An alginate lyase mutant, its preparation method and application, as well as a recombinant expression vector and a recombinant expression strain

Abstract

The present invention relates to an alginate lyase mutant, a preparation method and application thereof, as well as a recombinant expression vector and a recombinant expression strain, belonging to the technical field of genetic engineering. The present invention relates to an alginate lyase mutant, including alginate lyase mutant FlAlyA-H71K or alginate lyase mutant FlAlyA-H176D; the amino acid sequence of the alginate lyase mutant FlAlyA-H71K is as shown in SEQ ID NO: 1; the amino acid sequence of the alginate lyase mutant FlAlyA-H176D is as shown in SEQ ID NO: 3. The alginate lyase mutant of the present invention has improved thermal stability compared to the wild-type alginate lyase.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to an alginate lyase mutant, a preparation method and application thereof, a recombinant expression vector, and a recombinant expression strain. Background Art

[0002] Alginate is a linear acidic polysaccharide mainly produced by brown algae and bacteria, and is the main component of the cell walls of brown algae plants such as kelp and wakame. It is mainly composed of two monomers, β-D-mannuronic acid (M) and α-L-guluronic acid (G). The monomers are connected by 1→4 glycosidic bonds, and three different blocks can be formed in alginate: homopolymeric mannuronic acid fragment (polyM), homopolymeric guluronic acid fragment (polyG), and heterozygous fragment (polyMG) formed by alternating connection of the two monomers.

[0003] Alginate is difficult to be absorbed and utilized by the body due to its high degree of polymerization, large molecular weight, high viscosity, etc., which limits its application. Alginate oligosaccharides (AOs) are degradation products of alginate and have significant physiological activities such as immunomodulation, antibacterial, antioxidant, prebiotic, antihypertensive, antidiabetic, antitumor, and anticoagulant activities. At the same time, AOs can accelerate microbial metabolism, promote root growth, improve the yield and quality of plants, improve the disease resistance of plants, induce plant immunity, and can be used to prepare biological fertilizers.

[0004] The current methods for preparing AOs mainly include physical degradation method, chemical method, and enzymatic method. Compared with physical and chemical methods, the enzymatic production of AOs is more environmentally friendly, more energy-saving, and has great potential for sustainable development. Currently, alginate lyase has become a research hotspot. Alginate lyase belongs to the polysaccharide lyase family (PLs) and can degrade alginate to produce AOs through β-elimination. However, alginate lyase requires a high-temperature treatment step when producing AOs, which results in a significant reduction in enzyme activity. Therefore, it is necessary to develop an alginate lyase with excellent thermal stability to meet the market demand. Summary of the Invention

[0005] The purpose of the present invention is to provide an alginate lyase mutant, a preparation method and application thereof, a recombinant expression vector, and a recombinant expression strain.

[0006] In order to achieve the above invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides an alginate lyase mutant, including alginate lyase mutant FlAlyA-H71K or alginate lyase mutant FlAlyA-H176D;

[0008] The amino acid sequence of the alginate lyase mutant FlAlyA-H71K is shown in SEQ ID NO: 1;

[0009] The nucleotide sequence encoding the alginate lyase mutant FlAlyA-H71K is shown in SEQ ID NO: 2;

[0010] The amino acid sequence of the alginate lyase mutant FlAlyA-H176D is shown in SEQ ID NO: 3;

[0011] The nucleotide sequence encoding the alginate lyase mutant FlAlyA-H176D is shown in SEQ ID NO: 4.

[0012] The present invention also provides a method for preparing the alginate lyase mutant. The preparation method of the alginate lyase mutant FlAlyA-H71K is: mutating the 71st amino acid of the alginate lyase FlAlyA to obtain the alginate lyase mutant FlAlyA-H71K;

[0013] The preparation method of the alginate lyase mutant FlAlyA-H176D is: mutating the 176th amino acid of the alginate lyase FlAlyA to obtain the alginate lyase mutant FlAlyA-H176D.

