High-catalytic-activity artificial synthetic ulvan lyase a21 and application thereof

Abstract

The present application relates to a kind of high catalytic activity artificial synthesis of ulvan lyase A21 and application, belong to functional enzyme technical field, the amino acid sequence of the artificial synthesis of ulvan lyase A21 as shown in SEQ ID NO.1, the present application also provides the enzyme preparation comprising the artificial synthesis of ulvan lyase A21, and the recombination expression vector and engineering bacteria comprising the gene of the artificial synthesis of ulvan lyase A21 coding ulvan lyase A21.The present application also provides the application of the artificial synthesis of ulvan lyase A21 in the preparation of ulvan oligosaccharide of the cleavage of ulva lactuca.The artificial synthesis of ulvan lyase A21 compared with wild ulvan lyase has better stability, and its catalytic activity compared with wild enzyme JpPL40A is increased by nearly 4.5 times.

Description

Technical Field

[0001] This invention belongs to the field of functional enzyme technology, specifically relating to a highly catalytically active synthetic lyase A21 of Ulva polysaccharide and its applications. Background Technology

[0002] Ulva is a natural macromolecular linear polysaccharide extracted from the cell walls of green algae of the genus Ulva. It is one of the few naturally occurring polysaccharides with highly sulfated groups. The main monosaccharide monomers in the structure of Ulva are iduronicacid (IdoA), 3-sulfated rhamnose (Rha3S), xylose (Xyl), 2-sulfated xylose (Xyl2S), and glucuronic acid (GlcA). The structure of Ulva typically contains four disaccharide units: [→4) β -d-Xyl(1→4)- α -L-Rha3S(1→]、[→4) α -L-IdoA(1→4)-α-L-Rha3S(1→],[→4) β -d-GlcA(1→4)- α -L-Rha3S(1→] and [→4) β -d-Xyl2S(1→4)- α -L-Rha3S(1→). Due to the presence of the sulfate group, Ulva prostrata possesses various physiological activities such as antiviral, anti-inflammatory, anticoagulant, and antioxidant effects. Oligosaccharides obtained from Ulva prostrata through degradation treatment show significant improvements in solubility, stability, and various physiological activities compared to Ulva prostrata polysaccharides, representing an important direction for the high-value utilization of Ulva prostrata. Currently, methods for preparing Ulva prostrata oligosaccharides include physical, chemical, and biological methods. Acid hydrolysis has advantages such as rapid reaction and high substrate concentration, but the products after acid hydrolysis are complex and difficult to separate. Enzymatic hydrolysis, compared to acid hydrolysis, has advantages such as mild reaction, single product, and easy separation. Therefore, enzymatic methods are a green method with sustainable application prospects, and the discovery of Ulva prostrata polysaccharide lysins for the targeted preparation of Ulva prostrata oligosaccharides with specific structures is of great significance.

[0003] CN 120098981 A discloses the application of the PL40 family of Ulva polysaccharide lyase JpPL40A in the hydrolysis of Ulva polysaccharide to prepare oligosaccharides. However, it has two shortcomings that limit its industrial application: first, the enzyme's thermostability is insufficient, and it is completely inactivated after incubation at 40℃ for 30 minutes; second, its enzyme activity is low, limiting its hydrolysis of Ulva polysaccharide derived from Ulva lactuca. k cat The value is only 8.98 s -1Therefore, it is necessary to obtain a JpPL40A-based stonecrop polysaccharide lyase with better heat resistance and higher catalytic efficiency. SUMMARY

[0004] In view of the prior art, the present application provides a stonecrop polysaccharide lyase A21 and an amino acid sequence thereof, which can degrade stonecrop, is artificially synthesized, has better heat resistance, and has a wide application prospect.

[0005] The present application is realized by the following technical scheme: application of stonecrop polysaccharide lyase in degradation of stonecrop and in preparation of stonecrop oligosaccharide.

[0006] An artificially synthesized stonecrop polysaccharide lyase A21, wherein the amino acid sequence (SEQ ID NO: 1) of the stonecrop polysaccharide lyase A21 is as follows:

[0007]

[0008] The application also provides an enzyme preparation comprising the artificial Ulvan lyase A21.

