adapted alginate lyase with high activity at low temperatures and produces monosaccharides.

[0045] (2) The appropriate concentration of exo-acting alginate lyase VsAly7C pure enzyme was placed in a water bath at different temperatures (0, 20, 30, 40, 50, 60, 70, 80 °C) for 1 h, and then immediately placed in an ice bath for 5 min. 100 μL of enzyme solution was mixed with 900 μL of substrate and reacted at 30 °C for 10 min. The A 235 The value is calculated by taking the enzyme activity at 0 h as 100% to calculate the residual relative enzyme activity of the exo-algin lyase VsAly7C after incubation at different temperatures. Figure 3 As shown in Figure 2, the activity of the exo-algin lyase VsAly7C decreased when stored at temperatures above 0°C. After storage at 20°C for 1 h, the enzyme activity retained 80%, and after storage at 30°C for 1 h, the enzyme activity retained 60%.

[0046] 3. Substrate preference of exo-acting alginate lyase VsAly7C The enzyme activity of VsAly7C was determined according to the reaction conditions using 0.3% alginate, polyM, and polyG dissolved in 20 mM pH 8.0 PB buffer as substrates. The highest enzyme activity was taken as 100%, and the relative enzyme activities of other substrates were calculated. Figure 4 The results showed that the exo-acting alginate lyase VsAly7C had high catalytic activity against alginate, polyG, and polyM. VsAly7C had the highest degradation activity against polyM, followed by alginate, at 75.51%. It had the lowest degradation activity against polyM, at 34.61%.

[0047] 4. Degradation mode of exo-algin lyase VsAly7C TLC was used for detection. Alginate substrate (0.3%) was prepared using the optimal pH buffer and NaCl concentration described above. Pure enzyme was mixed with the substrate to achieve final enzyme activities of 1 U / mL, 3 U / mL, and 5 U / mL. Reactions were performed in a 28°C water bath. 1 mL samples were withdrawn at 1 min, 3 min, 5 min, 10 min, 20 min, 30 min, 1 h, 2 h, and 3 h. The appropriate amount of enzyme was added at 6 h of reaction. The reaction was then allowed to proceed overnight at 28°C. Samples were then withdrawn again.

[0048] After sampling, the sample was immediately boiled for 10 minutes to terminate the enzymatic hydrolysis reaction. Centrifuge at 12000 rpm for 10 minutes to remove impurities. Use a 2.5 μL pipette to take 0.3 μL each time, and add the reaction solution three times to the corresponding position on the TLC plate. Heat the TLC plate to dry after each addition. Obvious purple spots appear under UV light, and a mixture of unsaturated alginate disaccharides and trisaccharides is used as a control. After the sample is spotted, heat and dry, and place it in the pre-prepared developing agent (n-butanol: formic acid: water, 4:6:1, v / v / v). When the developing agent is about to reach the top of the TLC plate, take it out, dry it, and then develop and dry it again. Immerse the TLC plate in the color developer (aniline-diphenylamine solution) to wet it, quickly take it out and dry it after staining, and heat it at 150°C for 10 minutes to develop the color. The results are as follows. Figure 5 As shown, with the extension of time, only alginate oligosaccharides with a single degree of polymerization were produced, indicating that VsAly7C is an exo-type alginate lyase.

[0049] 5. Final degradation product of exo-algin lyase VsAly7C The reaction solution was filtered through a 0.22 μm filter membrane, and 500 μL was loaded onto a Superdex peptide 10 / 300 column for separation. 0.2 M NH4HCO3 was used as the mobile phase at a flow rate of 0.8 mL / min. The change in absorbance at 235 nm was detected and the separated products at the peak were collected to determine the degradation products of VsAly7C. Figure 6 As shown in A. This shows that the product of alginate degradation by alginate lyase VsAly7C is unsaturated alginate trisaccharide. The above-collected separated product was then mixed with equal volumes of acetonitrile, and the mixture was subjected to ESI-anion mass spectrometry to analyze the spectrum and determine the degradation product of VsAly7C. The results are shown in Figure 6 As shown in B, it shows that the product of alginate degradation by alginate lyase VsAly7C is unsaturated alginate trisaccharide.

[0050] 6. NaCl dependence of exo-algin lyase VsAly7C Prepare 0.3% alginate substrate, dissolve it in PB buffer (20 mM, pH=8.0), add different concentrations of NaCl, and measure the enzyme activity of VsAly7C; set the highest enzyme activity as 100%, and calculate the effect of different concentrations of NaCl on enzyme activity. Figure 7 As shown in the data, in the absence of NaCl, the enzyme activity is low. With the increase of NaCl concentration, the enzyme activity gradually increases and reaches the highest enzyme activity at 0.5 M NaCl concentration. Then, after the concentration is greater than 0.5 M, with the increase of NaCl concentration, the enzyme activity gradually decreases, indicating that the exo-acting alginate lyase VsAly7C has obvious NaCl dependence, and the enzyme activity is better at 0.4-0.8 M.

[0051] The above embodiments only represent the technical solutions of the present invention, rather than limiting the experiments. Although we have improved the experimental solutions, researchers in the same field can still make further improvements to the experimental solutions described above or make scientific equivalent replacements for the experimental steps. These changes do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. An exo-acting alginate lyase VsAly7C, characterized in that The amino acid sequence of the exo-acting alginate lyase VsAly7C is shown in SEQ ID No.

4.

2. The exo-acting alginate lyase VsAly7C according to claim 1, characterized in that The nucleotide sequence encoding the amino acid sequence of the exo-acting alginate lyase VsAly7C is shown in SEQ ID No.

1.

3. The exo-acting alginate lyase VsAly7C according to claim 1, characterized in that The suitable reaction temperature of the exo-acting alginate lyase VsAly7C is 24-28°C.

4. The exo-acting alginate lyase VsAly7C according to claim 1, characterized in that The suitable reaction pH of the exo-acting alginate lyase VsAly7C is 7.3-9.

6.

5. The exo-acting alginate lyase VsAly7C according to claim 1, characterized in that The suitable reaction NaCl concentration of the exo-acting alginate lyase VsAly7C is 0.4-0.8 M.

6. The exo-acting alginate lyase VsAly7C according to claim 1, characterized in that The exo-acting alginate lyase VsAly7C has the function of obviously degrading alginate, polyM and polyG.

7. The exo-acting alginate lyase VsAly7C according to claim 1, characterized in that The degradation mode of the exo-acting alginate lyase VsAly7C is exo-acting, and its final degradation product is unsaturated alginate trisaccharide.

8. A recombinant expression vector comprising the gene encoding the exo-acting alginate lyase VsAly7C according to claim 2.

9. A recombinant strain comprising the gene encoding the exo-acting alginate lyase VsAly7C according to claim 2, characterized in that: The recombinant strain is Escherichia coli BL21 (DE3).

10. Use of the exo-acting alginate lyase VsAly7C according to any one of claims 1 to 7 in the preparation of an enzyme preparation for degrading alginate, polyM and polyG.

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