[0014] Preferably, the 71st amino acid of the alginate lyase FlAlyA is histidine;

[0015] The 71st amino acid of the alginate lyase mutant FlAlyA-H71K is lysine;

[0016] The 176th amino acid of the alginate lyase FlAlyA is histidine;

[0017] The 176th amino acid of the alginate lyase mutant FlAlyA-H176D is aspartic acid.

[0018] The present invention also provides a recombinant expression vector, including the nucleotide sequence encoding the alginate lyase mutant FlAlyA-H71K or FlAlyA-H176D and the empty vector pET22b(+).

[0019] The present invention also provides a method for preparing the recombinant expression vector, including the following steps: ligating the nucleotide sequence encoding the alginate lyase mutant FlAlyA-H71K or FlAlyA-H176D with pET22b(+) to obtain the recombinant expression vector;

[0020] The linked site is the site after double digestion with restriction endonucleases Nde I and Xho I.

[0021] The present invention also provides a recombinant expression strain, comprising the recombinant expression vector and a starting strain;

[0022] The starting strain is Escherichia coli BL21 - CodonPlus - RIL.

[0023] The present invention also provides a method for preparing the recombinant expression strain, comprising the following steps: introducing the recombinant expression vector into the starting strain, and inducing expression to obtain the recombinant expression strain.

[0024] Preferably, the inducer for the induction is isopropyl - β - D - thiogalactoside (IPTG);

[0025] The final concentration of the isopropyl - β - D - thiogalactoside is 0.1 mM;

[0026] The temperature for the induction is 23 - 27 °C;

[0027] The time for the induction is 22 - 26 h.

[0028] The present invention also provides the application of the alginate lyase mutant in the preparation of products with high thermal stability.

[0029] The present invention also provides the application of the recombinant expression vector or the recombinant expression strain in the preparation of products with high thermal stability.

[0030] The present invention provides an alginate lyase mutant and its preparation method and application, as well as a recombinant expression vector and a recombinant expression strain.

[0031] The alginate lyase mutant of the present invention has the characteristics of high thermal stability and high yield, and can be used for industrial production.

[0032] The enzyme activity of the alginate lyase mutant FlAlyA - H71K provided by the present invention can reach 1.11×10 5 U / mg, the optimal pH is 8.0, and it has more than 80% enzyme activity under the condition of pH 7.0 - 9.0, showing good pH stability; the optimal temperature is 50 °C, and the enzyme activity still remains more than 80% when the temperature is 40 - 55 °C. Compared with the wild - type alginate lyase FlAlyA, ΔT m is 0.33 °C. The enzyme activity of the alginate lyase mutant FlAlyA - H176D can reach 0.95×10 5 U / mg, the optimal temperature is 50 °C, and the optimal pH is 8.0; compared with the wild - type alginate lyase FlAlyA, ΔT mIt is 1.26 °C and the half-life at 50 °C is significantly extended, indicating an improvement in its thermal stability. In addition, 600 ± 50 mg of pure FlAlyA-H71K and FlAlyA-H176D enzymes can be obtained from 1 L of fermentation broth, far higher than the existing level, providing the possibility for the industrial production of alginate lyase. Description of the Drawings

[0033] Figure 1 shows the enzyme activities of wild-type alginate lyase and alginate lyase mutants at different pH values (where ● represents FlAlyA-H71K, ■ represents wild-type alginate lyase AL, and ▲ represents FlAlyA-H176D).

[0034] Figure 2 shows the enzyme activities of wild-type alginate lyase and alginate lyase mutants at different temperatures (where ● represents wild-type alginate lyase AL, ■ represents FlAlyA-H71K, and ▲ represents FlAlyA-H176D).

[0035] Figure 3 of wild-type alginate lyase and alginate lyase mutants m determination.

