Alginate lyase OUC-AOSA0A2A and application thereof
By developing the new algae lysase OUC-AOSA0A2A, the problems of low lysis efficiency of algae and insufficient biological activity of the product in the prior art were solved, and the effect of efficient preparation of high-biologically active algae oligosaccharides was achieved.
Patent Information
- Application Number
- CN202510431692.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-08
- Publication Date
- 2025-06-27
AI Technical Summary
The existing algae lysases have low degradation efficiency and high cost in industrial applications, and the products are mainly oligosaccharides with polymerization degrees of 2 to 4, and lack high-biologically active algae oligosaccharides with polymerization degrees of 4 to 6.
A new algae lysase OUC-AOSA0A2A was developed. Its amino acid sequence has high enzyme activity and thermal stability, and can exhibit 6000U/ml enzyme activity at 55°C and pH 6.0-7.0. The prepared algae oligosaccharides are mainly polymerized by 4 to 6.
It realizes efficient degradation of brown algae, and prepares high concentrations of high-biologically active brown algae oligosaccharides, which reduces production costs and increases the commercial application value of the products.
Smart Images
Figure CN120210170A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to an alginate lyase OUC - AOSA0A2A and its application, belonging to the technical field of functional enzymes. Background Art
[0002] Alginate is a polysaccharide extracted from brown algae. It is mainly a high - molecular compound formed by the alternating connection of β - D - mannuronic acid (M) and α - L - guluronic acid (G) in different sequences. It is the general name of hydrophilic derivatives of alginic acid and widely exists in algae such as giant kelp, kelp, kombu, and Ascophyllum nodosum.
[0003] Alginate oligosaccharides have various biological activities, such as antiviral, anti - tumor, antioxidant, and promoting plant growth. Therefore, they can be widely applied in fields such as pharmaceuticals, health products, food additives, feed additives, and cosmetics. How to efficiently prepare alginate oligosaccharides has increasingly become a hot research topic.
[0004] The methods for preparing alginate oligosaccharides include physical methods, chemical methods, and enzymatic methods. Among them, physical methods mainly refer to the degradation of alginate by physical means such as ultrasound and microwave; chemical methods mainly include acid hydrolysis and oxidation degradation; currently, the most common degradation methods are acid hydrolysis and enzymatic hydrolysis. The enzymatic method has the advantages of mild reaction, single product, and easy separation compared with the acid method. Currently, relatively few varieties of alginate lyases reported for industrial production are available. The degradation products of high - activity alginate lyases mainly consist of oligosaccharides with a degree of polymerization of 2 - 4, and there are few alginate oligosaccharides with a degree of polymerization of 4 - 6. According to relevant literature reports, the biological activity of alginate oligosaccharides with a degree of polymerization of 4 - 6 is more obvious than that of 2 - 4. Summary of the Invention
[0005] In view of the above - mentioned prior art, the present invention provides a novel alginate lyase that can degrade alginate to prepare alginate oligosaccharides, which can efficiently degrade alginate and prepare high - concentration alginate oligosaccharides, solving the problems of low degradation efficiency and high cost in the industrial application of existing alginate lyases. The product mainly consists of alginate oligosaccharides with a degree of polymerization of 4 - 6, which has more commercial application value.
[0006] In order to achieve the above - mentioned object of the present invention, the present invention provides the following technical solutions:
[0007] In the first aspect of the present invention, an alginate lyase OUC - AOSA0A2A is provided, and its amino acid sequence is as shown below, as shown in SEQ ID NO.1.
