Jppl40a and use thereof

Abstract

The application relates to a stone moss polysaccharide cleavage enzyme JpPL40A and application thereof, and belongs to the technical field of functional enzymes. The amino acid sequence of the stone moss polysaccharide cleavage enzyme JpPL40A is shown as SEQ ID NO. 1, and the coding gene is shown as SEQ ID NO. 2 or SEQ ID NO. 3. The stone moss polysaccharide cleavage enzyme JpPL40A can hydrolyze stone moss to obtain unsaturated stone moss disaccharide, tetrasaccharide, hexose and / or octose.

Description

Technical Field

[0001] The invention belongs to the technical field of functional enzymes, and particularly relates to an ulva polysaccharide lyase JpPL40A and an application thereof. Background Art

[0002] Ulva is a naturally occurring, large linear polysaccharide extracted from the cell walls of the green algae Ulva. It is one of the few naturally occurring polysaccharides with a high sulfate group substitution. The main monosaccharide monomers in Ulva's structure are iduronic acid (IdoA), 3-sulfated rhamnose (Rha3S), xylose (Xyl), 2-sulfated xylose (Xyl2S), and glucuronic acid (GlcA). Four disaccharide units are commonly found in the Ulva structure: [→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 sulfate groups, Ulva has multiple physiological activities such as antiviral, anti-inflammatory, anticoagulant, and antioxidant. Oligosaccharides obtained through degradation of Ulva have significantly improved solubility, stability, and various physiological activities compared to Ulva polysaccharides, and are an important direction for the high-value utilization of Ulva.

[0003] Currently, methods for preparing Ulva oligosaccharides include physical, chemical, and biological methods. Acid hydrolysis offers advantages such as rapid reaction times and the ability to process high substrate concentrations. However, the resulting products are complex and difficult to separate. Enzymatic hydrolysis offers advantages over acid hydrolysis, such as milder reactions, more homogeneous products, and easier separation. Therefore, enzymatic methods are environmentally friendly and have promising sustainable applications. The discovery of Ulva polysaccharide lyases for the targeted preparation of Ulva oligosaccharides with specific structures is of great significance. Ulva polysaccharide lyases degrade Ulva to produce unsaturated oligosaccharides, typically designated as ∆-Rha3S. Currently, research on Ulva polysaccharide lyases has primarily focused on the PL24, PL25, and PL28 families, with the primary products being tetrasaccharides or disaccharides. Research on Ulva polysaccharide lyases that produce larger products is severely lacking, limiting the types of oligosaccharides that can be prepared. Therefore, further expression and characterization of unknown Ulva polysaccharide lyases and development of their potential for the preparation of Ulva polysaccharide lyases with different polymerization degrees are of great significance for in-depth exploration of the structure-activity relationship of oligosaccharides. SUMMARY

[0004] In view of the prior art, the present application provides a stone crack polysaccharide lyase JpPL40A which can degrade stone, and the main hydrolysis product is unsaturated stone six sugar, which can efficiently prepare unsaturated stone six sugar, and has important significance for solving the problem of cracking stone oligosaccharide with medium and long degree of polymerization, and has wide application prospect.

[0005] The present application is realized by the following technical solutions:

[0006] One of the purposes of the present application is to provide a stone crack polysaccharide lyase JpPL40A, and the amino acid sequence of the stone crack polysaccharide lyase JpPL40A is shown in SEQ ID NO. 1.

[0007] The second purpose of the present application is to provide an application of stone crack polysaccharide lyase JpPL40A, and the application is to crack stone by using the stone crack polysaccharide lyase JpPL40A to obtain unsaturated stone disaccharide, tetrasaccharide, hexose and / or octose, and the amino acid sequence of the stone crack polysaccharide lyase JpPL40A is shown in SEQ ID NO. 1.

[0008] The third purpose of the present application is to provide an application of stone crack polysaccharide lyase JpPL40A gene, and the application is to use the stone crack polysaccharide lyase JpPL40A gene to prepare an enzyme capable of cracking stone to obtain unsaturated stone disaccharide, tetrasaccharide, hexose and / or octose, and the stone crack polysaccharide lyase JpPL40A gene is shown in SEQ ID NO. 2 or SEQ ID NO. 3, and the coding protein of the stone crack polysaccharide lyase JpPL40A gene is shown in SEQ ID NO. 1.

[0009] As a preferred embodiment, the cracking stone condition is that the substrate concentration is 1-4 g / L, the enzyme addition amount is 0.02-0.32 U / mL, the enzyme hydrolysis temperature is 20-50 DEG C, the pH value is 6.0-10.0, and the enzyme hydrolysis time is more than 10 minutes. As a more preferred embodiment, the substrate concentration is 1-4 g / L, the enzyme addition amount is 0.08 U / mL, the enzyme hydrolysis temperature is 40 DEG C, the pH value is 8.0, and the enzyme hydrolysis time is 12 hours.

