A mutant for improving the catalytic activity and stability of α-agarase

Abstract

The present invention discloses a mutant for improving the catalytic activity and stability of α-agarase, belonging to the technical fields of genetic engineering and enzyme engineering. The present invention provides an α-agarase mutant, which is obtained by mutating the 469th amino acid of the agarase with the amino acid sequence shown in SEQ ID NO.1. The catalytic activity and stability of the α-agarase mutant G469S of the present invention are both improved compared with the wild-type enzyme, showing excellent heat resistance and freeze-thaw stability, which is beneficial to improving the production efficiency of continuous production of the enzyme in actual industrial applications and has good industrial application prospects.

Description

Technical Field

[0001] The present invention relates to a mutant for improving the catalytic activity and stability of α-agarase, belonging to the technical fields of genetic engineering and enzyme engineering. Background Art

[0002] Marine algae have received extensive attention because they can be used as food, materials and renewable energy sources. Among all commercial algae, red algae have the largest production. Agar is the main component of the cell wall of red algae and is usually used as a thickener and gelling agent in the food industry with relatively low added value. The oligosaccharides extracted from agar, however, have various beneficial biological activities, including anti-inflammatory, antioxidant, prebiotic, antibacterial and skin whitening effects, making agar oligosaccharides have application potential in the fields of food, medicine, agriculture, etc., greatly improving the economic benefits of agar.

[0003] The enzymatic method is considered to be the main method for sustainable commercial production of agar oligosaccharides because it can specifically hydrolyze agar and does not form harmful compounds. However, the research on agarase in China is still in its infancy. Among them, compared with β-agarase, α-agarase has fewer varieties, complex structures and generally poor stability, seriously hindering the large-scale application of α-agarase. The existing α-agarases include AgaA from Alteromonas agarlyticus GJ1B, AgaA33 from Thalassomonas sp. 44JAMB-A3, AgaD and AgaE from Thalassomonas sp. LD5, CaLJ96 from Catenovulum agarivorans, AgaWS5 from Catenovulum sediminis WS1-A, OUC-GaJJ96 from Gilvimarinus agarilyticus, Ce2834 and Ce2835 from Colwellia echini A3T, and Cm-AGA from Catenovulum maritimum STB14. Among them, the problems of low enzyme activity and poor thermal stability are common and difficult to meet the needs of industrial production.

[0004] Ca-AGA derived from Catenovulum agarivorans STB13 is currently the α-agarase with the highest enzyme activity expressed in the laboratory. It has strong substrate specificity, can produce agar oligosaccharides with different degrees of polymerization, and has great application potential. However, the optimal temperature of the vast majority of agarases from natural sources is between 30°C and 40°C, and agar forms a gel below 40°C, which greatly limits the hydrolysis efficiency of agarase and its actual industrial application. Therefore, improving the catalytic activity and stability of α-agarase Ca-AGA by means of genetic engineering and enzyme engineering is of great significance for the directed molecular modification of α-agarase and the high-efficiency and low-cost processing of agar. Summary of the Invention

[0005] To solve the problems such as poor stability of α-agarase in the prior art, the present invention provides an α-agarase mutant, which is obtained by mutating the 469th amino acid of the α-agarase with the amino acid sequence shown in SEQ ID NO.1.

[0006] In one embodiment of the present invention, the α-agarase is an α-agarase derived from the marine microorganism Catenovulum agarivorans STB13, and the nucleotide sequence encoding the α-agarase is shown in SEQ ID NO.2.

[0007] In one embodiment of the present invention, the α-agarase mutant is: mutating the glycine at the 469th position of the α-agarase with the amino acid sequence shown in SEQ ID NO.1 to serine, named: G469S.

[0008] Alternatively, the α-agarase mutant is: mutating the glycine at the 469th position of the α-agarase with the amino acid sequence shown in SEQ ID NO.1 to threonine, named: G469T.

