A mutant alginate lyase and its application in the degradation of Sargassum

Abstract

A mutant alginate lyase and its application in the degradation of Sargassum, belonging to the technical field of protein and enzyme engineering. On the one hand, the present invention provides a mutant alginate lyase, and on the other hand, it provides the use of the mutant alginate lyase. The present invention provides a mutant alginate lyase, whose thermal stability is increased from 40 °C to 45 °C after modification. This enzyme can cooperate with commercial enzymes such as cellulase to effectively catalyze the degradation of Sargassum, significantly improving its degradation efficiency and application scope, and is applicable to fields such as medicine, food industry, and environmental governance.

Description

Technical Field

[0001] The invention belongs to the technical field of protein and enzyme engineering, and particularly relates to a mutant alginate lyase and application thereof in the degradation of Sargassum. Background Art

[0002] my country is one of the main production areas of Sargassum, and its resources are very rich. Sargassum is the main feed ingredient for the cultivation of important marine delicacies such as sea cucumbers and abalone. At the same time, the seaweed polysaccharides and oligosaccharides it contains have good biological activities in anti-tumor, antibacterial, antioxidant, and immunomodulatory aspects, and can be used in agriculture, medicine, food and other industries. The resource development and application of brown algae polysaccharides and oligosaccharides have become one of the hot spots of current research. Among them, fucoidan is a sulfated polysaccharide composed of L-fucose and sulfate groups, and is now used as an ingredient in dietary supplements. At the same time, fucoidan exhibits a variety of physiological and biological activities, such as anti-inflammatory, antioxidant and anti-tumor effects, showing potential medicinal value.

[0003] Alginate lyase has a wide range of sources. So far, alginate lyases belonging to 8 different families have been identified from a variety of marine algae, marine mollusks (such as Littorina spp., Haliotis spp., Turbocornutus., etc.) and a series of marine and terrestrial microorganisms. However, due to the influence of factors such as its source environment, there are very few alginate lyases that can be used in industrial environments so far, and the properties of most alginate lyases often cannot meet the needs of industrial production. Therefore, it has become an important research hotspot to transform existing alginate lyases through genetic engineering and enzyme engineering to improve the catalytic performance of alginate lyases. Summary of the invention

[0004] .In view of the problems existing in the prior art, the purpose of the present invention is to design and provide a mutant alginate lyase which is heat-resistant and stable and can lyse alginate in Sargassum into brown algae oligosaccharides, thereby improving the hydrolysis rate of Sargassum; another purpose of the present invention is to design and provide a gene and amino acid sequence encoding the mutant alginate lyase, and a recombinant expression vector and host cell thereof; another purpose of the present invention is to design and provide a method for preparing the aforementioned mutant alginate lyase crude enzyme solution and pure enzyme solution; another purpose of the present invention is to design and provide a method for degrading Sargassum by using the aforementioned mutant alginate lyase in combination with commercial enzymes such as cellulase and hemicellulase.

[0005] The present invention is specifically implemented by the following technical solutions:

[0006] In the first aspect of the present invention, a mutant alginate lyase is provided. This mutant is based on the alginate lyase with the amino acid sequence shown in SEQ ID NO.1, and is obtained by mutating one or two sites among the 88th, 90th, 136th, 172nd, 180th, 181st, and 252nd positions.

[0007] The alginate lyase shown in SEQ ID NO.1 is derived from Flavobacterium nackdongense, and the original gene fragment of the alginate lyase FlAly is obtained by gene total synthesis.

[0008] Preferably: This mutant is based on the alginate lyase with the amino acid sequence shown in SEQ ID NO.1, and is obtained by mutating one or two sites among the 172nd, 180th, and 181st positions.

[0009] Furthermore, the amino acid mutation types at the mutation sites include: D88V or D88N; N90S or N90G; A136E; K172N; P180H; V181I or V181P or V181E; T252A.

[0010] Preferably: The lysine Lys at the 172nd position is mutated to asparagine Asn; the proline Pro at the 180th position is mutated to histidine His; the valine Val at the 181st position is mutated to isoleucine Ile or proline Pro or glutamic acid Glu.