[0008] Amino acid sequence of alginate lyase OUC - AOSA0A2A (SEQ ID NO.1): DQKNKKNVANIDLSHWTLTTPAQDPKKPGKTFDLNYPEIFDFASNETAKKYMYEDAKDKSIVFYAFPSGVSTANSHFSRSELRETMEIGNKNVNWTFAQGGYFKGTYAIEDVSKEADGKYSRVIIAQIHGILTDSQQELIGQKDKNAAPILKIFWDQGKIRVKTKVLKNQNASLKDMLPATAWGDDKGRDFKEKIDFNTKFTLEIKVSDGRLEVIMNGSESFVYEDINIKKWGVFENYFKAGNYFQSTNANTFAKVKIYDLQVSHGGGGSGGGGSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITK
[0009] The preparation method of the alginate lyase OUC - AOSA0A2A is as follows:
[0010] 1) Design and synthesize primers: The upstream primer is as shown in SEQ ID No.2, and the downstream primer is as shown in SEQ ID No.3, and perform PCR amplification;
[0011] 2) Vector construction: Transfer the ligation product into E.coli DH5a competent cells for culture to obtain a plasmid, and then transform it into the host E.coli BL21 competent cells to construct an engineering bacterium;
[0012] 3) Vector expression: Activate and culture the above - mentioned engineering bacterium, and induce expression to obtain alginate lyase.
[0013] In a second aspect of the present invention, there is provided the use of alginate lyase OUC-AOSA0A2A in degrading alginate and preparing alginate oligosaccharides.
[0014] Furthermore, the alginate oligosaccharides mainly with a degree of polymerization of 4 - 6 are obtained by preparing the alginate lyase.
[0015] The alginate lyase of the present invention has a specific enzyme activity up to 6000 U / ml under the conditions of 55 °C and pH = 6.0 - 7.0.
[0016] Furthermore, the conditions for the alginate lyase to prepare alginate oligosaccharides are as follows: the concentration of the alginate solution is 0.5%, reacting for 0.5 h - 3 h under the conditions of a temperature of 45 °C - 85 °C and a pH value of 4.0 - 10.0, and the volume ratio of the added enzyme to the solution volume is 1:3.
[0017] Furthermore, the preferred temperature for degradation is 55 °C - 75 °C, and the pH value is 7.0 - 8.0.
[0018] In a third aspect of the present invention, there is provided a coding gene for alginate lyase OUC-AOSA0A2A, characterized in that the nucleotide sequence of the coding gene is as shown in SEQ ID NO.2.
[0019] Nucleotide sequence (SEQ ID NO.2) encoding the OUC-AOSA0A2A gene:
[0020]
[0021] In the fourth aspect of the present invention, a vector is provided, characterized in that the nucleotide sequence of the vector comprises:
[0022] The nucleotide sequence SEQ ID No.2 contained in the coding gene of alginate lyase OUC-AOSA0A2A, or a nucleotide sequence with the same function obtained by substitution and / or deletion and / or addition of one or several nucleotide residues in the nucleotide sequence SEQ ID No.2 contained in the coding gene of alginate lyase OUC-AOSA0A2A.
[0023] Furthermore, the vector is a plasmid, phage, virus, host cell or genetically engineered bacterium.
[0024] In the fifth aspect of the present invention, an enzyme preparation is provided, and the enzyme preparation contains an effective dose of the alginate lyase OUC-AOSA0A2A described in claim 1.
[0025] Furthermore, the enzyme preparation also contains fillers, bovine serum albumin, calcium chloride, potassium sorbate, and sodium citrate.
[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0027] (1) The alginate lyase of the present invention has a relatively high enzyme activity and can rapidly degrade alginate to prepare alginate oligosaccharides. Its gene sequence has a low similarity with the reported gene sequences of the alginate lyase family, and there is no relevant report.
[0028] (2) The alginate lyase OUC-AOSA0A2A of the present invention has good thermal stability and can have a relatively high enzyme activity in the temperature range of 35°C - 85°C, and its activity is the highest at 65°C; the enzyme activity is relatively high in the range of pH 6.0 - 8.0.
[0029] (3) The alginate lyase of the present invention has a relatively high enzyme activity, and its enzymatic hydrolysis products are mainly low-molecular-weight alginate oligosaccharides with a polymerization degree of 4 - 6, which have higher biological activity compared with the reported high-activity enzymatic hydrolysis products with a polymerization degree of 2 - 4; at the same time, this enzyme has higher economic value in the preparation of high-concentration alginate oligosaccharides. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.
