A brown algin lyase and its use in preparation of guluronate oligosaccharides

Abstract

The application discloses a brown algae pectin lyase and application thereof in preparation of guluronate oligosaccharide, and belongs to the technical field of biotechnology, and particularly relates to a brown algae pectin lyase and application thereof in preparation of guluronate oligosaccharide. The protein of the application is any one of the following: 1) a protein with the amino acid sequence of sequence 1 in the sequence list; 2) a protein with the amino acid sequence of the 22th to 453th of sequence 1 in the sequence list. The application purifies a recombinant brown algae pectin lyase, and the specific enzyme activity of the purified enzyme is 86.1 U / mg, and the recovery rate is 76%. The optimal pH of the brown algae pectin lyase VaAly7 is 7.0, and the brown algae pectin lyase is stable between pH 4.0 and 9.5; the optimal temperature is 30 DEG C, and the brown algae pectin lyase is stable below 30 DEG C, and is a cold-adapted brown algae pectin lyase, and has the advantages of easy control and energy saving in production and application, and has application potential in production of guluronate oligosaccharide.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an alginate lyase and its application in the preparation of guluronic acid oligosaccharides. Background Technology

[0002] Alginate is a linear polysaccharide formed by the polymerization of β-D-mannuronate (M) and α-L-guluronate (G) through 1,4-glycosidic bonds. Based on monomer composition, alginate can be classified into three types: polymannuronate (poly M), polyguluronate (poly G), and heteropolysaccharides composed of both monomers (polyMG). Alginate oligosaccharides (AOS) are degradation products of alginate and possess various biological activities, such as antibacterial, antioxidant, antitumor, immunomodulatory, prebiotic activity, and plant growth promotion, showing great application potential in agriculture, biomedicine, and the food industry. Production methods for alginate oligosaccharides include physical, chemical, and enzymatic methods. The preparation of alginate oligosaccharides using alginate lyases has attracted widespread attention due to its advantages of high efficiency, environmental friendliness, and controllable product yield. Studies have shown that the unique functions of alginate oligosaccharides (AOS) are closely related to their specific structures, including molecular weight (Mw), degree of polymerization (DP), M / G ratio, monomer sequence, and terminal saturation. Currently, most research and applications of AOS are based on mixtures of AOS, with few reports on the preparation of guluronic acid oligosaccharides, leading to uncertainty regarding their bioactivity. Therefore, the targeted preparation of alginate oligosaccharides with specific monosaccharide compositions is crucial for studying their bioactivity.

[0003] Alginate lyases have been isolated from various organisms, but wild-type strains produce low yields, insufficient for industrial production. Heterologous expression has become a common method to increase enzyme production. Most alginate lyase genes are expressed in *E. coli*, such as those from... Rubrivirga marina The alginate lyase AlyRm1 has been successfully expressed in *E. coli*. Alteromonas portus The refractory-adaptive alginate lyase Alg2951 of HB161718T was successfully expressed in *E. coli*, with an extracellular enzyme activity of 63.6 U / mL. Pichia pastoris expression systems have significant advantages in the production of many recombinant proteins, such as high protein secretion capacity, simple product isolation, and absence of endotoxins; however, currently only a small number of alginate lyase genes have been successfully expressed in *Pichia pastoris*. Summary of the Invention

[0004] The main problem to be solved by this invention is how to efficiently degrade polyguluronic acid to prepare guluronic acid oligosaccharides.

[0005] To solve the above problems, the present application provides a protein.

[0006] The protein provided by the present application is any one of the following proteins:

[0007] 1) a protein with the amino acid sequence of SEQ ID NO: 1 in the sequence listing;

[0008] 2) a protein with the amino acid sequence of positions 22 to 453 of SEQ ID NO: 1 in the sequence listing;

[0009] 3) a protein obtained by substitution and / or deletion and / or addition of one or more amino acid residues in the protein of 1) or 2) and having the same function;

[0010] 4) a fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of 1) or 2) or 3).

[0011] To facilitate the purification or detection of the protein in 1), a tag protein can be connected to the amino-terminal or carboxyl-terminal end of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing.

[0012] The above-mentioned protein can be artificially synthesized, or a gene encoding the same can be synthesized first and then expressed biologically.