[0009] A recombinant expression vector carrying a gene encoding the Ulvan lyase A21.

[0010] A recombinant engineering bacterium carrying a gene encoding the Ulvan lyase A21 in the genome and capable of expressing the Ulvan lyase A21.

[0011] The application also provides application of the artificial Ulvan lyase A21 in preparation of Ulvan oligosaccharides by cracking Ulva lactuca.

[0012] Further, the enzymolysis conditions of the Ulvan lyase A21 are as follows: the substrate concentration is 5 g / L, the enzyme amount is 0.45 U / mL (1 U represents the enzyme required for releasing 1 micromole of reducing sugar in 1 minute), the enzymolysis temperature is 30-50 DEG C, preferably 40 DEG C, the pH value is 6.0-10.0, preferably 7.0, and the enzymolysis time is 10 minutes or more, preferably 40 minutes.

[0013] Compared with the prior art, the application has the following beneficial effects:

[0014] The Ulvan lyase A21 can degrade Ulva lactuca to prepare Ulvan oligosaccharides under the conditions of 40 DEG C and pH value of 7.0. The application constructs an expression vector containing the Ulvan lyase A21 gene and realizes the heterologous expression in Escherichia coli, thereby providing a good foundation for industrial large-scale production of the enzyme.

[0015] The optimal reaction temperature of the Ulvan lyase A21 is 40 DEG C, and the Ulva lactuca as the substrate can be kept in a solution state, thereby better realizing large-scale application of the Ulvan lyase A21. k cat / K m The catalytic constant of the Ulvan lyase A21 is 53.9 mL / mg / s, which is increased by nearly 4.5 times compared with the wild enzyme JpPL40A. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 : JpPL40A ancestral phylogenetic tree figure output by FireProt 2.0 online website;

[0017] Figure 2 : JpPL40A, A15, A20, A21, A22 and A23 and Ulvan octasaccharide ligand binding energy;

[0018] Figure 3: Two-dimensional graph of hydrogen bond interaction between wild enzyme JpPL40A and undecaose ligand;

[0019] Figure 4 : Two-dimensional graph of hydrogen bond interaction between A21 and undecaose ligand;

[0020] Figure 5 : SDS-PAGE electrophoretogram of purified stonecrop polysaccharide lyase A21 of the present application;

[0021] Figure 6 : Graph of influence of temperature change on hydrolytic enzyme activity of stonecrop polysaccharide lyase A21;

[0022] Figure 7 : Graph of influence of pH change on hydrolytic enzyme activity of stonecrop polysaccharide lyase A21;

[0023] Figure 8 : Comparison graph of stability of wild enzyme JpPL40A and A21 at 40℃;

[0024] Figure 9 : TLC graph of hydrolysis of stonecrop by stonecrop polysaccharide lyase A21 to generate oligosaccharides. DETAILED DESCRIPTION

[0025] The present application will be further described below in conjunction with examples. However, the scope of the present application is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the present application without departing from the spirit and scope of the present application.

[0026] The instruments, reagents, materials, etc. involved in the following examples, if not specifically stated, are all conventional instruments, reagents, materials, etc. already existing in the prior art, which can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, if not specifically stated, are all conventional experimental methods, detection methods, etc. already existing in the prior art.

[0027] The various terms and phrases used in the present application have the general meanings known to those skilled in the art.

[0028] The method of the present application will be further described below through specific examples.

[0029] Example 1: Obtaining potential high-activity and heat-resistant ancestral stonecrop polysaccharide lyase sequence based on JpPL40A

[0030] According to the description of CN 120098981 A, the inventors of the present application mined the marine bacteria Jejuia pallidiluteaA potential Ulva polysaccharide lyase JpPL40A fragment (WP_042240777.1) from the source, the gene contains 3162 bases, the sequence is shown in SEQ ID NO. 2, the encoded protein has 1053 amino acids, the sequence is shown in SEQ ID NO. 3. CN 120098981 A proves the application potential of JpPL40A in preparing Ulva oligosaccharides, but its heat resistance and catalytic efficiency are insufficient, and the present application attempts to take the amino acid sequence of JpPL40A as a template to obtain a potential ancestral Ulva polysaccharide lyase sequence with high catalytic activity and heat resistance.