[0011] Specifically, the alginate lyase mutant has one or two mutation sites. The alginate lyase mutant shown in SEQ ID NO.2 has a single mutation point of D88V; the alginate lyase mutant shown in SEQ ID NO.3 has a single mutation point of D88N; the alginate lyase mutant shown in SEQ ID NO.4 has a single mutation point of N90S; the alginate lyase mutant shown in SEQ ID NO.5 has a single mutation point of N90G; the alginate lyase mutant shown in SEQ ID NO.6 has a single mutation point of A136E; the alginate lyase mutant shown in SEQ ID NO.7 has a single mutation point of K172N; the alginate lyase mutant shown in SEQ ID NO.8 has a single mutation point of P180H; the alginate lyase mutant shown in SEQ ID NO.9 has a single mutation point of V181I; the alginate lyase mutant shown in SEQ ID NO.10 has a single mutation point of V181P; the alginate lyase mutant shown in SEQ ID NO.11 has a single mutation point of V181E; the alginate lyase mutant shown in SEQ ID NO.12 has a single mutation point of T252A; the alginate lyase mutant shown in SEQ ID NO.13 has two mutation points of K172N and P180H.

[0012] The second aspect of the present invention provides a gene encoding the above-mentioned mutant alginate lyase

[0013] Specifically, the present invention provides gene sequences encoding the foregoing alginate lyase mutants, wherein the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.1 are as shown in SEQ ID NO.14; the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.2 are as shown in SEQ ID NO.15; the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.3 are as shown in SEQ ID NO.16; the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.4 are as shown in SEQ ID NO.17; the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.5 are as shown in SEQ ID NO.18; the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.6 are as shown in SEQ ID NO.19; the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.7 are as shown in SEQ ID NO.20; the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.8 are as shown in SEQ ID NO.21; the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.9 are as shown in SEQ ID NO.22; the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.10 are as shown in SEQ ID NO.23; the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.11 are as shown in SEQ ID NO.24; the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.12 are as shown in SEQ ID NO.25; the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO.13 are as shown in SEQ ID NO.26.

[0014] The third aspect of the present invention provides a recombinant expression vector comprising the above gene.

[0015] Preferably, the expression vector is selected from any one of, but not limited to, pET-26b and pWB980, preferably the pET-26b plasmid.

[0016] The fourth aspect of the present invention provides a recombinant bacterium comprising the above recombinant expression vector.

[0017] Preferably, the host cell is selected from any one of, but not limited to, Escherichia coli and Bacillus subtilis. Since Escherichia coli cells grow and reproduce rapidly and BL21(DE3) is suitable for highly expressing foreign genes, Escherichia coli is preferably used as the host cell.

[0018] The fifth aspect of the present invention provides an application of the above mutant alginate lyase in degrading Sargassum thunbergii.

[0019] The sixth aspect of the present invention provides an application of the above-mentioned mutant alginate lyase in improving the degradation rate of Sargassum by combining with cellulase or hemicellulase.

[0020] The seventh aspect of the present invention provides a method for preparing a mutant alginate lyase enzyme solution. In this method, the above-mentioned recombinant bacterium is inoculated into a culture medium, an inducer is added for culturing, and the cells are collected by centrifugation; the cells are resuspended in phosphate buffer, ultrasonically disrupted, and centrifuged with flocculation to obtain the supernatant, thus obtaining the crude enzyme solution of the mutant alginate lyase;

[0021] Or the above-mentioned recombinant bacterium is inoculated into a culture medium, an inducer is added for culturing, and the cells are collected by centrifugation; the cells are resuspended in imidazole buffer, ultrasonically disrupted, and the supernatant is obtained by centrifugation, and then purified by nickel column affinity chromatography to obtain the pure enzyme solution of the mutant alginate lyase.

[0022] Furthermore, the recombinant bacterium is inoculated into TB liquid medium containing kanamycin, cultured at 37 °C and 150 rpm until OD600 reaches 1.5, IPTG with a final concentration of 0.1 mM is added for induction, and induction is carried out at 25 °C for 16 h.

[0023] The beneficial effects of the present invention are as follows: The present invention provides a mutant alginate lyase, and after modification, its thermal stability is increased from 35 °C to 40 °C. This enzyme can act synergistically with commercial enzymes such as cellulase, effectively catalyze the degradation of Sargassum, significantly improve its degradation efficiency and application scope, and is applicable to fields such as medicine, food industry, and environmental governance. Description of the Drawings

[0024] Figure 1 Construction of the FlAly-1 recombinant expression vector;

[0025] Figure 2 SDS-PAGE electrophoresis diagrams of FlAly-1 and FlAly-13 before and after purification, where M: standard protein; 1: crude enzyme of FlAly-1; 2: pure enzyme of FlAly-1; 3: crude enzyme of FlAly-13; 4: pure enzyme of FlAly-13;

[0026] Figure 3 Stability studies of FlAly-1 (A) and FlAly-13 (B) at different temperatures;

[0027] Figure 4 Stability studies of FlAly-1 (A) and FlAly-13 (B) at different pH values;

[0028] Figure 5 Nonlinear fitting results of the Michaelis constant of FlAly-1 (A) and FlAly-13 (B). Detailed Embodiments

[0029] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0030] In the present invention, each substitution is represented by a triplet: letter - number - letter, where the number represents the position of the mutant amino acid, the letter before the number corresponds to the amino acid involved in the mutation, and the letter after the number represents the amino acid used to replace the amino acid before the number.