[0013] The tag protein includes but is not limited to a GST (glutathione S-transferase) tag protein, a His6 tag protein (His-tag), an MBP (maltose binding protein) tag protein, a Flag tag protein, a SUMO tag protein, an HA tag protein, a Myc tag protein, an eGFP (enhanced green fluorescent protein), an eCFP (enhanced cyan fluorescent protein), an eYFP (enhanced yellow green fluorescent protein), an mCherry (monomeric red fluorescent protein), or an AviTag tag protein.

[0014] The above-mentioned protein is alginate lyase, named VaAly7.

[0015] A person of ordinary skill in the art can easily mutate the nucleotide sequence encoding the protein VaAly7 of the present application using known methods, such as methods of directed evolution or point mutation. Those nucleotides artificially modified, having 75% or more identity with the nucleotide sequence of the protein VaAly7 isolated from the present application, as long as they encode the protein VaAly7 and have the function of the protein VaAly7, are derived from the nucleotide sequence of the present application and equivalent to the sequence of the present application.

[0016] The above-mentioned 75% or more identity can be 80%, 85%, 90%, or 95% or more identity.

[0017] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.

[0018] In this document, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0019] In this document, the above 90% identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0020] In this article, the protein is derived from Vibrio alginolyticus (Villus alginolyticus). Vibrio alginolyticus ).

[0021] The present invention also provides nucleic acid molecules encoding the proteins described above.

[0022] The nucleic acid molecule may be a DNA molecule as described below:

[0023] 1) The nucleotide sequence is the DNA molecule of sequence 2 in the sequence listing;

[0024] 2) The nucleotide sequence is the DNA molecule with positions 64-1362 of sequence 2 in the sequence listing;

[0025] 3) A DNA molecule that has 85% or more identity with the nucleotide sequence defined in 1) and encodes the protein described above;

[0026] 4) A DNA molecule that hybridizes to the nucleotide sequence defined in 1) or 2) under strict conditions and encodes the protein described above.

[0027] In the present application, the sequence SEQ ID No. 2 consists of 1362 nucleotides, the sequence SEQ ID No. 1 consists of 453 amino acids, the nucleotide sequence shown in SEQ ID No. 2 in the sequence list encodes the amino acid sequence shown in SEQ ID No. 1 in the sequence list.

[0028] The present application also provides a biological material containing the nucleic acid molecule described above,

[0029] The biological material is any one of the following:

[0030] B1) an expression cassette containing the DNA molecule described above;

[0031] B2) a recombinant vector containing the DNA molecule described above, or a recombinant vector containing the expression cassette of B1);

[0032] B3) a recombinant microorganism containing the DNA molecule described above, or a recombinant microorganism containing the expression cassette of B1), or a recombinant microorganism containing the recombinant vector of B2).

[0033] In the above biological material, the recombinant vector is a recombinant vector obtained by inserting the nucleotide sequence from the 64th to the 1362nd nucleotide of sequence 2 into the multiple cloning site of the vector pPIC9K; the recombinant bacteria are recombinant Pichia pastoris obtained by introducing the recombinant vector into yeast; and the yeast is Pichia pastoris.

[0034] The recombinant bacteria can be obtained by introducing the recombinant vector into a host microorganism.

[0035] The host microorganism can be yeast, bacteria, algae or fungi. The yeast is Pichia pastoris GS115. Pichia pastoris )GS115.

[0036] The present application also provides a method for preparing the protein described above, which comprises introducing a DNA molecule capable of expressing the protein described above into a target microorganism, obtaining a recombinant microorganism, culturing the recombinant microorganism, collecting the supernatant of the fermentation broth, and obtaining the protein.

[0037] In the above method for preparing the protein, the culturing of the recombinant microorganism comprises the following steps: 1) inoculating the recombinant microorganism into a fermentation medium for culture, adding glycerol when the glycerol is completely consumed, and ensuring that the dissolved oxygen content in the fermentation system is 20%-80%; 2) stopping the addition of glycerol when the wet weight of the bacterial cells reaches 180-220 g / L; 3) starving for 30-60 min, and then adding 100% methanol to induce the culture, thereby obtaining the protein. In the above preparation method, the recombinant microorganism is recombinant Pichia pastoris, and the recombinant Pichia pastoris contains a recombinant vector capable of expressing the protein described above.

[0038] The application also provides a preparation method of guluronate oligosaccharide, which is prepared by using polyguluronate as a substrate and performing an enzymatic reaction on the substrate by using the protein as described above to obtain the guluronate oligosaccharide.

