Alginate lyase and application thereof in guluronic acid oligosaccharide preparation

Abstract

The invention discloses alginate lyase and application of the alginate lyase in preparation of guluronic acid oligosaccharide. The invention belongs to the technical field of biology, and particularly relates to alginate lyase and application thereof in guluronic acid oligosaccharide preparation. The protein disclosed by the invention is any one of the following proteins: 1) a protein with an amino acid sequence as a sequence 1 in a sequence table; and 2) the protein with the amino acid sequence from the 22nd site to the 453rd site of the sequence 1 in the sequence table. The recombinant alginate lyase pure enzyme obtained through purification has the specific enzyme activity of 86.1 U / mg and the recovery rate of 76%. The optimum pH value of the alginate lyase VaAly7 is 7.0, and the alginate lyase VaAly7 is kept stable when the pH value is 4.0-9.5; the algin lyase has the optimal temperature of 30 DEG C, keeps stable at the temperature of 30 DEG C or below, is cold-adapted algin lyase, has the advantages of easiness in control, energy conservation and the like in production and application, and has application potential in production of guluronic acid oligosaccharide.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an alginate lyase and application thereof in the preparation of guluronic acid oligosaccharides. Background Art

[0002] Alginate is a linear polysaccharide formed by the polymerization of β-D-mannuronic acid (β-D-mannuronate, M) and α-L-guluronate (α-L-guluronate, G) through 1,4-glycosidic bonds. According to the different monomer compositions, alginate can be divided into three categories: polymannuronic acid (poly M), polyguluronic acid (poly G) and heteropolysaccharides composed of two monomers (polyMG). Alginate oligosaccharides (AOS) are the degradation products of alginate. They have various biological activities, such as antibacterial, antioxidant, antitumor, immunomodulatory, prebiotic activity and plant growth promotion, and have great application potential in agriculture, biomedicine and food industry. The production methods of alginate oligosaccharides include physical method, chemical method and enzymatic method. The preparation of alginate oligosaccharides using alginate lyase has attracted wide attention due to its advantages such as high efficiency, environmental protection and product controllability. Studies have shown that the unique functions of brown algae oligosaccharides are closely related to specific structures, including molecular weight (Mw), degree of polymerization (DP), M / G ratio, monomer sequence and terminal saturation. At present, most of the research and application of AOS are based on AOS mixtures, and there are few reports on the preparation of guluronic acid oligosaccharides, resulting in uncertainty in biological activity. Therefore, the targeted preparation of brown algae oligosaccharides with specific monosaccharide composition is crucial for the study of their biological activity.

[0003] Alginate lyase has been isolated from different organisms, but the enzyme production of wild-type strains is low and cannot meet the needs of industrial production. Heterologous expression has become a common method to increase enzyme production. Most alginate lyase genes are expressed in Escherichia coli, such as Rubrivirga marina The alginate lyase AlyRm1 from Alteromonas portus The cold-adaptive alginate lyase Alg2951 of HB161718T was successfully expressed in E. coli, with an extracellular enzyme activity of 63.6 U / mL. The Pichia pastoris expression system has significant advantages in producing many recombinant proteins, such as high protein secretion capacity, simple product separation, and no endotoxin, but 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 the present invention is how to efficiently degrade polyguluronic acid to prepare guluronic acid oligosaccharides.

[0005] In order to solve the above problems, the present invention provides a protein.

[0006] The protein provided by the present invention is any of the following proteins: 1) The protein whose amino acid sequence is sequence 1 in the sequence list; 2) The amino acid sequence of the protein is from position 22 to position 453 of sequence 1 in the sequence listing; 3) Proteins obtained by replacing and / or deleting and / or adding one or more amino acid residues of the proteins in 1) or 2) and having the same function; 4) A fusion protein is obtained by connecting a protein tag to the N-terminus or / and the C-terminus of 1) or 2) or 3).

[0007] In order to facilitate purification or detection of the protein in 1), a tag protein may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in Sequence 1 in the sequence list.

[0008] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0009] The tag protein includes but is not limited to: GST (glutathione sulfhydryltransferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0010] The above protein is alginate lyase, named VaAly7.

[0011] A person skilled in the art can easily mutate the nucleotide sequence encoding the protein VaAly7 of the present invention by using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence of the protein VaAly7 isolated by the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein VaAly7 and have the function of the protein VaAly7.

[0012] The above-mentioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0013] Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching for the identity of a pair of amino acid sequences, the value of identity (%) can be obtained.

[0014] Herein, the 80% or greater identity may 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.

[0015] Herein, the 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0016] Herein, the protein is derived from Vibrio alginolyticus ( Vibrio alginolyticus ).

[0017] The present invention also provides a nucleic acid molecule encoding the protein described above.