[0031] Example 1: Mutant cloning

[0032] 1. According to the sequence of SEQ ID NO.1, Hangzhou Qingke Biotechnology Co., Ltd. was commissioned to perform total gene synthesis on the protein coding sequence (the nucleotide sequence of this gene is shown in SEQ ID NO.14), and it was cloned into the pET26b(+) vector to obtain the original enzyme expression vector pET26 - FlAly - 1 of alginate lyase, as shown in Figure 1 . After being verified correct by sequencing, it was introduced into Escherichia coli BL21(DE3) to construct an expression strain.

[0033] 2. Construction of mutant carbonyl reductase expression strains

[0034] Site - directed mutagenesis was performed on FlAly - 1 to obtain the following mutant groups:

[0035] (1) FlAly - 2: D88V, the amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.15.

[0036] (2) FlAly - 3: D88N, the amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.16.

[0037] (3) FlAly - 4: N90S, the amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.17.

[0038] (4) FlAly - 5: N90G, the amino acid sequence is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.18.

[0039] (5) FlAly - 6: A136E, the amino acid sequence is shown in SEQ ID NO.6, and the nucleotide sequence is shown in SEQ ID NO.19.

[0040] (6) FlAly - 7: K172N, the amino acid sequence is shown in SEQ ID NO.7, and the nucleotide sequence is shown in SEQ ID NO.20.

[0041] (7) FlAly-8: P180H, with the amino acid sequence shown in SEQ ID NO.8 and the nucleotide sequence shown in SEQ ID NO.21.

[0042] (8) FlAly-9: V181I, with the amino acid sequence shown in SEQ ID NO.9 and the nucleotide sequence shown in SEQ ID NO.22.

[0043] (9) FlAly-10: V181P, with the amino acid sequence shown in SEQ ID NO.10 and the nucleotide sequence shown in SEQ ID NO.23.

[0044] (10) FlAly-11: V181E, with the amino acid sequence shown in SEQ ID NO.11 and the nucleotide sequence shown in SEQ ID NO.24.

[0045] (11) FlAly-12: T252A, with the amino acid sequence shown in SEQ ID NO.12 and the nucleotide sequence shown in SEQ ID NO.25.

[0046] (12) FlAly-13: K172N, P180H, with the amino acid sequence shown in SEQ ID NO.13 and the nucleotide sequence shown in SEQ ID NO.26.

[0047] The above sequences were entrusted to Hangzhou Qingke Biotechnology Co., Ltd. to perform amino acid mutations on the respective encoded protein sequences to obtain their corresponding recombinant vectors. After being verified correct by sequencing, they were introduced into Escherichia coli BL21(DE3) to obtain the corresponding expression strains.

[0048] Example 2: Enzyme Activity Assay

[0049] Using anhydrous glucose as the standard, the enzyme activity was determined by the 3,5-dinitrosalicylic acid method (DNS). 50 μL of the enzyme solution was mixed with 450 μL of sodium alginate (0.6%, w / v, dissolved in PBS buffer solution), and the reaction was carried out at 45 °C for 10 min. Then 750 μL of DNS solution was added, and the reaction was terminated by boiling for 6 min. The absorbance was measured at 540 nm, and the enzyme activity unit (U) was defined as the amount of enzyme required to release 1 μg of reducing sugar per minute. The initial enzyme activity measured without 45 °C pretreatment was defined as 100%, and the enzyme activity after incubation was calculated as their corresponding relative values (%). Using bovine serum albumin (BSA) as the standard, the protein content was estimated by the Bradford method.

