A mutant enzyme of alginate lyase with improved thermal stability

Abstract

The present invention discloses a mutant enzyme of alginate lyase with improved thermal stability, belonging to the technical field of enzyme genetic engineering. The mutant enzyme provided by the present invention is obtained by mutating the lysine at the 48th position, tyrosine at the 106th position, serine at the 172nd position, and serine at the 190th position of the alginate lyase derived from Paenibacillus sp. YN15 into proline, glycine, valine, and glycine, respectively. The thermal stability and structural stability of the mutant enzyme are improved: the enzyme activity can still maintain more than 60% after being incubated at 55 °C for 8 h. The optimal temperature of the multi-site mutant rises to 65-70 °C, which is more than 10 °C higher than that of the wild-type enzyme. Under the optimal catalytic conditions, the relative enzyme activity of the enzyme catalyzing the substrate sodium alginate to produce alginate oligosaccharides does not decrease significantly, having important industrial application value.

Description

Technical Field

[0001] The present invention relates to a mutant enzyme of alginate lyase with improved thermal stability, belonging to the technical field of enzyme genetic engineering. Background Art

[0002] Sodium alginate, also known as alginic acid sodium, was initially extracted from alginate produced by brown algae. Industrially, it is usually a polymer formed by the polymerization of β-D-mannuronic acid (β-D-mannuronic acid, M) and α-L-guluronic acid (α-L-guluronic acid, G) through 1,4-glycosidic bonds. It has various uses in the food industry, such as thickening, gelling, and stabilizing systems. Alginate oligosaccharides (AOS) are molecules with a degree of polymerization of 2-10, decomposed from alginate, and have various functions such as regulating human immunity, antioxidation, anti-tumor, protecting the nervous system, promoting plant growth, and antibacterial. Due to its special chemical and biological properties, AOS has potential and extensive application value in the fields of agriculture, food, medicine, health products, cosmetics, metallurgy, and chemical industry. Currently, the decomposition methods of alginate include chemical decomposition method, physical decomposition method, and biological enzyme method. Among them, the biological enzyme method using alginate lyase has the advantages of less reaction by-products, low energy consumption, and the ability to generate highly bioactive oligosaccharides containing unsaturated double bonds, such as environmental protection, cleanliness, mild conditions, and high efficiency. It is inevitable that it will replace the physical and chemical methods as the mainstream method for preparing AOS.

[0003] So far, although domestic and foreign scholars have conducted a large amount of basic research on the sources, classification, enzymatic properties, enzyme structures, and enzyme catalytic mechanisms of alginate lyase, the existing alginate lyase still has problems such as low fermentation level, low enzyme activity, high price, poor stability, and not being suitable for large-scale use. On the international market, there is only the alginate lyase product A1603-100MG commercialized by Sigma Company in the United States. The enzyme activity is greater than 10000U / g, but the price is expensive and it is only sold in the form of a reagent.

[0004] Therefore, there is still a need in this field for an alginate lyase with strong enzyme stability. Based on the crystal structure of the existing alginate lyase, homologous modeling is carried out, the key site amino acid residues are analyzed, and the catalytic efficiency and thermal stability of Paenibacillus sp. YN15 Alyase are modified by site-directed mutagenesis. Non-conserved amino acid residues are selected for mutation to find mutants with improved thermal stability. Summary of the Invention

[0005] The object of the present invention is a mutant enzyme K48P / Y106G / S172V / S190G of alginate lyase with improved thermal stability, which has important practical significance for the synthesis of alginate oligosaccharides.

[0006] The first object of the present invention is to provide a mutant of alginate lyase with improved thermal stability, in which lysine at position 48 of the alginate lyase of Paenibacillus sp. YN15 is mutated to proline, tyrosine at position 106 is mutated to glycine, serine at position 172 is mutated to valine, and serine at position 190 is mutated to glycine.

[0007] In one embodiment, the amino acid sequence of the alginate lyase derived from the microorganism Paenibacillus sp. YN15 is as shown in SEQ ID NO.2.

[0008] In one embodiment, the amino acid sequence of the mutant enzyme K48P / Y106G / S172V / S190G is as shown in SEQ ID NO.4.