[0009] Alternatively, the α-agarase mutant is: mutating the glycine at the 469th position of the α-agarase with the amino acid sequence shown in SEQ ID NO.1 to lysine, named: G469K.

[0010] Alternatively, the α-agarase mutant is: mutating the glycine at the 469th position of the α-agarase with the amino acid sequence shown in SEQ ID NO.1 to arginine, named: G469R.

[0011] The present invention also provides a gene encoding the above mutant.

[0012] The present invention also provides a recombinant vector carrying the above gene.

[0013] In one embodiment of the present invention, the recombinant vector uses pET-28a(+) as the expression vector.

[0014] The present invention also provides a recombinant cell carrying the above gene or the above recombinant vector.

[0015] In one embodiment of the present invention, the recombinant cell uses bacteria or fungi as the host cell.

[0016] The present invention also provides a recombinant Escherichia coli expressing the above mutant, using Escherichia coli BL21(DE3) as the host and pET-28a(+) as the expression vector.

[0017] The present invention also provides a method for preparing the above mutant, and the steps of the method are as follows:

[0018] (1) According to the determined mutation site, design mutagenic primers for site-directed mutagenesis, and perform site-directed mutagenesis using the vector carrying the α-agarase encoding gene as the template; construct a vector containing the gene encoding the mutant.

[0019] (2) Transform the vector containing the gene encoding the mutant into a microbial cell.

[0020] (3) Select positive clones for fermentation culture, centrifuge to collect cells, and the supernatant after cell disruption is the crude enzyme solution of the α-agarase mutant.

[0021] The present invention also provides a method for improving the stability of α-agarase, by mutating the glycine at the 469th position of the α-agarase with the amino acid sequence shown in SEQ ID NO.1.

[0022] In one embodiment of the present invention, the method is to mutate the glycine at the 469th position of the α-agarase with the amino acid sequence shown in SEQ ID NO.1 into serine;

[0023] Or, mutate the glycine at the 469th position of the α-agarase with the amino acid sequence shown in SEQ ID NO.1 into threonine;

[0024] Or, mutate the glycine at the 469th position of the α-agarase with the amino acid sequence shown in SEQ ID NO.1 into lysine;

[0025] Or, mutate the glycine at the 469th position of the α-agarase with the amino acid sequence shown in SEQ ID NO.1 into arginine.

[0026] In one embodiment of the present invention, the stability includes thermal stability and freeze-thaw stability.

[0027] The present invention also provides the use of the mutant, the gene, the recombinant vector, the recombinant cell, or the recombinant Escherichia coli in the preparation of agarooligosaccharides.

[0028] Beneficial effects:

[0029] The present invention provides a mutant of an α-agarase with the amino acid sequence shown in SEQ ID NO.1, in which glycine at the 469th position is mutated to serine to obtain the mutant G469S. Compared with the wild type, the enzyme activity of the mutant G469S is improved, and it has higher freeze-thaw stability and thermal stability. After incubation at 37°C for 30 min, it retains 51.06% of the original enzyme activity, which is 1.29 times that of the wild enzyme, facilitating the improvement of the production efficiency of continuous production of the enzyme in actual industrial applications and having good industrial application prospects. Brief description of the drawings

[0030] Figure 1 : Agarose gel electrophoresis patterns of wild-type Ca-AGA and its mutants; where, in the figure, M: DNA standard molecular weight, 1: wild-type Ca-AGA plasmid, 2-5: mutant plasmids, namely G469K, G469T, G469S, and G469R respectively.

[0031] Figure 2 : SDS-PAGE patterns of wild-type Ca-AGA and its mutants; where, in the figure, M: protein standard molecular weight, 1: wild-type Ca-AGA, 2-5: mutants G469K, mutant G469T, mutant G469S, and mutant G469R.

[0032] Figure 3 : Enzyme activity characterization of wild enzyme Ca-AGA and its mutants.

[0033] Figure 4 : Thermal stability characterization of wild enzyme Ca-AGA and its mutants at 37°C.