[0039] In a specific embodiment, the preparation method can be:

[0040] 1) a method of adding enzyme first and then adding substrate, in which the alginate lyase VaAly7 is used to degrade polyguluronate to obtain an enzymatic hydrolysate;

[0041] 2) centrifuging the enzymatic hydrolysate to collect supernatant and precipitate respectively;

[0042] 3) collecting the supernatant obtained by centrifugation through a 0.22 μm microporous filter membrane and collecting the filtrate, and freeze-drying to constant mass.

[0043] Further, the conditions for degrading the polyguluronate are 30 ℃, 200 rpm, a substrate concentration of 10% (w / v), an enzyme amount of 100 U / g, and an enzymatic hydrolysis time of 6 h.

[0044] The application also provides applications of the protein and / or the biological material and / or the preparation method as described above in any one of the following:

[0045] 1) an application of preparing alginate lyase;

[0046] 2) an application of preparing a product containing alginate lyase activity;

[0047] 3) an application of preparing alginate oligosaccharide;

[0048] 4) an application of degrading polyguluronate;

[0049] 5) an application of preparing guluronate oligosaccharide.

[0050] In summary, the application provides an alginate lyase VaAly7, a coding gene thereof and related applications, the coding gene of the alginate lyase VaAly7 can be efficiently and stably expressed in a recombinant host (especially Pichia pastoris) by constructing a recombinant vector by using the coding gene and introducing the vector into the host, and the alginate lyase VaAly7 is obtained.

[0051] As an example of recombinant Pichia pastoris, the enzyme activity of the obtained enzyme can reach 413 U / mL, the protein content can reach 7.1 mg / mL, and the wet weight of the bacteria can reach 307.1 g / L. The purified recombinant alginate lyase has a specific enzyme activity of 86.1 U / mg, and the recovery rate is 76%. The optimal pH of alginate lyase VaAly7 is 7.0, and it is stable between pH 4.0-9.5; the optimal temperature is 30 DEG C, and it is stable below 30 DEG C, which is a cold-adapted alginate lyase. The new cold-adapted alginate lyase has application potential in the production of guluronate oligosaccharides. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 It is the enzyme production process chart of recombinant Pichia pastoris in 5 L fermenter high-density fermentation A and SDS-PAGE electrophoresis chart B. In A, (▲) enzyme activity, (■) protein concentration, (●) cell wet weight; in B, column M is low molecular weight standard protein, columns 1-8 are fermentation supernatant induced for 0, 24, 48, 72, 96, 120, 144 and 168 h, respectively.

[0053] Figure 2 It is the alginate lyase VaAly7 purification electrophoresis chart (column M: low molecular weight standard protein; column 1: crude enzyme liquid; column 2: pure enzyme liquid; column 3: alginate lyase treated by deglycosylation enzyme).

[0054] Figure 3 It is the optimal pH determination curve of alginate lyase VaAly7 (■: citrate buffer (pH 3.0-6.0), ●: PB buffer (pH 6.0-8.0), ▲: Tris-HCl buffer (pH 6.0-9.0), ▼: CHES buffer (pH 8.0-10.0), and ♦: CAPS buffer (pH 10.0-11.0)).

[0055] Figure 4 It is the pH stability determination curve of alginate lyase VaAly7 (■: citrate buffer (pH 3.0-6.0), ●: PB buffer (pH 6.0-8.0), ▲: Tris-HCl buffer (pH 6.0-9.0), ▼: CHES buffer (pH 8.0-10.0), and ♦: CAPS buffer (pH 10.0-11.0)).

[0056] Figure 5 It is the optimal temperature determination curve of alginate lyase VaAly7.

[0057] Figure 6The temperature stability curve of alginate lyase VaAly7 is shown in the figure.

[0058] Figure 7 The graph shows the half-life of the alginate lyase VaAly7.

[0059] Figure 8 The effect of NaCl on the alginate lyase VaAly7.

[0060] Figure 9 TLC analysis of alginate degradation (A), poly G and poly M (B) and guluronic acid oligosaccharides G2-G5 (C) by alginate lyase VaAly7.

[0061] Figure 10 This is a circular dichroic chromatogram of polyuronic acid.