[0031] Figure 1 Study on the enzyme activity of purified alginate lyase under different temperature conditions,
[0032] Figure 2 Study on the Enzyme Activity of Purified Alginate Lyase under Different pH Conditions
[0033] Figure 3 TLC Diagram of Alginate Oligosaccharides Prepared by Degrading Alginate with Purified Alginate Lyase
[0034] Figure 4 Liquid Chromatogram of Alginate Oligosaccharides Prepared by Degrading Alginate with Purified Alginate Lyase
[0035] Figure 5 TLC Diagram of 20% Concentration Alginate Oligosaccharides Prepared by Purified Alginate Lyase Specific Embodiment
[0036] The present invention will be further described below in conjunction with embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.
[0037] The alginate lyase gene, construction of engineering bacteria, research on enzymatic hydrolysis process, and application of enzyme preparation process of the present invention will be specifically described below.
[0038] Example 1 Cloning of Alginate Lyase OUC-AOSA0A2A Gene
[0039] Based on the reported high-yield alginate lyase in the Flavobacterium sp strain, the gene fragments in the NCBI database were compared and analyzed. Gene fragments with relatively high similarity were screened out and further analyzed through the Uniprot protein database. The sequences with similarity were used to simulate and construct a protein model through swiss-model for the fragment with relatively high similarity to the Flavobacterium sp strain. However, no relevant research reports on the related functions of this fragment were available. The inventor artificially synthesized this gene and expressed it in Escherichia coli through a suitable plasmid vector. The alginate lyase gene of the present invention contains 813 base sequences as shown in SEQ ID NO.2, encoding 271 amino acids as shown in SEQ ID NO.1:
[0040] Using the synthesized fragment as a template, primers for seamless connection were designed at the upstream and downstream of the gene, and PCR was performed to amplify the alginate lyase gene fragment.
[0041] The sequences of the primers are as follows:[[]]
[0042] Upstream primer: 5’-GACCAGAAGAACAAGAAGAACGTGGCCAACATCGACCTGA-3’, as shown in SEQ ID NO.3;
[0043] Downstream primer: 5’-GGACGAGGCCCTGAAGGACGCCCAGACCAGGATCACCAAG-3’, as shown in SEQ ID NO.4.
[0044] The PCR reaction system is: 2×PCR Buffer 25 μl, dNTP 10 μl, each primer 1.5 μl, template 1 μl, KOD Fx enzyme 1 μl, sterile water 10 μl, and the total volume is 50 μl.
[0045] The reaction conditions of PCR are: pre-denaturation at 94°C for 5 min, denaturation at 95°C for 20 s, annealing at 65°C for 30 s, extension at 72°C for 60 s, 30 cycles of reaction, and post-extension at 72°C for 10 min.
[0046] Example 2 Construction of an expression vector containing the alginate lyase OUC-AOSA0A2A gene
[0047] The gene fragment was ligated to the pET-21a cloning vector using seamless cloning technology. The ligation product was transformed into competent E. coli DH5a cells and spread on a solid LB medium plate (containing 100 μg / ml sodium ampicillin). After culturing in an incubator at 37°C for 16 hours, single colonies were picked and transferred to an LB liquid medium containing 100 μg / ml sodium ampicillin, and cultured overnight in a shaker at 37°C with a rotation speed of 200 rpm. After positive verification of the single colonies, they were sequenced and named pET21a-AOSA0A2A. The plasmid was stored at -20°C for later use.
[0048] Example 3 Construction of a recombinant plasmid and engineering bacteria containing the alginate lyase OUC-AOSA0A2A gene
[0049] The plasmid extracted in Example 2 was transformed into competent host E. coli BL21 cells, and the constructed engineering bacteria grew on a sodium ampicillin resistance plate, obtaining recombinant expression strains.
[0050] Example 4 Preparation of recombinant alginate lyase using E. coli engineering bacteria
[0051] After activation of the E. coli recombinant strain in 5 ml of LB liquid medium (containing 100 μg / ml ampicillin), it was inoculated into an LB medium containing 100 μg / ml ampicillin at an inoculation amount of 1%, and cultured at 37°C and 200 rpm for 6 h. When the OD(600) value of the bacterial solution was 0.6, 1‰ IPTG (100 mM / L) was added, and induction was carried out at 20°C for 20 h to express alginate lyase.