[0034] Figure 5 : Freeze-thaw stability characterization of wild enzyme Ca-AGA and its mutants. Specific embodiments

[0035] The embodiments of the present invention are only for further illustration of the content of the present invention and cannot be used as the limiting content or scope of the present invention.

[0036] The culture media involved in the following examples are as follows:

[0037] LB liquid medium: yeast powder 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH 7.0.

[0038] LB solid medium: 5 g / L yeast powder, 10 g / L tryptone, 10 g / L NaCl, pH 7.0, 1.5% (w / v) agar.

[0039] Fermentation liquid medium: 12 g / L yeast powder, 24 g / L tryptone, 5 g / L sucrose, KH 2 PO 4 17 mM, K 2 HPO 4 72 mM, pH 9.0.

[0040] The detection methods involved in the following examples are as follows:

[0041] Detection of α-agarase activity:

[0042] The hydrolysis activity of α-agarase was characterized by measuring the change in the content of reducing sugar using the 3,5-dinitrosalicylic acid (DNS) method. An agarose substrate solution of 0.15% (w / w) was prepared with Tris-HCl buffer (20 mM, pH 8.0). 0.1 mL of the enzyme solution was directly pipetted into 0.9 mL of the agarose solution, and after reacting at 37 °C for 10 min, 1 mL of DNS was added to terminate the reaction. After boiling for 5 min and then placing in an ice bath, 2 mL of deionized water was added for dilution, and the absorbance value was measured at 540 nm. The inactivated enzyme was used as a control, and galactose was used as a standard to draw a standard curve.

[0043] Definition of enzyme activity (U / mL): Under the above conditions, the volume of the enzyme required to catalyze the production of 1 μmol of reducing sugar (calculated as galactose) per minute was defined as one enzyme activity unit (U).

[0044] The specific enzyme activity of α-agarase refers to: Under specific conditions, the number of enzyme activity units per unit weight (mg) of protein. Specific enzyme activity (U / mg) = enzyme activity (U / mL) / enzyme concentration (mg / mL).

[0045] Example 1: Preparation of mutants

[0046] The specific steps are as follows:

[0047] (1) Chemically synthesize α-agarase Ca-AGA with the nucleotide sequence shown in SEQ ID NO.2, and clone it into the plasmid pET-28a(+) using the restriction enzymes Nco I and Xho I to obtain the recombinant plasmid pET-28a(+)-ca-aga;

[0048] (2) Using the recombinant plasmid pET-28a(+)-ca-aga prepared in step (1) as a template, complementary primer strands required for the experiment were designed (see Table 1), and the primers were synthesized by Genewiz Biotechnology Co., Ltd. Site-directed mutagenesis was carried out according to the method described in the instruction manual of TaKaRa's PrimerSTAR GXL kit; Recombinant plasmids containing the genes of mutants G469K, G469T, G469S, and G469R were respectively prepared.

[0049] Table 1 Introduction of α-agarase mutation sites

[0050]

[0051] 1 Underlined bases correspond to the corresponding mutant amino acids

[0052] The PCR reaction system was set according to the conditions in the instruction manual of the PrimerSTAR GXL kit: 5×PrimeSTAR GXL Buffer 10 μL, dNTP Mixture (2.5 mM each) 4 μL, upstream primer (10 μM) 1 μL, downstream primer (10 μM) 1 μL, DNA template 0.5 μL, PrimeSTAR GXL DNA Polymerase 1 μL, and ddH 2 O 32.5 μL.

[0053] The PCR amplification conditions were: pre-denaturation at 98°C for 3 min; then denaturation at 98°C for 10 s, annealing at 58°C for 15 s, and extension at 68°C for 10 min as one cycle, for a total of 30 cycles; final extension at 68°C for 10 min, and finally incubation at 4°C.