[0062] Figure 11 Composition (A) and molecular weight (B) of polyuronic acid degradation products. Detailed Implementation

[0063] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0064] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0065] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0066] In the embodiments of the present invention, the enzyme activity of alginate lyase was determined using the 3,5-dinitrosalicylic acid (DNS) method. Unless otherwise specified, the determination steps are as follows: 100 μL of appropriately diluted enzyme solution was added to 900 μL of 0.3% alginate solution (prepared using alginate and pH 7.0, 50 mM Tris-HCl buffer), reacted at 30 °C for 10 min, 750 μL of DNS was added to terminate the reaction, the mixture was boiled for 10 min, and the absorbance was measured at 540 nm. Using glucose as a standard, the amount of reducing sugar produced was calculated. Under the above reaction conditions, the amount of enzyme required to produce 1 μmol of reducing sugar per minute was defined as one enzyme activity unit (U).

[0067] The following examples use Origin 2022 statistical software to process the data, and the experimental results are expressed as mean ± standard deviation.

[0068] Example 1: Alginate lyase gene VaAly7 Cloning and expression

[0069] one, VaAly7 PCR amplification of genes

[0070] alginate lyase gene VaAly7 The coding region is 1299 bp, and its sequence is shown as positions 64 to 1362 of SEQ ID No. 2. The amino acid sequence encoding alginate lyase (VaAly7) is shown as positions 22 to 453 of SEQ ID No. 1.

[0071] artificial synthesis VaAly7 The gene was amplified by PCR using primers designed with positions 64 to 1362 of SEQ ID No. 2 as templates. The PCR conditions were: 94 °C pre-denaturation for 5 min; 94 °C denaturation for 30 s, 55 °C annealing for 30 s, 72 °C extension for 90 s, 30 cycles; and 72 °C extension for 5 min. The PCR product VaAly7 was recovered by 1% agarose gel electrophoresis.

[0072] II. Construction of the Recombinant Carrier

[0073] The alginate lyase gene obtained in step one VaAly7 The expression vector pPIC9K of Pichia pastoris (purchased from Invitrogen, USA) was used via... EcoR I and Not After double digestion with enzyme I, the recombinant plasmid pPIC9K-VaAly7 was obtained by ligation with T4 DNA ligase.

[0074] The recombinant plasmid pPIC9K-VaAly7 was transformed into *E. coli* DH5α for amplification and verification. The structure of the recombinant plasmid pPIC9K-VaAly7 is described below: it is derived from the originating vector pPIC9K... EcoR I and Not The recombinant vector is obtained by inserting a DNA fragment from positions 64 to 1362 of SEQ ID No. 2 between the two restriction sites, while keeping the other sequences of the pPIC9K vector unchanged. The pPIC9K-VaAly7 vector can express the VaAly7 protein, whose amino acid sequence is from positions 22 to 453 of SEQ ID No. 1.

[0075] The obtained recombinant vector pPIC9K-VaAly7 was used with restriction endonuclease SacAfter linearization, *Pichia pastoris* GS115 (purchased from Invitrogen, USA, catalog number C18100) was electroporated to form the recombinant strain GS115 / pPIC9K-VaAly7. The electroporation conditions were: voltage 2 kV, capacitance 25 μF, resistance 200 Ω, and electroporation time 5–10 ms. The electroporation process was as follows: 80 μL of pre-cooled *Pichia pastoris* was thawed on ice... P. pastoris Add approximately 10 μg of linearized recombinant plasmid to GS115 competent cells and mix well. Place the cells in an ice-cold electroporation cuvette for 5 min, insert the cuvette into an electroporator and electroporate for approximately 5 ms. Then add approximately 1 mL of pre-cold 1 mM sorbitol solution and mix quickly to obtain the recombinant strain GS115 / pPIC9K-VaAly7.

[0076] III. Recombinant bacteria expressing alginate lyase VaAly7

[0077] 1. Inoculate the recombinant bacteria GS115 / pPIC9K-VaAly7 obtained in step two above onto MD plates (1.34% YNB, 4×10⁻⁶). -5 His was obtained by culturing in a solution of 1% biotin and 2% glucose at 30 °C for 3-5 days. + Transformer.

[0078] 2. After completing step 1, scrape the transformants with sterile water and take 100 μL to spread on YPD plates with different G418 concentrations (1% yeast extract, 2% tryptone, 2% glucose, G418 concentrations of 1, 2, 3 and 4 mg / mL, respectively), and incubate at 30 ℃ for 3-5 days.

[0079] 3. After completing step 2, inoculate the transformants into BMGY medium (1% yeast extract, 2% tryptone, 100 mM pH 6.0 phosphate buffer, 1.34% YNB, 4×10⁻⁶ ppm). -5 Incubate with 1% biotin and 1% glycerol at 30°C and 200 rpm for 16-18 hours on a shaker.