[0018] The nucleic acid molecule may be a DNA molecule as described below: 1) The nucleotide sequence of the DNA molecule is sequence 2 in the sequence list; 2) The nucleotide sequence is the DNA molecule at positions 64-1362 of sequence 2 in the sequence list; 3) A DNA molecule that has 85% or more identity with the nucleotide sequence defined in 1) and encodes the protein described above; 4) A DNA molecule that hybridizes with the nucleotide sequence defined in 1) or 2) under stringent conditions and encodes the protein described above.

[0019] In the present invention, the sequence SEQ ID No. 2 consists of 1362 nucleotides, the sequence SEQ ID No. 1 consists of 453 amino acids, and the nucleotide sequence shown in SEQ ID No. 2 in the sequence listing encodes the amino acid sequence shown in SEQ ID No. 1 in the sequence listing.

[0020] The present invention also provides a biological material containing the nucleic acid molecule described above, The biological material is any one of the following: B1) an expression cassette containing the DNA molecule described above; B2) a recombinant vector containing the DNA molecule described above, or a recombinant vector containing the expression cassette described in B1); B3) A recombinant microorganism containing the DNA molecule described above, or a recombinant microorganism containing the expression cassette described in B1), or a recombinant microorganism containing the recombinant vector described in B2).

[0021] In the above biological materials, the recombinant vector is a recombinant vector obtained by inserting the nucleotide sequence from positions 64 to 1362 of sequence 2 into the multiple cloning site of vector pPIC9K; the recombinant bacteria is a recombinant Pichia pastoris obtained by introducing the recombinant vector into yeast; and the yeast is Pichia pastoris.

[0022] The recombinant bacteria can be obtained by introducing the corresponding recombinant vector into a host microorganism.

[0023] The host microorganism can be yeast, bacteria, algae or fungi. The yeast is Pichia pastoris ( Pichia pastoris )GS115.

[0024] The present invention 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 to obtain a recombinant microorganism, culturing the recombinant microorganism, collecting the fermentation broth supernatant, and obtaining the protein.

[0025] In the above protein preparation method, culturing the recombinant microorganism comprises the following steps: 1) inoculating the recombinant microorganism into the fermentation medium for cultivation, and adding glycerol after the glycerol is completely consumed to ensure that the dissolved oxygen content in the fermentation system is 20%-80%; 2) stopping the addition of glycerol when the wet weight of the bacteria reaches 180-220 g / L; 3) starving for another 30-60 minutes, and starting to add 100% methanol by volume for induction culture to obtain the protein. In the above preparation method, the recombinant microorganism is a recombinant Pichia pastoris, and the recombinant Pichia pastoris contains a recombinant vector capable of expressing the protein described above.

[0026] The present invention also provides a method for preparing guluronic acid oligosaccharides, which comprises taking polyguluronic acid as a substrate, using the above-mentioned protein to perform enzymatic hydrolysis reaction on the substrate, and obtaining the guluronic acid oligosaccharides.

[0027] In a specific embodiment, the preparation method may be: 1) adding the enzyme first and then the substrate, using the alginate lyase VaAly7 to degrade polyguluronic acid to obtain an enzymatic hydrolysate; 2) centrifuging the enzymatic hydrolysate to collect the supernatant and the precipitate respectively; 3) Take the supernatant obtained by centrifugation and filter it through a 0.22 μm microporous filter membrane, collect the filtrate, and lyophilize it to a constant mass.

[0028] Furthermore, the conditions for degrading the polyguluronic acid are 30°C, 200 rpm, substrate concentration of 10% (w / v), enzyme addition amount of 100 U / g, and enzymatic hydrolysis time of 6 h.

[0029] The present invention also provides the use of the above-mentioned protein, and / or biological material and / or preparation method in any of the following: 1) Application of preparing alginate lyase; 2) Application in the preparation of products containing alginate lyase activity; 3) Application in the preparation of brown algae oligosaccharides; 4) Application in degradation of polyguluronic acid; 5) Application in the preparation of guluronic acid oligosaccharides.

[0030] In summary, the present invention provides an alginate lyase VaAly7 and its encoding gene and related applications. The encoding gene can be constructed into a recombinant vector and introduced into a host such as Pichia pastoris. The encoding gene of the alginate lyase VaAly7 of the present invention can be efficiently and stably expressed in the recombinant host (especially Pichia pastoris) to obtain the alginate lyase VaAly7.