[0036] Figure 4 shows the residual enzyme activities of wild-type alginate lyase and alginate lyase mutants at different temperatures (where ● represents wild-type alginate lyase AL, ■ represents FlAlyA-H71K, and ▲ represents FlAlyA-H176D). Detailed Embodiments

[0037] The present invention provides an alginate lyase mutant, including alginate lyase mutant FlAlyA-H71K or alginate lyase mutant FlAlyA-H176D; the amino acid sequence of the alginate lyase mutant FlAlyA-H71K is as shown in SEQ ID NO: 1; the nucleotide sequence encoding the alginate lyase mutant FlAlyA-H71K is as shown in SEQ ID NO: 2; the amino acid sequence of the alginate lyase mutant FlAlyA-H176D is as shown in SEQ ID NO: 3; the nucleotide sequence encoding the alginate lyase mutant FlAlyA-H176D is as shown in SEQ ID NO: 4.

[0038] The present invention also provides a method for preparing the alginate lyase mutant. The method for preparing the alginate lyase mutant FlAlyA-H71K is: mutating the 71st amino acid of alginate lyase FlAlyA to obtain the alginate lyase mutant FlAlyA-H71K; the method for preparing the alginate lyase mutant FlAlyA-H176D is: mutating the 176th amino acid of alginate lyase FlAlyA to obtain the alginate lyase mutant FlAlyA-H176D.

[0039] In the present invention, the 71st amino acid of the alginate lyase FlAlyA is histidine; the 71st amino acid of the alginate lyase mutant FlAlyA-H71K is lysine; the 176th amino acid of the alginate lyase FlAlyA is histidine; the 176th amino acid of the alginate lyase mutant FlAlyA-H176D is aspartic acid.

[0040] The present invention also provides a recombinant expression vector, comprising a nucleotide sequence encoding the alginate lyase mutant FlAlyA-H71K or FlAlyA-H176D and an empty vector pET22b(+).

[0041] The present invention also provides a method for preparing the recombinant expression vector, comprising the following steps: ligating the nucleotide sequence encoding the alginate lyase mutant FlAlyA-H71K or FlAlyA-H176D with pET22b(+) to obtain a recombinant expression vector; the ligation site is the site after double digestion with restriction endonucleases Nde I and Xho I.

[0042] The present invention also provides a recombinant expression strain, comprising the recombinant expression vector and a starting strain; the starting strain is Escherichia coli BL21-CodonPlus-RIL.

[0043] The present invention also provides a method for preparing the recombinant expression strain, comprising the following steps: introducing the recombinant expression vector into the starting strain and inducing expression to obtain a recombinant expression strain. In the present invention, the inducer for induction is isopropyl-β-D-thiogalactoside (IPTG); the final concentration of isopropyl-β-D-thiogalactoside is 0.1 mM; the induction temperature is 23-27 °C; the induction time is 22-26 h.

[0044] The present invention also provides the application of the alginate lyase mutant in the preparation of products with high thermal stability. In the present invention, the application is to construct a recombinant expression vector with the alginate lyase mutant, introduce the recombinant expression vector into a starting strain to obtain a recombinant expression strain, culture the recombinant expression strain, collect the bacterial cells, break the bacterial cells to obtain a crude extract of the alginate lyase mutant, purify the crude extract of the alginate lyase mutant with an Ni column, and collect the eluate to obtain the alginate lyase mutant enzyme solution.

[0045] The present invention also provides the application of the recombinant expression vector or the recombinant expression strain in the preparation of products with high thermal stability.

[0046] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0047] The LB liquid medium described in the embodiments of the present invention comprises components with the following concentrations: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L.

[0048] The LB solid medium described in the embodiments of the present invention comprises components with the following concentrations: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L, agar 2 g / L.

[0049] Example 1

[0050] Cloning of the alginate lyase mutant gene sequence

[0051] Using the sequence of the wild-type alginate lyase FlAlyA gene as shown in SEQ ID NO:6 as a template, primers FlAlyA-H71K-F and FlAlyA-H71K-R were designed, and the mutant FlAlyA-H71K gene sequence as shown in SEQ ID NO:2 was obtained by PCR amplification; using the wild-type alginate lyase FlAlyA gene as a template, primers FlAlyA-H176D-F and FlAlyA-H176D-R were designed, and the mutant FlAlyA-H176D gene sequence as shown in SEQ ID NO:4 was obtained by PCR amplification; the amino acid sequence of the wild-type alginate lyase FlAlyA encoded by SEQ ID NO:6 is as shown in SEQ ID NO:5.