[0050] Example 3: Preliminary Screening of the Thermal Stability of Mutant Enzymes

[0051] The recombinant strains FlAly-1 to FlAly-12 were respectively inoculated into TB liquid medium containing 50 μg / mL kanamycin, cultured at 37°C and 150 rpm until the OD600 reached about 1.5, and IPTG with a final concentration of 0.1 mM was added for induction, and induced at 25°C for 16 h. Then, the cells were collected by centrifugation at 10000 rpm for 10 min. 1 g of the cells was resuspended in 10 mL of phosphate buffer (0.1 mol / L, pH 7.0), sonicated, and then centrifuged at 10000 rpm for 5 min, and the supernatant was taken as the crude recombinant enzyme solution. After the crude enzyme solution of the mutant enzyme was placed at 45°C for 30 min, 50 μL of the enzyme solution was taken for enzyme activity determination, and the relative enzyme activity was calculated. As can be seen from Table 1, after being placed at 45°C for 30 min, the best enzyme activity and relative enzyme activity were those of FlAly-8 (P180H) and FlAly-7 (K172N).

[0052] To further improve the thermal stability of FlAly-1, the double mutant enzyme FlAly-13 containing K172N and P180H was constructed. After the crude enzyme solutions of FlAly-1, FlAly-7, FlAly-8 and FlAly-13 were placed at 45°C for 30 min, 50 μL of the enzyme solution was taken for enzyme activity determination, and the relative enzyme activity was calculated. As can be seen from Table 2, after being placed at 45°C for 30 min, compared with FlAly-8 and FlAly-7, the enzyme activity and relative enzyme activity of the double mutant enzyme FlAly-13 were further improved.

[0053] Table 1 Preliminary screening results of thermal stability

[0054]

[0055] Table 2 Secondary screening results of thermal stability

[0056]

[0057] Example 4: Preparation of recombinant pure enzyme solution

[0058] The recombinant crude enzyme solution containing His-tag was purified by metal chelate affinity chromatography (nickel column affinity). Deionized water with a volume of 3 times the column volume was used to remove ethanol. The Ni2+-Chelating Sepharose Fast Flow affinity gel was pre-equilibrated in the equilibration buffer (10 mM imidazole, 20 mM Tris-HCl, 500 mM sodium chloride, pH 7.5). 4 mL of the crude enzyme solution was loaded and the flow-through was collected. Then, it was equilibrated again with the equilibration buffer, and subsequently washed with the washing buffer (30 mM imidazole, 20 mM Tris-HCl, 500 mM sodium chloride, pH 7.5) for 3 - 4 times the column volume. Finally, the target protein was eluted with the elution buffer (150 mM imidazole, 20 mM Tris-HCl, 500 mM sodium chloride, pH 7.5), and the eluate was collected. The purity and molecular weight of the enzyme were detected by SDS-PAGE electrophoresis, as shown in Figure 2 . Clear protein bands were presented between 25 - 35 kDa for the target protein sample, which was consistent with the theoretical relative molecular mass of FlAly-1 (32 kDa), and the purity of the purified protein sample was > 95%.

[0059] Example 5: Study on the thermal stability and pH stability of the double mutant enzyme FlAly-13

[0060] To evaluate the thermal stability of FlAly-13, the enzyme was incubated in 50 mM phosphate buffer (pH 6.0) at 35 °C, 40 °C and 45 °C for 180 min respectively. The enzyme solution was taken out at regular intervals for enzyme activity determination, as shown in Figure 3 . After FlAly-1 enzyme was incubated at 35 °C for 180 min, the enzyme activity remained stable. However, when the temperature was raised to 40 °C and incubated for 180 min, the activity of FlAly-1 enzyme decreased significantly, only 43.9% of the initial activity, and the half-life was 148.8 min. When incubated at 45 °C for 15 min, the activity of FlAly-1 enzyme was basically lost, and the half-life was 11.59 min, confirming its poor thermal stability. In contrast, after FlAly-13 enzyme was incubated at 40 °C for 180 min, the enzyme activity remained stable, showing good thermal stability. When the temperature was raised to 45 °C and incubated for 30 min, the activity of FlAly-13 enzyme could still be maintained above 50%, and the half-life reached 41.46 min, proving that its thermal stability was significantly improved compared with FlAly-1.

[0061] To evaluate the pH stability of FlAly-13, after incubating in different buffers (at a concentration of 50 mM) including citrate-sodium citrate (pH 5.0 - 6.0), disodium hydrogen phosphate-potassium dihydrogen phosphate (pH 6.0 - 7.5), Tris-HCl (pH 7.5 - 9.0), and glycine-sodium hydroxide (pH 9.0 - 10.0) at 4 °C for 24 h, the residual activity was measured to evaluate the pH stability of the enzyme, as shown in Figure 4 . The activity of FlAly-1 enzyme is relatively sensitive to pH value and remains stable only in the range of pH 6.0 to 7.0, while the FlAly-13 mutant shows a wider adaptability. Its activity has no obvious fluctuation in the range of pH 5.5 to 8.0 and basically remains above 70%. This indicates that the FlAly-13 mutant has a significantly improved adaptability in terms of pH value compared to the original enzyme FlAly-1.