[0080] 4. After completing step 3, centrifuge the culture medium at 8000 rpm for 5 min, collect the bacterial cells, and transfer them to BMMY medium (1% yeast extract, 2% tryptone, 100 mM pH 6.0 phosphate buffer, 1.34% YNB, 4×10⁻⁶ ppm). -5 Resuspend the bacterial cells to OD500 using 1% biotin and 0.5% methanol. 600 The concentration was approximately 1.0. The culture was carried out at 30 °C and 200 rpm, with methanol added every 24 h to a final concentration of 0.5% (v / v). After 72 h, 1 mL of culture was aspirated and centrifuged at 10000 rpm for 10 min.

[0081] 5. After step 4, take the supernatant to determine the enzyme activity.

[0082] IV. High-density fermentation of recombinant Pichia pastoris to produce alginate lyase

[0083] 1. Seed liquid culture: inoculate the recombinant Pichia pastoris strain GS115 / pPIC9K-VaAly7 with the highest enzyme production level obtained in step three above into 150 mL YPD medium, and cultivate at 30°C and 200 rpm for overnight until the OD 600 is 8-10 to obtain the seed liquid.

[0084] 2. Basic culture: inoculate the seed liquid of step 1 into a 5 L fermenter containing 1.35 L basic medium. The basic medium formula is: CaSO40.93 g / L, K2SO418.2 g / L, MgSO4·7H2O 14.9 g / L, KOH 4.13 g / L, glycerol 40 g / L, 85% H3PO426.7 mL / L, and PTM1 salt 4.375 mL / L. The medium temperature is 30°C, the pH is 4.0, the rotation speed is 600 rpm, and the glycerol is added when the dissolved oxygen is increased to more than 80% to indicate that the glycerol is completely consumed.

[0085] 3. Glycerol feeding culture: feed the glycerol with a mass concentration of 50% (w / v) at a temperature of 30°C and a pH of 5.0, and keep the dissolved oxygen at 20%-80% by adjusting the feeding speed for 4-8 h; stop feeding the glycerol when the wet weight of the bacteria reaches 180-220 g / L.

[0086] 4. Methanol feeding culture: stop feeding the glycerol, starve for 30-60 min, and then feed 100% methanol for induction at a rotation speed of 800 rpm, a culture temperature of 28-30°C, and a pH of 6.0, and keep the dissolved oxygen at 20%-80%.

[0087] 5. Take samples at different time points during the methanol feeding process of step 4, and determine the wet weight of the bacteria, the protein content, and the enzyme activity of the alginate lyase.

[0088] The results are shown in Table A. Figure 1 The enzyme activity of the alginate lyase reaches the highest value at 168 h of methanol induction culture, the enzyme activity of the fermentation supernatant is 413 U / mL, the protein concentration is 7.1 mg / mL, and the wet weight of the bacteria is 307.1 g / L.

[0089] Electrophoretically detect the supernatant, and the results are shown in Table B. Figure 1 The target band gradually deepens with the extension of the fermentation time, and the content of the alginate lyase gradually increases.

[0090] Example 2, Purification and Enzymatic Properties of Recombinant Alginate Lyase VaAly7

[0091] I. Purification of Recombinant Alginate Lyase VaAly7

[0092] 1. The methanol-induced 168 h fermentation supernatant of Example 1 above was dialyzed against 20 mM PB pH 8.0 buffer solution (buffer solution A) for 12 h, and centrifuged at 4 °C, 10000 rpm for 10 min, and the supernatant was reserved for use.

[0093] 2. The enzyme solution obtained in step 1 was purified by QSFF strong anion exchange column (1.0 x 10 cm), using AKTA protein purification system, and the loading flow rate was 0.5 mL / min.

[0094] 3. After step 2 was completed, the flow rate was set to 1 mL / min, and buffer solution A was used for elution until the eluate OD 280 <0.05.

[0095] 4. After step 3 was completed, buffer solution A containing 100-500 mM NaCl was used for linear elution within 90 min until the eluate OD 280 <0.05.

[0096] 5. The eluate obtained in step 4 was collected, and the protein purity was tested by SDS-PAGE.

[0097] The results, as shown in Figure 2 , the recombinant alginate lyase VaAly7 was electrophoretically pure enzyme, and the corresponding molecular weight was 44 kDa. The enzyme recovery rate was 76%, and the specific enzyme activity of the pure enzyme was 86.1 U / mg.