[0010] The fourth purpose of the present application is to provide an enzyme preparation containing stone crack polysaccharide lyase JpPL40A, and the amino acid sequence of the stone crack polysaccharide lyase JpPL40A is shown in SEQ ID NO. 1.

[0011] The fifth purpose of the present application is to provide the application of the enzyme preparation in hydrolyzing stone, and the application is to use the enzyme preparation to prepare unsaturated stone disaccharide, tetrasaccharide, hexose and / or octose.

[0012] The amino acid sequence of the Ulva pertusa polysaccharide lyase JpPL40A is as follows (SEQ ID NO: 1):

[0013]

[0014] The nucleotide sequence of the gene encoding the Ulva polysaccharase JpPL40A (direction 5'-3') (SEQ ID NO: 2):

[0015]

[0016] Compared with the prior art, the application has the beneficial effects: through experimental research, it is found that the stonecrop polysaccharide lyase JpPL40A can degrade stonecrop under the condition of 40 DEG C and pH value of 8.0, and the main product composition is unsaturated stonecrop hexasaccharide, and the unsaturated stonecrop hexasaccharide can be specifically prepared.

[0017] The application constructs an expression vector containing the stonecrop polysaccharide lyase JpPL40A gene, realizes the heterologous expression in E. coli, and provides a good foundation for the industrialized large-scale production of the enzyme. The optimal reaction temperature of the stonecrop polysaccharide lyase JpPL40A is 40 DEG C, and the stonecrop serving as the substrate can keep a solution state at the temperature, so that the large-scale application of the stonecrop polysaccharide lyase can be better realized. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is an SDS-PAGE electrophoretogram for purification of the stonecrop polysaccharide lyase JpPL40A of the application, wherein lane M represents a protein Maker, and lane P represents a pure enzyme;

[0019] Figure 2 It is a line graph for influence of temperature change on hydrolytic enzyme activity of the stonecrop polysaccharide lyase JpPL40A;

[0020] Figure 3 It is a line graph for influence of pH change on hydrolytic enzyme activity of the stonecrop polysaccharide lyase JpPL40A;

[0021] Figure 4 It is a line graph for residual enzyme activity of incubation for 1 hour under different temperatures;

[0022] Figure 5 It is an HPLC graph for stonecrop product hydrolyzed by 0.02 U / mL stonecrop polysaccharide lyase JpPL40A;

[0023] Figure 6 It is an HPLC graph for stonecrop product hydrolyzed by 0.08 U / mL stonecrop polysaccharide lyase JpPL40A;

[0024] Figure 7 It is an HPLC graph for separation of stonecrop product hydrolyzed by the stonecrop polysaccharide lyase JpPL40A;

[0025] Figure 8 It is a mass spectrum graph for separation product T1 of stonecrop hydrolyzed by the stonecrop polysaccharide lyase JpPL40A;

[0026] Figure 9 It is a mass spectrum graph for separation product T2 of stonecrop hydrolyzed by the stonecrop polysaccharide lyase JpPL40A;

[0027] Figure 10Mass spectrum of the product T3 separated from Ulva lactuca by the Ulva lactuca polysaccharide lyase JpPL40A;

[0028] Figure 11 Mass spectrum of the product T4 separated from Ulva lactuca by the Ulva lactuca polysaccharide lyase JpPL40A;

[0029] Figure 12 Mass spectrum of the product T5 separated from Ulva lactuca by the Ulva lactuca polysaccharide lyase JpPL40A. DETAILED DESCRIPTION

[0030] The application will be further described in conjunction with the following examples. However, the scope of the 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 application without departing from the spirit and scope of the application.

[0031] The instruments, reagents, materials and the like involved in the following examples are all conventional instruments, reagents, materials and the like existing in the prior art if not specifically stated, which can be obtained through regular commercial channels. The experimental methods, detection methods and the like involved in the following examples are all conventional experimental methods, detection methods and the like existing in the prior art if not specifically stated. Various terms and phrases used in the application have the general meanings known to those skilled in the art.

[0032] The method of the application will be further described in conjunction with the following specific examples.