[0031] The nucleotide sequence of the artificially synthesized Ulva polysaccharide lyase JpPL40A coding gene is as follows (direction 5'-3') (as shown in SEQ ID NO. 2):

[0032]

[0033] Amino acid sequence of the stone-wool polysaccharide lyase JpPL40A (SEQ ID NO: 3):

[0034]

[0035] Firstly, the amino acid sequence of wild enzyme Ulvan lyase JpPL40A was input into the online website of FireProt 2.0, the website address is: https: / / loschmidt.chemi.muni.cz / fireprotweb / , and the phylogenetic tree of its ancestral sequences was obtained Figure 1 ).

[0036] According to the phylogenetic tree of ancestral sequences, we obtained A15, A20, A21, A22 and A23 sequences belonging to the same evolutionary branch as JpPL40A;

[0037] Table 1 A15, A20, A21, A22 and A23 amino acid sequence table

[0038] .

[0039] The complex structures of JpPL40A, A15, A20, A21, A22 and A23 with Ulvan octasaccharide ligand were predicted by AlphaFold3, respectively. Then the binding energy between the receptor and the ligand was evaluated by UniDock software. As shown in Figure 2 , the absolute value of the binding energy of A21 with octasaccharide complex is greater than that of wild enzyme JpPL40A, and is the largest among the absolute values of the binding energy of the five ancestral sequences, indicating that the binding between them is easier, revealing its potential high catalytic activity. Further analysis of the hydrogen bond interaction between wild enzyme and A21 and ligand found that wild enzyme JpPL40A and ligand formed 7 hydrogen bond interactions Figure 3 , while A21 and ligand formed 9 hydrogen bond interactions Figure 4 , indicating that the binding between A21 and ligand is more firm, further revealing its potential high catalytic activity. Through the above results, we finally selected A21 for further exploration, which proved that its catalytic activity and thermal stability were indeed improved compared with the original enzyme.

[0040] Example 2 Cloning of Ulvan lyase A21

[0041] The protein encoding A21 has 1164 amino acids, and the sequence is SEQ ID NO. 1. According to the codon bias of the host Escherichia coli, the inventors optimized the DNA sequence of the gene except the 5' end 60 bp encoding signal peptide fragment, and the optimized gene sequence is shown as SEQ ID NO. 8.

[0042] The nucleotide sequence of the artificially synthesized Ulvan lyase A21 encoding gene is as follows (direction 5'-3')(as shown in SEQ ID NO. 8):

[0043]

[0044] The gene fragment shown in SEQ ID NO. 8 was artificially synthesized. The artificial gene fragment was used as a template for PCR amplification. The specific primers used in PCR are as follows:

[0045] Forward primer: 5'-CTGCAGCATCCGGTTATTTG-3', as shown in SEQ ID NO. 9;

[0046] Reverse primer: 5'-TTTCACAATCAGTTTCTGG-3', as shown in SEQ ID NO. 10.

[0047] Example 3 Construction of an expression vector carrying Ulva polysaccharide lyase A21

[0048] The target fragment amplified in Example 2 was ligated with the linearized pCold II vector using a seamless ligation kit at 50°C for 5 minutes, and then transformed into E. coli DH5a competent cells. The cells were plated on LB solid resistance plates containing 100 μg / mL ampicillin. After overnight culture in a 37°C incubator, single colonies were selected for positive clone verification. The single colonies with correct band sizes were sent to a sequencing company for sequencing. After the sequencing results were completely correct, a recombinant plasmid was obtained and named pCold- Ulva polysaccharide lyase A21, which was stored in a -20°C refrigerator for future use. al21

[0049] Example 4 Construction of an engineered bacterium containing Ulva polysaccharide lyase A21

[0050] The recombinant plasmid carrying the Ulva polysaccharide lyase gene obtained in Example 3 was transformed into E. coli BL21(DE3) competent cells using a 42°C heat shock transformation method. The cells were plated on LB solid resistance plates containing 100 μg / mL ampicillin. After overnight culture in a 37°C incubator, single colonies were selected for positive clone verification. The single colonies with correct band sizes were cultured in liquid LB medium (containing 100 μg / mL ampicillin) overnight. The bacterial liquid was preserved in 10% glycerol and stored at -80°C for long-term preservation.