[0052] After fermentation, 8000g centrifugation for 10 minutes to collect the cells, and the cells were resuspended in 50 mM citric acid-sodium citrate buffer with pH = 6.0. Then, it was ultrasonically disrupted in an ice-water bath for 10 min (600W, 3s on, 3s interval), and then centrifuged at 8000g for 15 min again. The supernatant was collected, which was the crude enzyme solution. Based on the His-tagged fusion protein, the crude enzyme solution was purified by affinity chromatography using a Ni-NTA column. The column was equilibrated with a low-concentration 10 mM imidazole solution (500 mM NaCl, 50 mM Tris-HCl), and then weakly bound impurities were eluted with a 20 mM imidazole solution (500 mM NaCl, 50 mM Tris-HCl), and the target protein was eluted with a 200 mM imidazole solution. The buffer elution components of this part were collected to obtain a purified recombinant alginate lyase solution. Its molecular weight was calculated and predicted to be approximately 30KDa.
[0053] Example 5 Determination of specific enzyme activity of recombinant alginate lyase OUC-AOSA0A2A
[0054] The standard method for determining the activity of recombinant alginate lyase OUC-AOSA0A2A-N4 is as follows: In a 200-μL reaction system, it contains 50 μL of enzyme solution and 150 μL of 0.3% alginate. The reaction is carried out at 50 °C for 30 min. The reaction sample is mixed with 300 μL of DNS reagent and boiled in a boiling water bath for 5 min for color development, and its absorbance is detected at OD540. The enzyme activity is defined as the amount of enzyme required to produce 1 μmol of reducing sugar per minute under standard conditions, and the specific enzyme activity reaches 6000 U / ml.
[0055] Example 6 Determination of the optimal reaction conditions of alginate lyase OUC-AOSA0A2A
[0056] The purified alginate lyase obtained in Example 4 was reacted at different temperatures and pH values to determine the effects of temperature and pH on enzyme activity. Temperatures of 35 °C, 45 °C, 55 °C, 65 °C, 75 °C, 85 °C, and 95 °C were selected, and the optimal temperature was determined by reacting for 30 min according to the method for determining the specific enzyme activity of alginate lyase OUC-AOSA0A2A in Example 5. The results are as Figure 1 . At 50 °C, buffers with pH values ranging from 4.0 to 10.0 were used as different assay pH buffers for the enzyme reaction. According to the enzyme activity of alginate lyase OUC-AOSA0A2A, the optimal pH of alginate lyase OUC-AOSA0A2A was determined. Taking the highest enzyme activity as 100%, the relative enzyme activity results under different conditions were calculated as Figure 2 shown.
[0057] Alginate lyase OUC-AOSA0A2A has good thermal stability. In the temperature range of 35°C - 85°C, the activity of alginate lyase shows an upward trend. The activity of alginate lyase is the highest at 65°C. Although the activity of alginate lyase shows a downward trend with the increase of temperature, it indicates that this alginate lyase has a wide temperature range of action and still has a high enzyme activity at a relatively high temperature, belonging to a high-temperature tolerant alginate lyase. When the pH is between 7.0 and 8.0, the enzyme activity is relatively high, and the optimal pH value for action is 7.0. In the pH range of 4.0 - 6.0, the enzyme activity shows an upward trend and has strong hydrolysis activity.
[0058] Example 7 Determination of the degradation products of alginate lyase OUC-AOSA0A2A
[0059] The purified alginate lyase OUC-AOSA0A2A obtained in Example 4 was reacted with 0.5% alginate at 50°C for 30 min, and then analyzed by TLC plate spotting as Figure 3 shown. On the left, 2, 3, 4, and 6 are disaccharide, trisaccharide, tetrasaccharide, and hexasaccharide standards. The pair is the degradation product of alginate lyase on the market, and the sample is the normal enzymatic hydrolysis product. The results show that the main products are tetrasaccharide, pentasaccharide, and hexasaccharide. The degradation products were analyzed by high-performance liquid chromatography as Figure 4 It can be seen that most of the products are alginate oligosaccharides with a degree of polymerization of 4 - 6, and their Mn is 1687 g / mol.