[0033] Example 1 Cloning of Ulva lactuca polysaccharide lyase JpPL40A

[0034] The inventors of the present application mined a potential Ulva lactuca polysaccharide lyase fragment (WP_042240777.1) from a marine bacterium Jejuia pallidilutea According to phylogenetic tree and multiple sequence comparison analysis, it was found that the Ulva lactuca polysaccharide lyase expressed by the gene fragment belongs to the PL40 family, and the gene fragment has no homology with the Ulva lactuca polysaccharide lyases disclosed in the prior art, which illustrates the novelty of the mined sequence of the present application. The gene comprises 3162 bases, and the sequence is shown in SEQ ID NO. 2. The encoded protein has 1053 amino acids, and the sequence is shown in SEQ ID NO. 1. The inventors optimized the DNA sequence of the gene except for the 5' end 60 bp encoding signal peptide fragment according to the codon bias of the host Escherichia coli. The optimized gene sequence is shown in SEQ ID NO. 3.

[0035] The nucleotide sequence of the artificially synthesized Ulva lactuca polysaccharide lyase JpPL40A encoding gene is shown below (direction 5'-3') (as shown in SEQ ID NO. 3):

[0036]

[0037] The gene fragment shown in SEQ ID NO. 3 was artificially synthesized. PCR amplification was performed using the artificially synthesized gene fragment as a template. The specific primers used in PCR are as follows:

[0038] Forward primer: 5'-CTGGAACATCCGGTTATTTGG-3', as shown in SEQ ID NO. 4.

[0039] Reverse primer: 5'-GTCAACAATCAGTTTCTGGG-3', as shown in SEQ ID NO. 5.

[0040] Example 2 Construction of an expression vector carrying the Ulva polysaccharide lyase JpPL40A

[0041] The target fragment obtained by amplification in Example 1 was transformed into E. coli DH5a competent cells after reaction at 50°C for 5 minutes using a seamless assembly kit, and then coated on LB solid resistance plates containing 100 μg / mL ampicillin. After overnight culture in a 37°C incubator, single colonies were picked 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, named pCold- JpPL40A, and stored in a -20°C refrigerator for standby use. jpPL40A

[0042] Example 3 Construction of an engineering bacterium containing Ulva polysaccharide lyase JpPL40A

[0043] The recombinant plasmid carrying the Ulva polysaccharide lyase gene obtained in Example 2 was transformed into E. coli BL21(DE3) competent cells using a 42°C heat shock transformation method. The cells were coated on LB solid resistance plates containing 100 μg / mL ampicillin. After overnight culture in a 37°C incubator, single colonies were picked 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.

[0044] Example 4 Preparation and purification of Ulva polysaccharide lyase JpPL40A

[0045] ​The bacteria liquid saved in Example 3 was activated by overnight culture in LB liquid medium (containing 100 μg / mL ampicillin) at 37 ℃, then transferred to 100 mL LB flask (containing 100 μg / mL ampicillin) for culture at 37 ℃, 200 rpm until OD600 was about 0.6, then IPTG was added to a final concentration of 0.1 mM, and the culture was shifted to 18 ℃ for 16 hours to express the Ulva polysaccharide lyase JpPL40A.

[0046] After fermentation, the bacteria were collected by centrifugation at 8000 rpm for 5 minutes, washed with a certain amount of sterile water, and then collected again by centrifugation at 8000 rpm for 5 minutes. The bacteria were resuspended in Tris-HCl buffer at pH 8.0 and then broken by ultrasonic treatment (320 W, 3 seconds on and 3 seconds off for 30 minutes) in an ice bath. After complete breakage, the supernatant was collected by centrifugation at 8000 rpm and 4 ℃ for 15 minutes, which was the crude enzyme. The expressed target gene contained a His purification tag, so we used Ni-NTA affinity chromatography for purification. Different concentrations of imidazole (10, 20, 50, 80, 120, 200, and 500 mM) were used to elute the target protein, and the Akta purification instrument was used for purification. The SDS-PAGE results showed that a relatively single target protein band could be obtained by gradient concentration of imidazole Figure 1 ). The protein concentration of the enzyme solution after purification was determined to be 0.04 g / L by the R250 Coomassie brilliant blue method. The enzyme solution was concentrated to a protein concentration of 1.0 g / L using a 50 kDa ultrafiltration tube and ultrapure water as the displacement solvent to obtain pure enzyme for determination of enzymatic properties.

[0047] Example 5 Determination of the optimal reaction conditions of Ulva polysaccharide lyase JpPL40A

[0048] The JpPL40A pure enzyme obtained in Example 4 was used to determine the effect of temperature on its hydrolytic enzyme activity at different temperatures (20, 30, 40, 50, 60, and 70 ℃). The reaction substrate was 4 g / L Ulva, and the pH value was 7.0. The reaction was terminated after boiling for 10 minutes. The released reducing sugar was determined by the pHBAH method, i.e. 100 μL of 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 for determination of the absorbance value at 405 nm. 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, JpPL40A showed the maximum hydrolytic activity at 40 ℃ Figure 2 ). Further determination of the optimal pH of JpPL40A at pH 3-10 showed that the optimal reaction pH was 8.0 as shown in Figure 3 .