[0052] FlAlyA-H71K-F(SEQ ID NO:7): 5'-CACGGCTAATACGAAGTATTCTCGTTCTGAGCTAAGAGAGACA-3'

[0053] FlAlyA-H71K-R (SEQ ID NO:8): 5'-ACTTCGTATTAGCCGTGGTCACTCCCGAAGGA-3'

[0054] FlAlyA-H176D-F (SEQ ID NO:9): 5'-TTCAGAAGATGCTTGGGGTGATGATGAAGGTC-3'

[0055] FlAlyA-H176D-R (SEQ ID NO:10): 5'-CCCAAGCATCTTCTGAAAGCATTTCTTTATAAGGCG-3'

[0056] The gene sequence of the wild-type alginate lyase FlAlyA described in this example was completed by Shanghai Sangon Biotech Co., Ltd.

[0057] Example 2

[0058] Expression of Alginate Lyase Mutants

[0059] The gene fragment of the alginate lyase mutant FlAlyA-H71K and the empty vector pET22b(+) were double digested with the restriction enzymes Nde I and Xho I respectively to obtain a vector pET22b(+) fragment with sticky ends; the alginate lyase gene fragment and the vector pET22b(+) fragment with the same sticky ends were ligated by homologous recombination to obtain a recombinant expression vector of alginate lyase, which was then transformed into Escherichia coli DH5α, selected with ampicillin, and the clones were verified by sequencing. After correct sequencing, the recombinant vector pET22b(+)-FlAlyA containing the alginate lyase gene was obtained. H71K 。

[0060] The recombinant vector pET22b(+)-FlAlyA containing the alginate lyase mutant FlAlyA-H176D was constructed by the same method as above. H176D 。

[0061] The recombinant vector pET22b(+)-FlAlyA H71K and the recombinant vector pET22b(+)-FlAlyA H176D were respectively introduced into the starting strain BL21-CodonPlus-RIL to obtain the recombinant expression strains BL21-pET22b(+)-FlAlyA H71K and BL21-pET22b(+)-FlAlyA H176D 。

[0062] Pick monoclonal recombinant expression strain BL21-pET22b(+)-FlAlyA H71K and BL21-pET22b(+)-FlAlyA H176D Inoculate them separately into LB solid medium, incubate overnight at 37°C with 220 rpm shaking, then transfer to LB liquid medium, and shake at 37°C with 220 rpm until the OD 600 reaches 0.8. Then add IPTG to a final concentration of 0.1 mM, and induce at 25°C with 220 rpm shaking for 24 h. After the induction is completed, centrifuge at 8000 rpm for 20 min at 4°C to collect the bacterial cells. Resuspend 1 g of wet bacterial cells with 25 mL of a mixture (20 mM Tris, 300 mM NaCl (pH 7.0)) to obtain a bacterial suspension. Transfer 50 mL of the bacterial suspension to a 100 mL beaker, and under ice-water bath conditions, sonicate at a power of 600 W for 2 s with a 2 s interval for 10 min to lyse the bacterial cells. After centrifugation at 7200 rpm for 15 min at 4°C, discard the precipitate, and the supernatant is the crude extract enzyme solution of the alginate lyase mutant. Resuspend the crude extract enzyme solution with the above-mentioned mixture to obtain a crude enzyme solution. Purify the crude enzyme solution with a Ni column purchased from Shanghai Yisheng, wash away impurities with 20 mM imidazole, elute with 500 mM imidazole, collect the eluate, and perform protein SDS-PAGE electrophoresis detection. Obtain the purified alginate lyase mutant enzyme solution.

[0063] Example 3

[0064] Expression of alginate lyase mutant

[0065] The method for preparing the substrate for the determination of the enzyme activity of the alginate lyase mutant is as follows: Weigh 0.5 g of sodium alginate into 100 mL of 10 mM sodium phosphate buffer, heat and stir at 100°C until dissolved, then add NaCl to a final concentration of 100 mM, and add BSA to a final concentration of 0.1 mg / mL.

[0066] One enzyme activity unit is the amount of enzyme required to increase the absorbance by 0.01 per minute of hydrolysis.