[0054] The PCR product obtained after the reaction was digested with DpnI at 37°C for 1 - 2 h, and then the treated PCR product was transformed into E. coli JM109 according to the method for transforming E. coli JM109 competent cells to prepare transformants. The transformants were spread on kanamycin (20 μg / mL) LB solid medium and incubated at 37°C in an inverted position for 12 h. Positive monoclonal colonies were picked into LB liquid medium containing kanamycin (20 μg / mL) and cultured at 37°C and 200 rpm for 10 - 12 h. Plasmids were extracted and sent to the company for sequencing. Recombinant plasmids containing mutant genes were respectively obtained: pET-28a(+)-G469K, pET-28a(+)-G469T, pET-28a(+)-G469S, pET-28a(+)-G469R. The verification results of plasmid extraction are as Figure 1 shown.

[0055] Example 2: Construction of genetically engineered bacteria and expression of mutants

[0056] The specific steps are as follows:

[0057] (1) Transfer the recombinant plasmids pET-28a(+)-ca-aga, pET-28a(+)-G469K, pET-28a(+)-G469T, pET-28a(+)-G469S, and pET-28a(+)-G469R obtained in Example 1 into E. coli BL21(DE3) respectively to prepare the recombinant strains E. coli BL21(DE3) / pET-28a(+)-ca-aga, E. coli BL21(DE3) / pET-28a(+)-G469K, E. coli BL21(DE3) / pET-28a(+)-G469T, E. coli BL21(DE3) / pET-28a(+)-G469S, and E. coli BL21(DE3) / pET-28a(+)-G469R;

[0058] (2) Streak the recombinant strains on LB solid medium containing kanamycin (20 μg / mL) and culture them in a constant temperature incubator at 37°C for 12 h. Pick positive monoclonal colonies into LB liquid medium containing kanamycin (20 μg / mL) and culture them at 37°C and 200 rpm for 10 - 12 h to prepare the seed solution;

[0059] (3) Inoculate the seed solution into the fermented liquid medium containing kanamycin (20 μg / mL) for shake flask fermentation at an inoculation amount of 2% (v / v). When the OD 600 value reaches 0.6 - 0.8, add isopropyl-β-D-thiogalactoside (IPTG, 10 μL / 50 mL, final concentration 0.005 mM), and culture in a shake flask at 25°C and 200 rpm for 16 h; obtain the fermentation broth. After centrifuging the fermentation broth at 4°C and 10000 rpm for 20 min, collect the cells, resuspend the cells by adding lysis buffer (20 mM Tris-HCl, pH 8.0) in equal proportion, and after ultrasonic disruption, collect the supernatant by high-speed centrifugation to obtain the crude intracellular enzyme solution. The SDS-PAGE gel electrophoresis pattern is as Figure 2 shown, and the results show that both the original enzyme and the mutant enzyme are expressed.

[0060] Example 3: Purification and Enzyme Activity Assay of Enzyme

[0061] The specific steps are as follows:

[0062] (1) Protein purification and ultrafiltration concentration:

[0063] Use HisTrap TMPurification by nickel column affinity chromatography using an HP column (5 mL): ① Pretreatment of the enzyme solution: Add 500 mM NaCl and 20 mM imidazole to 50 mL of the crude enzyme solution, and filter through a 0.45 μm aqueous filter membrane to remove macromolecular impurities; ② First, wash the pump with ultrapure water and clean the pipeline; ③ Equilibrate the nickel column (about 5 column volumes) with buffer A (500 mM NaCl, 50 mM Tris-HCl, 20 mM imidazole, pH 8.0) at a flow rate of 2 mL / min, then load the sample at a rate of 1.5 mL / min. After loading, equilibrate the nickel column with buffer A again. ④ Elution of the nickel column: Elute the sample with eluent B (500 mM NaCl, 50 mM Tris-HCl, 500 mM imidazole, pH 8.0) in the best isocratic elution mode (80% A, 20% B, 2 mL / min), and collect the eluate (containing the target protein) in fractions during the peak elution.