[0049] Example 6 Determination of the specific enzyme activity of Ulva polysaccharide lyase JpPL40A

[0050] The enzyme activity was determined by a 200 μL reaction system containing 50 mM Tris-HCl buffer, pH 8.0, 4 g / L Ulva, and 1.0 mg of pure JpPL40A (prepared in Example 4). The reaction was incubated at 40°C for 10 minutes, then boiled for 10 minutes to terminate the reaction. The reducing sugars released were determined using the pHBAH method (see Example 5).

[0051] Enzyme activity is defined as the amount of enzyme required to convert 1 μmol of reducing sugar into sugar in 1 minute under optimal reaction conditions.

[0052] The specific enzyme activity of Ulva polysaccharide lyase JpPL40A in hydrolyzing Ulva was determined to be 0.64 U / mg.

[0053] Example 7 Determination of the Thermal Stability of Ulva Polysaccharide Lyase JpPL40A

[0054] The pure JpPL40A enzyme obtained in Example 4 was incubated at 20, 30, 40, 50, 60, 70 and 80°C for 60 minutes and then the residual enzyme activity was measured. Figure 4 As shown in Figure 3, the results showed that JpPL40A still maintained a relative enzyme activity of about 68.92% after incubation at 30 °C for 60 min.

[0055] Example 8 Determination of the hydrolysis products of Ulva salsa by JpPL40A

[0056] The pure JpPL40A enzyme obtained in Example 4 was reacted with 4 g / L of Ulva at 40°C and pH 8.0 for 12 hours and the hydrolysis product was determined by HPLC. Figure 5 As shown in the figure, no oligosaccharide products were generated at 0 minutes. As the reaction proceeded, peaks P2 and P3 were generated first and gradually accumulated, and finally five oligosaccharide peaks P1, P2, P3, P4 and P5 were generated, of which P2 and P3 were the main products. Figure 6 As shown in the figure, no oligosaccharide products were generated at 0 minutes, and six oligosaccharide peaks of P1, P2, P3, P4, P5 and P6 were finally produced, among which P2 and P3 were still the main products.

[0057] Due to the lack of Ulva oligosaccharide standards on the market, it is impossible to determine the specific oligosaccharides of the six oligosaccharide peaks P1, P2, P3, P4, P5 and P6. We used gel chromatography to separate them, such as Figure 7As shown, five tubes of oligosaccharides were obtained in total, T1 corresponds to P1, T2 contains both P2 and P3, T3 corresponds to P4, T4 corresponds to P5, and T5 corresponds to P6. Further, mass spectrometry was used to detect T1, T2, T3, T4 and T5: as shown, Figure 8 T1 is an unsaturated octasaccharide; as shown, Figure 9 T2 is an unsaturated hexasaccharide; as shown, Figure 10 and Figure 11 T3 and T4 are both unsaturated tetrasaccharides; as shown, Figure 12 T5 is an unsaturated disaccharide. Thus, it is shown that JpPL40A cleaves Ulva lactuca to produce unsaturated disaccharides, tetrasaccharides, hexasaccharides and octasaccharides, among which the unsaturated hexasaccharide is the main product.

Claims

1. An application of Ulva polysaccharide lyase JpPL40A, characterized in that: The application is to use the ulva polysaccharide lyase JpPL40A to lyse ulva to obtain unsaturated ulva disaccharides, tetrasaccharides, hexasaccharides and / or octasaccharides. The amino acid sequence of the ulva polysaccharide lyase JpPL40A is shown in SEQ ID NO.

1.

2. Use of a gene encoding Ulva polysaccharide lyase JpPL40A, characterized in that: The application is to use the Ulva polysaccharide lyase JpPL40A gene to prepare an enzyme capable of lysing Ulva to obtain unsaturated Ulva disaccharides, tetrasaccharides, hexasaccharides and / or octasaccharides. The Ulva polysaccharide lyase JpPL40A gene is shown as SEQ ID NO.2 or SEQ ID NO.3, and the protein encoded by the Ulva polysaccharide lyase JpPL40A gene is shown as SEQ ID NO.

1.

3. The use according to claim 1 or 2, characterized in that The conditions for cracking Ulva are: substrate concentration of 1-4 g / L, enzyme amount of 0.02-0.32 U / mL, enzymatic hydrolysis temperature of 20-50 °C, and pH of 6.0-10.

0.

4. The use according to claim 3, characterized in that The substrate concentration was 1-4 g / L, the enzyme dosage was 0.08 U / mL, the enzymatic hydrolysis temperature was 40°C, the pH value was 8.0, and the enzymatic hydrolysis time was 12 hours.

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