[0098] II. Enzymatic Properties of Recombinant Alginate Lyase VaAly7

[0099] 1. Determination of Optimal Reaction pH

[0100] The alginate lyase VaAly7 pure enzyme solution obtained in step one above was used as the test solution, and its enzyme activity was determined at 30 °C in different buffer solution systems, and the relative enzyme activity was calculated based on the highest enzyme activity. Various buffer solution systems are as follows: citrate buffer (pH 3.0-6.0), PB buffer (pH 6.0-8.0), Tris-HCl buffer (pH 6.0-9.0), CHES buffer (pH 8.0-10.0), and CAPS buffer (pH 10.0-11.0).

[0101] The results, as shown in Figure 3 , the optimal pH of alginate lyase VaAly7 was pH 7.0.

[0102] 2. Determination of pH stability

[0103] The VaAly7 pure enzyme solution was diluted in different buffer solution systems, placed in a 25 ℃ water bath for 30 min, and then quickly cooled in ice water for 30 min. The residual enzyme activity was measured at 30 ℃. The various buffer solution systems were as follows: citrate buffer (pH 3.0-6.0), PB buffer (pH 6.0-8.0), Tris-HCl buffer (pH 6.0-9.0), CHES buffer (pH 8.0-10.0), and CAPS buffer (pH 10.0-11.0). The residual enzyme activity was calculated as a percentage of the untreated enzyme solution, which was taken as 100%.

[0104] The results are shown in Table 1. Figure 4 As shown in Table 1, VaAly7 has good pH stability, and retains more than 80% of the enzyme activity after incubation at pH 4.0-9.5 for 30 min.

[0105] 3. Determination of optimum temperature

[0106] The VaAly7 pure enzyme solution was diluted with 50 mM Tris-HCl pH 7.0 buffer solution, and the enzyme activity was measured at different temperatures (15-40 ℃). The relative enzyme activity was calculated based on the highest enzyme activity, which was taken as 100%.

[0107] The results are shown in Table 2. Figure 5 As shown in Table 2, the optimum temperature of VaAly7 is 30 ℃.

[0108] 4. Determination of temperature stability

[0109] The VaAly7 pure enzyme solution was diluted with 50 mM Tris-HCl pH 7.0 buffer solution, and then incubated at different temperatures (15-40 ℃) for 30 min. The solution was quickly cooled in ice water for 30 min, and then the residual enzyme activity was measured at 30 ℃. The residual enzyme activity was calculated as a percentage of the untreated enzyme solution, which was taken as 100%.

[0110] The results are shown in Table 3. Figure 6 As shown in Table 3, VaAly7 has good stability below 30 ℃.

[0111] 5. Determination of half-life

[0112] The enzyme solution was appropriately diluted with 50 mM Tris-HCl pH 7.0 buffer and incubated at 25, 30, and 35 °C for 4 h. Samples were taken at different time points and immediately incubated in an ice-water bath for 30 min. The residual enzyme activity was measured under optimal conditions. The enzyme activity of the untreated enzyme solution was taken as 100%, and the percentage of residual enzyme activity was calculated. The time required for the enzyme activity to decrease to 50% was the half-life at the corresponding temperature.

[0113] The results are as follows Figure 7 As shown, the half-lives of VaAly7 at 25 ℃, 30 ℃, and 35 ℃ are 878 min, 207 min, and 15 min, respectively.

[0114] 6. Effect of NaCl on the activity of alginate lyase VaAly7

[0115] Prepare a 0.3% (w / v) alginate solution (50 mM Tris-HCl pH 7.0), add NaCl to make the NaCl concentration in the reaction system 0-1 M, and determine the alginate lyase activity at different NaCl concentrations. With the enzyme activity without NaCl as 100%, calculate the relative enzyme activity at different NaCl concentrations.

[0116] The results are as follows Figure 8 As shown, the enzyme activity was highest when the NaCl concentration was 400 mM, reaching about 8 times that of the control group.

[0117] 7. Substrate specificity

[0118] The VaAly7 pure enzyme solution was appropriately diluted with 50 mM Tris-HCl pH 7.0 buffer, and the alginate lyase activity of different substrates was measured. The substrates were poly G (Qingdao Bozhi Huili Biotechnology Co., Ltd., MAPG6-8K), poly M (Qingdao Bozhi Huili Biotechnology Co., Ltd., MAPM6-8K), and sodium alginate (Sinopharm Chemical Reagent Co., Ltd., 9005-38-3), and the concentration of each substrate in the reaction system was 10 mg / mL. Using the enzyme activity of VaAly7 against sodium alginate as 100%, the relative and specific enzyme activities of the enzyme against poly G and poly M were calculated.