[0067] The method for determining the enzyme activity is as follows: Mix 900 μL of 0.5% substrate + 100 μL of diluted enzyme solution (0.01 mg / mL), react at 30°C for 10 min, then boil at 100°C for 10 min to terminate the reaction, and obtain a sample. Dilute the sample 20 times and measure the absorbance at a wavelength of 235 nm with an enzyme-labeling instrument, and convert it into the corresponding enzyme activity unit.

[0068] Take the purified alginate lyase mutant enzyme solution obtained in Example 2, and under the condition of 30°C, measure the enzyme activity of the alginate lyase mutant at pH values of 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0 respectively. The measurement results are asFigure 1 as shown

[0069] Figure 1 It is shown that the optimal pH values of the alginate lyase mutants FlAlyA-H71K and FlAlyA-H176D are both 8.0. The mutant FlAlyA-H71K has more than 80% enzyme activity under the conditions of pH 7.0 - 9.0, showing good pH stability.

[0070] Taking the purified alginate lyase mutant enzyme solution obtained in Example 2, when the pH value is 8.0, the enzyme activities of the alginate lyase mutants are respectively detected at 30, 40, 45, 50, 55, 60, and 70 °C, and the measurement results are as Figure 2 shown

[0071] Figure 2 shown. The optimal temperature of the mutant FlAlyA-H71K is 50 °C, and the enzyme activity remains above 80% at 40 - 55 °C. The optimal reaction temperature of FlAlyA-H176D is 50 °C. Thus, it can be seen that the mutants FlAlyA-H71K and FlAlyA-H176D can maintain relatively high enzyme activities at higher temperatures.

[0072] Taking the purified alginate lyase mutant enzyme solution obtained in Example 2 and diluting it to 0.4 mg / mL, the change in ellipticity of the protein at 216 nm is measured by a circular dichroism spectrometer. After fitting with the Boltzman equation, the T m value is obtained. The parameters of the circular dichroism spectrometer are set as follows: temperature range: 20 - 90 °C, temperature interval: 5 °C, wavelength range: 190 - 260 nm, wavelength interval: 1 nm. The results are as Figure 3 shown

[0073] Figure 3 shown. The T m of the mutant FlAlyA-H71K is 50.57 °C, and the T m of the mutant FlAlyA-H176D is 51.50 °C. Thus, it can be seen that the alginate lyase mutant strains of the present invention have good thermal stability.

[0074] Taking the purified alginate lyase mutant enzyme solution obtained in Example 2, when the temperature is 50 °C, incubate for 5, 10, 15, 20, 25, and 30 min, and respectively detect the residual enzyme activities of the alginate lyase mutants. The measurement results are as Figure 4 shown

[0075] Figure 4It shows that at a temperature of 50 °C and with different heat preservation times, the residual enzyme activities of the mutants FlAlyA-H71K and FlAlyA-H176D are higher, indicating enhanced temperature stability.

[0076] Example 4

[0077] The protein concentration was measured using a Thermo Fisher NanoDrop 2000C ultra-micro spectrophotometer. After detection, 1 L of the recombinant expression strain BL21-pET22b(+)-FlAlyA H71K and BL21-pET22b(+)-FlAlyA H176D fermentation broth can obtain 600 ± 50 mg of pure enzymes of FlAlyA-H71K and FlAlyA-H176D after separation and purification. This is much higher than other alginate lyases, providing the possibility for the industrial production of alginate lyases.

[0078] Comparative Example 1

[0079] The wild-type alginate lyase recombinant expression vector and recombinant expression strain were set up using the scheme of Example 2, and the pH stability, temperature stability, T m and the residual enzyme activity within different time periods of the wild-type alginate lyase were compared with the alginate lyase mutants in Example 3. The results are as Figures 1 to 4 shown.

[0080] Figures 1 to 4 As described, the optimal pH of the wild-type alginate lyase is 8.0, and within the temperature range of 30 - 70 °C, the enzyme activity of the wild-type alginate lyase is lower than that of the alginate lyase mutant FlAlyA-H71K. The T m of the wild-type alginate lyase is 50.24 °C, which is lower than that of the alginate lyase mutant.