[0062] Example 6: Determination of kinetic parameters of the enzyme

[0063] Sodium alginate solutions with different concentrations (0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%) were prepared with 50 mM phosphate buffer (pH 6.0). Equal amounts of enzyme solution were added to the sodium alginate solutions with different concentrations, and the enzyme activity was measured. According to the Michaelis-Menten kinetic equation, a graph with substrate concentration as the abscissa and enzyme activity as the ordinate was plotted, and the Michaelis constant (Km), maximum reaction rate (Vmax), and catalytic constant (kcat) were calculated by non-linear fitting when sodium alginate was used as the substrate. See specifically Figure 5 and Table 3. The specific activity of the FlAly-13 mutant enzyme is 2139.8 U / mg, slightly higher than 2023.1 U / mg of the original enzyme FlAly-1. The Kcat value of the FlAly-13 mutant enzyme is 52.4 s-1, higher than 49.3 s-1 of the original enzyme FlAly-1, showing a higher catalytic efficiency.

[0064] The Michaelis-Menten kinetic equation is as follows:

[0065] Km - the substrate concentration at half of the maximum reaction rate Vmax;

[0066] Vmax - the maximum reaction rate;

[0067] S - the substrate concentration.

[0068] Table 3 Comparison of kinetic parameters of FlAly-1 and FlAly-13

[0069]

[0070] Example 7: Degradation of Sargassum

[0071] The Sargassum sample was washed twice with water and dried in an oven at 65 °C overnight. The Sargassum powder was obtained by a grinder for use. 50 g of Sargassum powder was weighed and fully swollen in PBS (0.1 mM; PH 7.0-7.5) buffer at a solid-liquid ratio of 1:20. Recombinant alginate lyase FlAly-13, cellulase, hemicellulase and xylanase (4:3:3) were added respectively and reacted at 40 °C for 6 h. 0.1% trypsin was added to the total system for 1 h. The enzyme was inactivated by boiling for 10 min, and the supernatant was collected by centrifugation. The Sargassum hydrolyzate powder was obtained by rotary evaporation and freeze-drying. The hydrolysis rate is shown in Table 4.

[0072] The hydrolysis rate of Sargassum is calculated as follows: Hydrolysis rate (%) = M2 / M1*100

[0073] M1 is the weight of dry Sargassum powder; M2 is the weight of Sargassum hydrolyzate powder.

[0074] Table 4 Comparison of hydrolysis rates of Sargassum by different enzyme combinations

[0075]

Claims

1. A mutant alginate lyase, characterized in that: The mutant is based on the alginate lyase with an amino acid sequence as shown in SEQ ID NO.1, and is obtained by mutation at two sites, 172 and 180. The amino acid mutation types of the mutation sites are K172N and P180H. Its amino acid sequence is shown in SEQ ID NO.

13.

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

3. A recombinant expression vector comprising the gene according to claim 2. A recombinant bacterium comprising the recombinant expression vector according to claim 3.

5. Use of the mutant alginate lyase according to claim 1 in degrading Sargassum.

6. Use of the mutant alginate lyase as claimed in claim 1 in combination with cellulase or hemicellulase to improve the degradation rate of Sargassum.

7. A method for preparing a mutant alginate lyase solution, characterized in that: The recombinant bacteria of claim 4 are inoculated into a culture medium, an inducer is added for culture, and the bacterial bodies are collected by centrifugation; the bacterial bodies are resuspended in a phosphate buffer, ultrasonically disrupted, flocculated and centrifuged to obtain the supernatant, thereby obtaining the crude enzyme solution of the mutant alginate lyase; Or the recombinant bacteria according to claim 4 are inoculated into a culture medium, an inducer is added for culture, and the bacteria are collected by centrifugation; the bacteria are resuspended in an imidazole buffer, ultrasonically disrupted, and the supernatant is obtained by centrifugation, and then affinity purified by a nickel column to obtain the pure enzyme solution of the mutant alginate lyase.

8. The method for preparing a mutant alginate lyase solution according to claim 7, characterized in that: The recombinant bacteria were inoculated into TB liquid medium containing kanamycin and cultured at 37°C and 150 rpm until OD 600 To 1.5, add IPTG to a final concentration of 0.1 mM for induction, and induce at 25°C for 16 h.

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