[0009] Compared with the wild enzyme, the optimal pH of the multi-site mutant enzyme K48P / Y106G / S172V / S190G of Paenibacillus sp. YN15 Alyase has not changed, and the optimal temperature has increased by more than 10 °C; the relative enzyme activity has not changed significantly either. At the same time, the thermal stability has been improved: the enzyme activity can still maintain more than 60% after being maintained at 55 °C for 8 h. Under the optimal catalytic conditions, the relative enzyme activity of the enzyme catalyzing the substrate sodium alginate to produce alginate oligosaccharides has no obvious decrease, and this finding has important research value for the industrial preparation of alginate oligosaccharides.

[0010] The second object of the present invention is to provide a gene encoding the above-mentioned mutant of alginate lyase.

[0011] In one embodiment, the nucleotide sequence of the gene is as shown in SEQ ID NO.3.

[0012] The third object of the present invention is to provide a recombinant expression plasmid carrying the above gene.

[0013] In one embodiment, the recombinant expression plasmid uses a pET series plasmid as the expression vector

[0014] In one embodiment, the pET series plasmids include pET-28a or pET-22b(+).

[0015] The fourth object of the present invention is to provide a cell containing the above gene or the above recombinant expression plasmid.

[0016] The fifth object of the present invention is to provide a recombinant Escherichia coli containing the above gene or the above recombinant expression plasmid.

[0017] In one embodiment, the recombinant Escherichia coli uses E. coli BL21(DE3) as an expression host.

[0018] The sixth object of the present invention is to provide a method for improving the thermal stability of alginate lyase, which is to mutate lysine at position 48 of alginate lyase with the amino acid sequence shown in SEQ ID NO.2 into proline, tyrosine at position 106 into glycine, serine at position 172 into valine, and serine at position 190 into glycine.

[0019] The present invention also provides a method for preparing alginate oligosaccharides, adding the above alginate lyase mutant or the above recombinant Escherichia coli to a reaction system containing sodium alginate for reaction.

[0020] The present invention also provides the application of the above alginate lyase mutant, or the above gene, or the above recombinant expression plasmid, or the above recombinant Escherichia coli in the preparation of products containing alginate oligosaccharides.

[0021] Beneficial effects:

[0022] The present invention provides a mutant enzyme K48P / Y106G / S172V / S190G of Paenibacillus sp. YN15 Alyase, whose thermal stability and structural stability are improved: the enzyme activity can still maintain more than 60% after maintaining for 8 h at 55°C. The optimal temperature of the multi-site mutant rises to 65 - 70°C, which is more than 10°C higher than that of the wild-type enzyme. Under the optimal catalytic conditions, the relative enzyme activity of the enzyme catalyzing the substrate sodium alginate to produce alginate oligosaccharides does not decrease significantly. This discovery has important research value for the industrial preparation of alginate oligosaccharides. Description of the drawings

[0023] Figure 1 Comparison of residual enzyme activities of wild enzyme and mutant enzyme;

[0024] Figure 2 Comparison of the optimal reaction temperatures of wild enzyme and mutant enzyme under the optimal reaction conditions. Detailed implementation manners

[0025] The culture media involved in the following examples:

[0026] LB culture medium: yeast extract 5 g / L; tryptone 10 g / L; sodium chloride 10 g / L, and the pH of the culture medium is 7.0.

[0027] The detection methods involved in the following examples:

[0028] Alginate lyase activity assay method:

[0029] Use 0.35% sodium alginate as substrate, add 7μg / mL pure enzyme, 50mmol / L NaCl, react for 5min at 65℃, pH 8.0, add two times volume of DNS solution to inactivate, boil, cool and dilute 4 times, measure absorbance.

[0030] Definition of enzyme activity (U): The amount of enzyme required to release 1 μmol of reducing sugar per minute under standard reaction conditions, where the content of reducing sugar is measured by DNS (dinitrosalicylic acid) colorimetry.

[0031] Example 1: Preparation method of Paenibacillus sp. YN15Alyase enzyme mutant

[0032] (1) The alginate lyase from Paenibacillus sp. YN15 (GenBank accession number: WP_113020684.1, the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2) was ligated into the multiple cloning site of pET-22b(+) to obtain the wild-type plasmid pET-22b(+)-payn.