[0051] Example 5 Preparation and purification of Ulva polysaccharide lyase A21

[0052] After the bacterial liquid stored in Example 4 was activated by overnight culture in LB liquid medium (containing 100 μg / mL ampicillin) at 37°C, it was transferred to a 100 mL LB flask (containing 100 μg / mL ampicillin) and cultured at 37°C and 200 rpm until the OD600 was about 0.6. Then, IPTG was added to a final concentration of 0.1 mM, and the culture was transferred to 16°C for 16 hours to express Ulva polysaccharide lyase A21. ​

[0053] After fermentation, the bacteria were collected by centrifugation at 8000 rpm for 5 min, and then washed with a certain amount of sterile water. The bacteria were collected again by centrifugation at 8000 rpm for 5 min. The bacteria were resuspended in Tris-HCl buffer at pH 8.0, and then ultrasonically broken (320 W, 3 seconds on and 3 seconds off for 30 minutes) in an ice bath. After complete breaking, the supernatant was collected by centrifugation at 8000 rpm and 4°C for 15 min, and was the crude enzyme. The expressed target gene contained a His purification tag, and therefore, Ni-NTA affinity chromatography was used for purification. Different concentrations (10, 40, 80, 200 and 500 mM) of imidazole were used for elution of the target protein. The SDS-PAGE results showed that a relatively single target protein band could be obtained by using gradient concentrations of imidazole Figure 5 ). The protein concentration of the enzyme solution after purification was determined by the R250 Coomassie brilliant blue method to be 0.50 g / L. The enzyme solution was concentrated to a protein concentration of 7.0 g / L by using a 30 kDa ultrafiltration tube and ultra-pure water as the displacement solvent, and the purified enzyme was obtained and used for determination of enzymatic properties.

[0054] Example 6 Determination of the optimal reaction conditions of Cladophora polysaccharide lyase A21

[0055] The purified enzyme JpPL40A obtained in Example 4 was used to determine the effect of temperature on its hydrolytic enzyme activity at different temperatures (30, 40, 50, 60, 70 and 80°C). The reaction substrate was 5 g / L of self-extracted Cladophora polysaccharide from Cladophora and Ulva, the pH value was 7.0, and the reaction was terminated by boiling for 10 minutes after 15 minutes of reaction. The released reducing sugar was determined by the pHBAH method, i.e., 100 μL of the reaction solution was added with 300 μL of pHBAH after termination of the reaction, and then boiled for 5 minutes for color development. After cooling to room temperature, 200 μL was taken and measured at 410 nm for the absorbance value. The production of reducing sugar was calculated according to the standard curve (D-galactose was used as the standard substance to draw the standard curve). According to the determination results, A21 showed the maximum hydrolytic activity at 40°C Figure 6 . Further, the optimal pH of A21 was determined at pH 3-10, and the results are shown in Figure 7 , indicating that the optimal reaction pH of A21 was 7.0.

[0056] Example 7 Comparison of the catalytic constants of Cladophora polysaccharide lyase A21 and wild enzyme JpPL40A in hydrolysis of Cladophora polysaccharide

[0057] The determination method of Michaelis kinetics constant of A21 is as follows: the reaction system is 200 μL, containing pH 7.0, 50 mM Tris-HCl buffer, different concentrations of stonecrop polysaccharide (0.1-10 g / L), 5 μL of pure A21 (prepared in Example 5), and the reaction is terminated by boiling for 10 minutes after reaction at 40 ℃ for 8 minutes. The released reducing sugar is determined by pHBAH method (see Example 6). The reaction rate at different concentrations is calculated according to the reaction results.

[0058] The determination method of Michaelis kinetics constant of wild enzyme JpPL40A is as follows: the reaction system is 200 μL, containing pH 8.0, 50 mM Tris-HCl buffer, different concentrations of stonecrop polysaccharide (0.1-10 g / L), 25 μL of pure JpPL40A (prepared in Example 4 of CN 120098981A), and the reaction is terminated by boiling for 10 minutes after reaction at 40 ℃ for 10 minutes. The released reducing sugar is determined by pHBAH method (see Example 6). The reaction rate at different concentrations is calculated according to the reaction results.