[0119] The results are shown in Table 1. VaAly7 is a bifunctional enzyme that can degrade sodium alginate, poly M and poly G. It has the highest specific enzyme activity for poly G, which is 162.7 U / mg; followed by sodium alginate, with a specific enzyme activity of 86.1 U / mg; and specific enzyme activity for poly M is 84.4 U / mg.

[0120] Table 1. Substrate specificity of alginate lyase VaAly7

[0121]

[0122] 8. Hydrolysis properties

[0123] The 1% (w / v) alginate, poly M, poly G and guluronan oligosaccharides (G2-G5) were prepared respectively, and the enzyme amount was 1 U / mL, hydrolysis was carried out at 30 ℃ for 12 h, and samples were taken at different time points. The samples were inactivated by boiling for 5 min, centrifuged at 10000 rpm for 5 min, and the composition of the products in the supernatant was analyzed by thin layer chromatography. The developing agent was n-butanol: formic acid: water (2:1:1, V / V / V).

[0124] The results are shown in Table 1. Figure 9 As shown in Table 1, alginate oligosaccharides with DP2-4 and a series of oligosaccharides with higher polymerization degree were produced in the initial stage of alginate degradation by VaAly7. With the extension of reaction time, the oligosaccharides with higher polymerization degree were gradually degraded, and the alginate oligosaccharides with lower polymerization degree increased (Table 1, middle A). Figure 9 Alginate lyase VaAly7 could produce guluronan oligosaccharides with DP2-4 by degrading poly G, and mainly produce a series of oligosaccharides with higher polymerization degree by degrading poly M (Table 1, middle B). Figure 9 VaAly7 could not hydrolyze G2, G3 and G4, but could degrade G5 to produce guluronan disaccharide (Table 1, middle C). Figure 9

[0125] Example 3, Preparation of guluronan oligosaccharides by recombinant alginate lyase VaAly7 degrading poly guluronate

[0126] I. Isolation and preparation of substrate poly guluronate

[0127] A 2% (w / v) alginate solution was prepared and stirred with an electric blender to swell. Concentrated hydrochloric acid was added to make the HCl concentration of the system 0.5 mol / L, and acid hydrolysis was carried out at 100 ℃ for 10 h. The supernatant was discarded by centrifugation at 4000 rpm for 10 min. The precipitate was dissolved with 8% (w / v) NaHCO3 solution, and the pH was adjusted to 2.85 with 0.3 mol / L dilute hydrochloric acid to produce a large amount of white flocculent precipitate. The precipitate was centrifuged at 4000 rpm for 10 min. The precipitate was diluted with water to 6%, and the pH was adjusted to neutral with 10% (w / v) NaOH solution to dissolve the precipitate. The filtrate was obtained by filtration, and the pH was adjusted to 2.85 with 0.3 mol / L dilute hydrochloric acid to obtain the precipitate. The above operation was repeated 2-3 times to obtain a crude product with high purity of poly guluronate.

[0128] ​The M / G ratio of poly G was determined by measuring the circular dichroism spectrum using a J-815 circular dichroism spectrometer. A 0.1% (w / v) solution was prepared, and a quartz cell with an optical path length of 1 mm was used to scan the wavelength range of 190 nm-250 nm, with water as the reference, and the scanning temperature was 25°C.

[0129] The results are shown in Figure 10 Figure 1, and the M / G ratio of the prepared poly guluronate was 0.21, and the purity was 82%, according to the ratio of the peak height to the wave depth.

[0130] II. Efficient preparation of guluronate oligosaccharides

[0131] A 10% (w / v) poly G solution was prepared, and the enzyme VaAly7 was used to hydrolyze the solution at 30°C for 6 h, with an enzyme amount of 100 U / g. The enzyme activity was inactivated by boiling at 100°C for 5 min, and the solution was centrifuged at 4000 rpm for 10 min. The supernatant was collected to obtain the guluronate oligosaccharides.