[0033] (2) Construction of pET-22b(+)-K48P / Y106G / S172V / S190G single-point mutation plasmid: The experiment used the QuikChange TM The site-directed mutagenesis method was used. The single-point mutants to be constructed included: K48P, Y106G, S172V, and S190G. Sequencing verification results showed that no random mutations occurred except for the desired mutation sites, and the mutant plasmids pET-22b(+)-K48P, pET-22b(+)-Y106G, pET-22b(+)-S172V, and pET-22b(+)-S190G were successfully constructed.

[0034] Using the recombinant plasmid pET22b-payn as a template, forward and reverse primers were designed. The mutation primers are as follows, with capital letters indicating mutation points:

[0035] K48P-R: 5'-ggcagacgaggtgccgttttttgc-3'

[0036] K48P-F: 5'-gcacctcgtctgccCCAattacgattaaagg-3'

[0037] Y106G-F:5'-ggctggaaGGActggatgtgtacaacatcg-3'

[0038] Y106G-R: 5'-ccagTCCttccagccaattgttgttggcatg-3'

[0039] S172V-F: 5'-GTTttcggtcccaatgttcgggctgag-3'

[0040] S172V-R: 5'-tgggaccgaaAACtacagccgagatccggttattg-3'

[0041] S190G-R: 5'-ggtGCCgctcgatccttccttaatatcg-3'

[0042] S190G-F: 5'-gatcgagcGGCaccatcgttgagaattg-3'

[0043] PCR amplification: The total volume of the reaction system is 20 μL. The reaction program is as follows: 95°C, 2 min; 95°C, 15 s; 56°C, 15 s; 72°C, 3 min 15 s; cycle 26 times; 72°C, 5 min; 4°C.

[0044] Table 1 Composition of the PCR reaction system

[0045]

[0046] (3) Verification of PCR product nucleic acid electrophoresis and digestion of the template: Verify whether the size of the PCR product band is correct by agarose gel electrophoresis. After verification, add 1 μL of Q.cut DpnI and 2 μL of Q.cut Buffer (10×) to the PCR product system, and perform an enzymatic digestion reaction at 37°C for 1 h to remove the wild-type recombinant plasmid in the system. Then pick positive clones for plasmid extraction and DNA sequencing. Introduce the successfully sequenced mutant plasmid into E.coli BL21(DE3) competent cells to construct a mutant gene recombinant bacterium for the induced expression of the mutant enzyme.

[0047] (4) Construction of pET-28a-K48P / Y106G / S172V / S190G single-point mutant plasmid: Using the four single-point mutant recombinant plasmids pET-22b(+)-K48P, pET-22b(+)-Y106G, pET-22b(+)-S172V, and pET-22b(+)-S190G constructed in step (2) as templates, forward and reverse primers were designed, and the Golden Gate Cloning technique was used to combine the target fragments containing four mutation sites to obtain the stacked mutant plasmid pET-28a-K48P / Y106G / S172V / S190G.

[0048] The mutant primers are as follows:

[0049] 1: 5’-TTGGTCTCAAATGGCGTCCGTAACTTGCAGCACG-3’

[0050] 11: 5’-TTGGTCTCTCGTAATTGGGGCAGACGAGGTGCCGTTTTTTG-3’

[0051] 2: 5’-TTGGTCTCATACGATTAAAGGAGCAAGCTCCACGAATAAG-3’

[0052] 22: 5’-TTGGTCTCTATCCAGTCCTTCCAGCCAATTGTTGTTGGCATGATC-3’

[0053] 3: 5’-TTGGTCTCAGGATGTGTACAACATCGGGGAAGAAGGCG-3’

[0054] 4: 5’-TTGGTCTCTAAGCTTAATGGTGATGGTGATGGTGATTCACA-3’

[0055] 44: 5’-TTGGTCTCTAAGCTTAATGGTGATGGTGATGGTGATTCACA-3’

[0056] var-f: 5’-GTTTTCGGTCCCAATGTTCGGGCTGAGCATATCGATATTAAGGA-3’

[0057] var-r: 5’-GGGACCGAAAACTACAGCCGAGATCCGGTTATTGTTCGTCTCCTTC-3’

[0058] Table 2 Composition of the PCR reaction system

[0059]

[0060] For seamless cloning using Golden Gate Cloning, the reaction system was prepared according to Table 2 with a total volume of 25 μL. The reaction procedure was as follows: 37°C for 2 min; 16°C for 5 min; 50 cycles; 50°C for 5 min; 80°C for 10 min; 4°C. Then, sequencing verification and transformation of the combinatorial mutant plasmids were carried out, as well as induction expression and isolation purification of the combinatorial mutant enzymes.