[0060] Example 8 Preparation of high-concentration alginate oligosaccharides using alginate lyase
[0061] The alginate lyase prepared in Example 4 was used for the preparation of high-concentration alginate oligosaccharides. The specific process is as follows: Alginate was prepared into a 5% solution, and 1 ml of crude alginate lyase solution was added to the solution at 50°C. After reacting for 0.5 h, the solution became thinner; 5% alginate was continuously added to the solution, and 2 ml of crude alginate lyase solution was added again and reacted for 1 h; the viscosity of the solution was measured. After the solution became a thin solution, 5% alginate was continuously added, and 2 ml of crude alginate lyase solution was added and reacted for 1 h, and then the solution became thinner. 5% alginate was added again for an overnight reaction. After the reaction ended, the solution became thinner, and most of the alginate was degraded into small molecules. The results of TLC were like the Figure 5 samples. On the left, 2, 3, 4, and 6 are disaccharide, trisaccharide, tetrasaccharide, and hexasaccharide standards. The degradation products are mainly alginate oligosaccharides with a degree of polymerization of 4 - 6.
[0062] Example 9 Preparation of enzyme preparation using alginate lyase OUC-AOSA0A2A
[0063] Preparing an enzyme preparation using the alginate lyase OUC-AOSA0A2A prepared in Example 4: Replace imidazole in the alginate lyase solution after fermentation and disruption with purified water, select starch as a filler with an addition amount of 15%, bovine serum albumin 1.5%, calcium chloride 1.2%, potassium sorbate 0.1%, sodium citrate 0.01% and other additives, and preserve the enzyme powder after drying. The activity of the enzyme powder can still be about 92% after being stored for 6 months.
[0064] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, for those of ordinary skill in the art, it is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions required to be protected by the present invention.
Claims
1. An alginate lyase OUC-AOSA0A2A, whose amino acid sequence is shown in SEQ ID No.
1.
2. Use of the alginate lyase OUC-AOSA0A2A according to claim 1 in degrading alginate and preparing brown algal oligosaccharides.
3. The use of the alginate lyase OUC-AOSA0A2A according to claim 2 in degrading alginate and preparing brown algal oligosaccharides, characterized in that: The brown algae oligosaccharide is mainly brown algae oligosaccharide with a polymerization degree of 4 to 6.
4. The use of the alginate lyase OUC-AOSA0A2A according to claim 3 in degrading alginate and preparing brown algal oligosaccharides, characterized in that: The degradation conditions are as follows: the concentration of the alginate solution is 0.5% to 20%, the mass to volume ratio is 0.5g to 20g / 100ml, and the reaction is carried out at a temperature of 35°C to 85°C and a pH value of 4.0 to 10.0 for 0.5h to 3h. Preferably, the temperature is 55°C to 75°C and the pH value is 6.0 to 8.
0.
5. The use of the alginate lyase OUC-AOSA0A2A according to claim 4 in degrading alginate and preparing brown algal oligosaccharides, characterized in that: The degradation process is: 1) Prepare alginate into a solution, add alginate lyase OUC-AOSA0A2A, make the mass and volume ratio of alginate to enzyme be 5g:1ml, and react for 0.5h; 2) Continue to add alginate lyase to make the mass and volume ratio of alginate to enzyme be 5g:1ml, and react for 1 hour; repeat 1 to 3 times, and then react overnight to obtain the product.
6. A gene encoding alginate lyase OUC-AOSA0A2A, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO.
2.
7. A carrier, characterized in that The nucleotide sequence of the vector contains: 1) the nucleotide sequence SEQ ID No. 2 according to claim 6, or 2) A nucleotide sequence having the same function as the nucleotide sequence SEQ ID No. 2 according to claim 6, wherein one or more nucleotide residues are substituted and / or deleted and / or added.
8. The carrier according to claim 7, characterized in that The vector is a plasmid, a bacteriophage, a virus, a host cell or a genetically engineered bacterium.
9. An enzyme preparation, characterized in that The method comprises an effective dose of the alginate lyase OUC-AOSA0A2A according to claim 1.
10. The enzyme preparation according to claim 9, characterized in that The enzyme preparation also contains fillers, bovine serum albumin, calcium chloride, potassium sorbate and sodium citrate.
Citation Information
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