[0132] The sample was filtered through a 0.22 μm filter, and the composition of the guluronate oligosaccharides was determined by ESI-MS. The detection was performed in the negative ion mode, with an ESI ionization source, a spray voltage of 4 kV, a capillary temperature of 350°C, a tube lens of 120 V, a sheath gas pressure of 45 AU, and a molecular weight screening range of 150-1500 m / z.

[0133] High-performance gel permeation chromatography (HPGPC) was used to analyze the molecular weight of alginate and poly G before and after enzymatic hydrolysis. The determination conditions were as follows: TSK-gel G3000PWXL chromatographic column, mobile phase 0.1 M NaNO3, column temperature 30°C, detector temperature 30°C, and flow rate 1 mL / min.

[0134] The results are shown in Figure 11 Figure 2, and the composition of the hydrolysis product was analyzed by ESI-MS, mainly including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and pentasaccharides Figure 11 Figure 2A). The HPGPC analysis results showed that the molecular weight of alginate was 2327 kDa, the molecular weight of the prepared poly guluronate was 11.5 kDa, and the molecular weight of the guluronate oligosaccharides was 2.1 kDa Figure 11 Figure 2B).

[0135] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the application.

Claims

1. A protein, which is any one of the following: 1) a protein having the amino acid sequence of SEQ ID NO: 1 in the Sequence Listing; 2) a protein having the amino acid sequence of positions 22 to 453 of SEQ ID NO: 1 in the Sequence Listing; The protein is derived from Vibrio alginolyticus (V. alginolyticus) Vibrio alginolyticus ).

2. A nucleic acid molecule encoding a protein, which is a DNA molecule of 1) or 2) as follows: 1) a DNA molecule having the nucleotide sequence of SEQ ID NO: 2 in the Sequence Listing; 2) a DNA molecule having the nucleotide sequence of positions 64 to 1362 of SEQ ID NO: 2 in the Sequence Listing.

3. Biomaterials containing the nucleic acid molecule according to claim 2, characterized in that, The biological material is any one of the following: B1) an expression cassette containing the nucleic acid molecule of claim 2; B2) a recombinant vector containing the nucleic acid molecule of claim 2, or a recombinant vector containing the expression cassette of B1); B3) a recombinant microorganism containing the nucleic acid molecule of claim 2, or a recombinant microorganism containing the expression cassette of B1), or a recombinant microorganism containing the recombinant vector of B2).

4. The biomaterial of claim 3, wherein, The recombinant vector is a recombinant vector obtained by inserting the nucleotide sequence of positions 64 to 1362 of SEQ ID NO: 2 into the multiple cloning site of vector pPIC9K; the recombinant microorganism is a recombinant Pichia pastoris obtained by introducing the recombinant vector into a yeast; and the yeast is Pichia pastoris.

5. A method of producing the protein of claim 1, characterized by, The method is introducing a DNA molecule capable of expressing the protein of claim 1 into a microorganism of interest to obtain a recombinant microorganism, culturing the recombinant microorganism to obtain the protein; and the nucleic acid molecule expressing the protein is a DNA molecule of 1) or 2) as follows: 1) a DNA molecule having the nucleotide sequence of SEQ ID NO: 2 in the Sequence Listing; 2) a DNA molecule having the nucleotide sequence of positions 64 to 1362 of SEQ ID NO: 2 in the Sequence Listing.

6. The production method according to claim 5, wherein Culturing the recombinant microorganism comprises the following steps: 1) inoculating the recombinant microorganism into a fermentation medium for culture, adding glycerol when the glycerol is completely consumed, and ensuring that the dissolved oxygen content in the fermentation system is 20%-80%; 2) stopping the addition of glycerol when the wet weight of the bacterial cells reaches 180-220 g / L; and 3) starving for another 30-60 min, and then starting to add 100% methanol to induce culture to obtain the protein.

7. The production method according to claim 5 or 6, characterized by: The recombinant microorganism is a recombinant Pichia pastoris containing a recombinant vector capable of expressing the protein of claim 1.

8. A process for the preparation of a guluronate oligosaccharide, characterized in that: Using the protein of claim 1 to perform enzymatic reaction on polyuronic acid as a substrate to obtain the guluronan oligosaccharide.

9. Use of the protein of claim 1, or the biological material of claim 3 or 4, or the method of claim 5 or 6 in any one of the following: 1) use in preparing alginate lyase; 2) use in preparing a product containing alginate lyase activity; 3) use in preparing alginate oligosaccharide; 4) use in degrading polyuronic acid; 5) use in preparing guluronan oligosaccharide.

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