[0061] Example 2: Method for expression and purification of mutant enzymes of Paenibacillus sp. YN15 Alyase.

[0062] The mutant plasmid pET-28a-K48P / Y106G / S172V / S190G and the wild-type plasmid pET22b-payn after sequencing verification in Example 1 were respectively transformed into Escherichia coli BL21(DE3) cells. Positive transformants were picked and cultured overnight at 37°C and 200 rpm in LB medium, and then inoculated into LB medium and cultured at 37°C until the OD 600 value reached 0.6 - 0.8. The temperature was lowered to 28°C, and IPTG was added to a final concentration of 1 mM and induced for 6 h to obtain the fermentation broth.

[0063] The fermentation broth was centrifuged at 4°C and 8000 rpm for 5 min to collect the cells. 15 mL of buffer (50 mM Tris-HCl, 100 mM NaCl, adjusted to pH about 7.5) was added to fully resuspend the cells, and then the centrifuge tube was placed in an ice bath and put into an ultrasonic cell disruptor. The conditions for ultrasonic disruption were: working time 1 s, stopping time 2 s, for a total of 15 min. The obtained disrupted solution was centrifuged at low temperature and high speed, at 4°C and 8000 rpm for 15 min to obtain the crude enzyme solution. It was filtered through a 0.45 μm microporous membrane and reserved to obtain the crude enzyme solution of the alginate lyase mutant K48P / Y106G / S172V / S190G and the crude enzyme solution of the wild-type alginate lyase.

[0064] Prepare a nickel ion affinity chromatography column. First, use a constant flow pump to pump deionized water into the column to wash the column (about 6 - 12 times the column volume), and then balance the column environment with a buffer solution of low salt concentration (500 mmol / L NaCl, 50 mM Tris - HCl, adjust the pH to about 7.5). Wait until the reading of the ultraviolet detector for the effluent at the lower end of the column stabilizes (about 5 times the column volume of the buffer solution), and then add the obtained crude enzyme solution to the column. First, wash the impurity proteins to baseline balance with a buffer solution containing low - concentration imidazole (500 mmol / L NaCl, 50 mmol / L imidazole, 50 mM Tris - HCl, adjust the pH to about 7.5), and then elute with an eluent containing high - concentration imidazole (500 mmol / L NaCl, 500 mmol / L imidazole, 50 mM Tris - HCl, adjust the pH to about 7.5). Collect the eluent of the absorption peak, and dialyze at 4°C to remove imidazole and residual metal ions to obtain the target protein. The mutants K48P / Y106G / S172V / S190G of the purified alginate lyase Paenibacillus sp. YN15 Alyase and the wild - type enzyme reach electrophoretic purity, and the enzyme activities are 104.8 U / mg and 96.8 U / mg respectively.

[0065] The mutants K48P, Y106G, S172V, and S190G of the purified alginate lyase Paenibacillus sp. YN15 Alyase were prepared by the same method.

[0066] Example 3: Comparison of the thermal stability of the enzyme before and after mutation

[0067] After diluting the enzyme solution obtained in Example 2 to a concentration of about 48 μg / mL, incubate it at 55°C, 60°C, and 65°C respectively, and take samples at regular intervals for lysis reactions under the optimal conditions of the enzyme to observe the change of the enzyme activity of the alginate lyase of the present invention with time at different temperatures. Use the untreated enzyme solution as a control (i.e., 100%), and the results are shown in Table 3.

[0068] The present invention studies the thermal stability and structural stability of the mutant K48P / Y106G / S172V / S190G. The thermal stability (t 1 / 2) has been greatly improved. At 55 °C, after 8 h of storage, the relative enzyme activity of the mutant K48P / Y106G / S172V / S190G increased from 2.9% of the wild-type enzyme to 63.0%; at 65 °C, after 1 h of storage, the relative enzyme activity of the mutant K48P / Y106G / S172V / S190G increased from 6.6% of the wild-type enzyme to 71.1%. At 65 °C, after 0.5 h of storage, the relative enzyme activity of the mutant K48P / Y106G / S172V / S190G was 85.3%, which was significantly higher than that of other single-point mutants. The thermostability of the mutant enzyme K48P / Y106G / S172V / S190G of Paenibacillus sp. YN15 Alyase was significantly improved, making it more suitable for industrial production requirements (see Figure 1 ).