[0081] From the above examples, it can be seen that the present invention provides an alginate lyase mutant, its preparation method and application, as well as a recombinant expression vector and a recombinant expression strain. The enzyme activity of the alginate lyase mutant FlAlyA-H71K can reach 1.11×10 5 U / mg, with an optimal pH of 8.0; the optimal temperature is 50 °C, and it has more than 80% enzyme activity under the conditions of pH 7.0 - 9.0, showing good pH stability; the optimal temperature is 50 °C, and the enzyme activity remains above 80% at 40 - 55 °C. Compared with the wild-type alginate lyase FlAlyA, ΔT m is 0.33 °C. The enzyme activity of the alginate lyase mutant FlAlyA-H176D can reach 0.95×10 5 U / mg, with an optimal pH of 8.0. Compared with the wild-type alginate lyase FlAlyA, ΔTm It was 1.26 °C, indicating an improvement in its thermal stability. In addition, 600 ± 50 mg of pure FlAlyA-H71K and FlAlyA-H176D enzymes could be obtained from 1 L of fermentation broth, far higher than the existing level, providing the possibility for the industrial production of alginate lyase.

[0082] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An alginate lyase mutant, characterized in that, Comprising the alginate lyase mutant FlAlyA-H71K or the alginate lyase mutant FlAlyA-H176D; The amino acid sequence of the alginate lyase mutant FlAlyA-H71K is shown in SEQ ID NO: 1; The nucleotide sequence encoding the alginate lyase mutant FlAlyA-H71K is shown in SEQ ID NO: 2; The amino acid sequence of the alginate lyase mutant FlAlyA-H176D is shown in SEQ ID NO: 3; The nucleotide sequence encoding the alginate lyase mutant FlAlyA-H176D is shown in SEQ ID NO:

4.

2. A method for preparing the alginate lyase mutant according to claim 1, characterized in that, The preparation method of the alginate lyase mutant FlAlyA-H71K is: mutating the 71st amino acid of the alginate lyase FlAlyA to obtain the alginate lyase mutant FlAlyA-H71K; The preparation method of the alginate lyase mutant FlAlyA-H176D is: mutating the 176th amino acid of the alginate lyase FlAlyA to obtain the alginate lyase mutant FlAlyA-H176D; The nucleotide sequence of the alginate lyase FlAlyA is shown in SEQ ID NO:

6.

3. A recombinant expression vector, characterized in that, Comprising the nucleotide sequence encoding the alginate lyase mutant FlAlyA-H71K or FlAlyA-H176D as claimed in claim 1 and the empty vector pET22b(+).

4. The method for preparing the recombinant expression vector according to claim 3, characterized in that, Comprising the following steps: ligating the nucleotide sequence encoding the alginate lyase mutant FlAlyA-H71K or FlAlyA-H176D as claimed in claim 1 with pET22b(+) to obtain a recombinant expression vector; The site of the ligation is the site after double digestion with the restriction endonucleases Nde I and Xho I.

5. A recombinant expression strain, characterized in that, Comprising the recombinant expression vector as claimed in claim 3 and a starting strain; The starting strain is Escherichia coli BL21-CodonPlus-RIL.

6. The preparation method of the recombinant expression strain according to claim 5, characterized in that, Comprising the following steps: introducing the recombinant expression vector as claimed in claim 3 into the starting strain and inducing expression to obtain a recombinant expression strain.

7. The preparation method according to claim 6, characterized in that, The inducer for the induction is isopropyl-β-D-thiogalactoside (IPTG); The final concentration of the isopropyl-β-D-thiogalactoside is 0.1 mM; The temperature for the induction is 23-27 °C; The time for the induction is 22-26 h.

8. Use of the alginate lyase mutant as claimed in claim 1 in the preparation of a product containing an alginate lyase with high thermal stability.

9. Use of the recombinant expression vector as claimed in claim 3 or the recombinant expression strain as claimed in claim 5 in the preparation of a product containing an alginate lyase with high thermal stability.

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