[0059] The catalytic constant of stonecrop polysaccharide lyase A21 and wild enzyme JpPL40A is shown in Table 2. The value of A21 is 53.9 mL / mg / s, which is nearly 4.5 times higher than that of wild enzyme; k cat / K m

[0060] Table 2 Kinetic constants of stonecrop polysaccharide lyase A21 and wild enzyme JpPL40A

[0061] .

[0062] Example 8 Comparison of thermal stability of stonecrop polysaccharide lyase A21 and wild enzyme JpPL40A

[0063] The A21 pure enzyme obtained in Example 5 was incubated at 40 ℃ for 30, 40 and 60 minutes respectively to determine the residual enzyme activity. As shown in Table 3, the results show that A21 still maintains about 31.79% relative enzyme activity after incubation at 40 ℃ for 30 minutes, while the original enzyme JpPL40A is completely inactivated after incubation at 40 ℃ for 30 minutes, indicating that the thermal stability of A21 is also improved compared with the wild enzyme. Figure 8

[0064] Example 9 TLC determination of oligosaccharide products of A21 hydrolysis of stonecrop

[0065] ​​The A21 purified enzyme obtained in Example 5 was reacted with 5 g / L of Ulva lactuca at 40 °C and pH 7.0 for 10, 30, and 40 minutes, respectively. The hydrolysis products were then determined by TLC. The TLC conditions were as follows:

[0066] Developing solvent: Prepare 36 ml of developing solvent in a fume hood according to the ratio of isopropanol / ammonia / ultrapure water = 7 / 2 / 3 (v / v / v);

[0067] Color developer: Prepare 55 ml of color developer in a fume hood according to the ratio of anhydrous ethanol / sulfuric acid = 10 / 1 (v / v).

[0068] After spotting the sample onto the TLC aluminum plate, develop it in the developing agent for 1.5 hours, then dry it with a hair dryer, spray it with a color developer, dry it again, and then develop it in a 100℃ oven for 2 minutes.

[0069] like Figure 9 As shown, A21 can hydrolyze Ulva polysaccharide into oligosaccharide in 10 minutes, and oligosaccharide accumulates and polysaccharide degrades with the reaction until the polysaccharide is almost completely degraded into Ulva oligosaccharide in 40 minutes.

[0070] The amino acid sequence of ancestral sequence A15 (SEQ ID NO:4):

[0071]

[0072] The amino acid sequence of ancestral sequence A20 (SEQ ID NO: 5):

[0073]

[0074] The amino acid sequence of ancestral sequence A22 (SEQ ID NO: 6):

[0075]

[0076] The amino acid sequence of ancestral sequence A23 (SEQ ID NO: 7):

[0077]

Claims

1. A synthetic Ulva polysaccharide lyase A21, characterized in that, The amino acid sequence of the artificial Ulva polysaccharide lyase A21 is shown as SEQ ID NO:

1.

2. An enzyme preparation, characterized in that, The enzyme preparation comprises the artificial Ulva polysaccharide lyase A21 according to claim 1.

3. A recombinant expression vector, characterized in that, The expression vector carries a gene encoding the artificial Ulva polysaccharide lyase A21 according to claim 1.

4. A recombinant engineered bacterium, characterized in that, The recombinant engineering bacteria carry a gene encoding the artificial Ulva polysaccharide lyase A21 according to claim 1.

5. The use of the artificially synthesized Ulvan lyase A21 according to claim 1, characterized in that, The application is to use the artificial Ulva polysaccharide lyase A21 to hydrolyze Ulva to prepare Ulva oligosaccharide.

6. Use according to claim 5, characterized in that, The enzymolysis conditions of the artificial Ulva polysaccharide lyase A21 are as follows: the substrate concentration is 5 g / L, the enzyme amount is 0.45 U / mL, the enzymolysis temperature is 30-50 DEG C, the pH value is 6.0-10.0, and the enzymolysis time is more than 10 minutes.

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