[0069] Table 3 Comparison of t 1 / 2 and T m values of wild enzyme and mutant enzyme

[0070]

[0071] Table 4 Comparison of residual enzyme activity and T m values of wild enzyme and mutant enzyme after 0.5 h of storage at 65 °C

[0072]

[0073]

[0074] Example 4: Comparison of the Optimal pH of the Enzyme before and after Mutation

[0075] Buffers with different pH values of 50 mmol / L were selected (sodium phosphate buffer, pH 6.0 - 7.5; Tris-HCl buffer, pH 7.5 - 9.0; glycine-NaOH buffer, pH 9.0 - 10.5). According to the DNS method, the alginate lyase activity at different pH values was measured, and taking the maximum enzyme activity as 100%, the relative enzyme activities under different pH conditions were calculated respectively. This invention studied the effects of pH on the catalytic activity and stability of the mutant enzyme after thermostability modification. After the wild-type enzyme of Paenibacillus sp. YN15 Alyase mutated into the multi-point mutant K48P / Y106G / S172V / S190G, the optimal pH did not change, and the optimal pH was 8.0 (Tris-HCl). In the weakly alkaline range of pH 7.5 - 8.5, the catalytic activity of the mutant enzyme was relatively high, and the relative enzyme activity was greater than 60%.

[0076] Example 5: Comparison of the Optimal Temperature of the Enzyme before and after Mutation

[0077] This invention studies the effect of temperature on the catalytic activity of mutant enzymes. The mutant enzyme K48P / Y106G / S172V / S190G prepared in Example 2 and the wild-type enzyme are respectively subjected to enzymatic reactions at different temperatures (40 - 80 °C) at pH 8.0. The optimal temperature of the multi-site mutant K48P / Y106G / S172V / S190G rises to 65 - 70 °C, which is more than 10 °C higher than that of the wild-type enzyme. At the same time, the relative enzyme activity of the mutant at high temperatures of 75 and 80 °C also increases significantly (see Figure 2 ).

[0078] Table 5 Comparison of the optimal reaction temperatures of wild-type enzyme and mutant enzyme under optimal reaction conditions:

[0079]

[0080] 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 alterations 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 alginate lyase mutant with improved thermal stability, characterized in that, Mutate the lysine at position 48, tyrosine at position 106, serine at position 172, and serine at position 190 of the alginate lyase derived from Paenibacillus sp. YN15 to proline, glycine, valine, and glycine, respectively; The amino acid sequence of the alginate lyase derived from Paenibacillus sp. YN15 is shown in SEQ ID NO.

2.

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

3. A recombinant expression plasmid carrying the gene according to claim 2.

4. The recombinant expression plasmid according to claim 3, characterized in that, Using pET series plasmids as expression vectors.

5. A cell containing the gene according to claim 2 or the recombinant expression plasmid according to claim 3 or 4.

6. A recombinant Escherichia coli, characterized in that, The recombinant Escherichia coli contains the gene according to claim 2 or the recombinant expression plasmid according to claim 3 or 4.

7. The recombinant Escherichia coli according to claim 6, wherein Using E. coli BL21(DE3) as an expression host.

8. A method for improving the thermal stability of alginate lyase, characterized in that, The method is to mutate the lysine at position 48, tyrosine at position 106, serine at position 172, and serine at position 190 of the alginate lyase with the amino acid sequence shown in SEQ ID NO.2 to proline, glycine, valine, and glycine, respectively.

9. A method for preparing fucoidan oligosaccharide, characterized in that, Add the alginate lyase mutant according to claim 1 or the recombinant Escherichia coli according to claim 6 or 7 to a reaction system containing sodium alginate and carry out the reaction.

10. Use of the alginate lyase mutant according to claim 1, the gene according to claim 2, the recombinant expression plasmid according to claim 3 or 4, or the recombinant Escherichia coli according to claim 6 or 7 in the preparation of a product containing alginate oligosaccharides.

Images