[0064] The ultrafiltration concentration steps are as follows: Add the purified enzyme solution to an ultrafiltration centrifuge tube and centrifuge at low speed. Add an appropriate amount of Tris-HCl buffer (pH 8.0) and repeat 5 times to remove imidazole and NaCl.

[0065] Pure enzyme solutions containing wild-type agarase and mutant enzymes G469K, G469T, G469S, and G469R were prepared respectively.

[0066] (2) Enzyme activity assay:

[0067] Determine the specific enzyme activities of the above wild-type agarase and mutant enzymes G469K, G469T, G469S, and G469R. Set the wild-type enzyme activity as 100%, and detect the relative enzyme activities of the mutant enzymes. The results are as Figure 3 shown. Exclude the mutant G469R with a significantly reduced initial enzyme activity, and screen out the mutants G469K, G469T, and G469S with enzyme activities similar to or higher than that of the wild-type for subsequent experiments.

[0068] Example 4: Determination of enzyme stability

[0069] The specific steps are as follows:

[0070] (1) Thermal stability of the enzyme:

[0071] Thermal stability experiment: Incubate the pure enzyme solution obtained in Example 3 at 37 °C for 30 min, then terminate the incubation, and detect the enzyme activity of the enzyme solution before and after incubation. The results are as Figure 4 shown.

[0072] The experimental results show that after the wild enzyme was incubated at 37 °C for 30 min, only 41.67% of the original enzyme activity was retained; while after the mutant G469S was incubated at 37 °C for 30 min, 51.06% of the original enzyme activity was retained (1.29 times that of the wild enzyme); after the mutant G469T was incubated at 37 °C for 30 min, 50% of the original enzyme activity was retained; after the mutant G469K was incubated at 37 °C for 30 min, 40.52% of the original enzyme activity was retained. In summary, the thermal stabilities of mutants G469S and G469T were both improved compared to the wild type, while G469K showed average performance.

[0073] (2) Freeze-thaw stability of the enzyme:

[0074] Freeze-thaw stability experiment: The pure enzyme solution obtained in Example 3 was frozen at -20 °C for 0 - 8 days. The enzyme solution was taken out at different time points and thawed at 4 °C for 1 day, and then the enzyme activity was detected. The results are as Figure 5 shown.

[0075] The changes in the enzyme activities of the wild type and mutants after being frozen at -20 °C for 0 - 8 days were measured. The results show that for both the wild type and mutants, the activity of the enzyme solution after freezing increased significantly, indicating good cold adaptability. The activity of mutant G469S increased by 296.42% compared to before freezing, and the highest enzyme activity could reach 95.62 U / mg. Mutants G469K and G469T showed average performance.

[0076] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and variations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. An α-agarase mutant, characterized in that The following mutations were performed on the basis of the parent enzyme whose amino acid sequence is shown in SEQ ID NO.1: Mutate glycine at position 469 to serine; Or mutate the glycine at position 469 to threonine.

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

3. A recombinant vector carrying the gene according to claim 2.

4. A recombinant cell carrying the gene according to claim 2 or the recombinant vector according to claim 3.

5. The recombinant cell according to claim 4, characterized in that Bacteria or fungi are used as host cells.

6. A recombinant Escherichia coli, characterized in that The mutant according to claim 1 is expressed using Escherichia coli BL21 (DE3) as a host and pET-28a (+) as an expression vector.

7. Use of the mutant according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3, the recombinant cell according to claim 4 or 5, or the recombinant Escherichia coli according to claim 6 in the preparation of agar-oligosaccharides.

8. A method for improving the stability of α-agarase, characterized in that: The glycine at position 469 of the α-agarase with the amino acid sequence shown in SEQ ID NO.1 is mutated to serine; the stability is thermal stability and freeze-thaw stability.

9. A method for improving the stability of α-agarase, characterized in that: The glycine at position 469 of the α-agarase with the amino acid sequence shown in SEQ ID NO.1 is mutated to threonine; and the stability is thermal stability.

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