[0031] Taking recombinant Pichia pastoris as an example, after high-density fermentation, the obtained enzyme activity can reach 413 U / mL, the protein content can reach 7.1 mg / mL, and the wet weight of the bacteria is 307.1 g / L. The recombinant alginate lyase pure enzyme was purified, with an enzyme activity of 86.1 U / mg and a recovery rate of 76%. The optimal pH of alginate lyase VaAly7 is 7.0, and it remains stable between pH 4.0-9.5; the optimal temperature is 30 ℃, and it remains stable below 30 ℃. It is a cold-adapted alginate lyase, which has unique advantages such as easy control and energy saving in production applications. The new cold-adapted alginate lyase described in the present invention has application potential in the production of guluronic acid oligosaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1Figure A and SDS-PAGE electrophoresis diagram B show the enzyme production process of recombinant Pichia pastoris in a 5 L fermenter with high density. In A, (▲) enzyme activity, (■) protein concentration, (●) cell wet weight; in B, column M is a low molecular weight standard protein, and columns 1-8 are the fermentation supernatants induced at 0, 24, 48, 72, 96, 120, 144 and 168 h, respectively.

[0033] Figure 2 This is the electrophoresis diagram of the purified alginate lyase VaAly7 (column M: low molecular weight standard protein; column 1: crude enzyme solution; column 2: pure enzyme solution; column 3: alginate lyase treated with deglycosylation enzyme).

[0034] Figure 3 This 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), ◆: CAPS buffer (pH 10.0-11.0)).

[0035] Figure 4 This 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), ◆: CAPS buffer (pH 10.0-11.0)).

[0036] Figure 5 This is the optimum temperature determination curve of alginate lyase VaAly7.

[0037] Figure 6 This is a curve diagram for determining the temperature stability of alginate lyase VaAly7.

[0038] Figure 7 This is a half-life determination curve of alginate lyase VaAly7.

[0039] Figure 8 This is the effect of NaCl on alginate lyase VaAly7.

[0040] Fig. 9 TLC analysis of the degradation of alginate (A), poly G and poly M (B) and guluronic acid oligosaccharides G2-G5 (C) by alginate lyase VaAly7.

[0041] Fig.10 This is the circular dichroism spectrum of polyguluronic acid.

[0042] Fig.11 Composition (A) and molecular weight (B) analysis of polyguluronic acid degradation products. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0044] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0045] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0046] In the embodiment of the present invention, the enzyme activity of alginate lyase is determined by 3,5-dinitrosalicylic acid (DNS) method. If there is no special instruction, the determination steps are as follows: 100 μL of appropriately diluted enzyme solution is added to 900 μL of 0.3% alginate solution (prepared with alginate and pH 7.0, 50 mM Tris-HCl buffer), reacted at 30 ° C for 10 min, 750 μL of DNS is added to terminate the reaction, boiled for 10 min and the absorbance at 540 nm is measured. With glucose as the standard, the amount of reducing sugar generated by the reaction is calculated. Under the above reaction conditions, the amount of enzyme required to generate 1 μmol of reducing sugar per minute is defined as one enzyme activity unit (U).

[0047] The following examples use origin 2022 statistical software to process the data, and the experimental results are expressed as mean ± standard deviation.

[0048] Example 1. Alginate lyase gene VaAly7 Cloning and expression of one, VaAly7 PCR amplification of genes Alginate lyase gene VaAly7 The coding region is 1299 bp, and its sequence is shown in positions 64 to 1362 of SEQ ID No.2, encoding an alginate lyase (VaAly7) with an amino acid sequence as shown in positions 22 to 453 of SEQ ID No.1.

[0049] Synthesis VaAly7The 64th to 1362th position of SEQ ID No.2 was used as a template, and primers were designed for PCR amplification to obtain PCR products. 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; 72°C extension for 5 min. The PCR amplification product VaAly7 was recovered by 1% agarose gel electrophoresis.

[0050] 2. Construction of recombinant vector The alginate lyase gene obtained in step 1 VaAly7 and the Pichia pastoris expression vector pPIC9K (purchased from Invitrogen, USA) EcoR I and Not After double enzyme digestion with I, the cells were ligated with T4 DNA ligase to obtain the recombinant plasmid pPIC9K-VaAly7.

[0051] The recombinant plasmid pPIC9K-VaAly7 was transferred into E. coli DH5α for amplification and verification. The structure of the recombinant plasmid pPIC9K-VaAly7 is described as follows: EcoR I and Not A recombinant vector is obtained by inserting a DNA fragment having a sequence of positions 64 to 1362 of SEQ ID No.2 between the two restriction sites I, and keeping other sequences of the vector pPIC9K unchanged. The pPIC9K-VaAly7 vector can express VaAly7 protein, whose amino acid sequence is positions 22 to 453 of SEQ ID No.1.

[0052] The obtained recombinant vector pPIC9K-VaAly7 was cloned and cloned with restriction endonuclease Sac After linearization, it was electroporated into Pichia pastoris GS115 (purchased from Invitrogen, USA, catalog number C18100) to construct the recombinant bacteria GS115 / pPIC9K-VaAly7. The electroporation conditions were: voltage 2 kV, capacitance 25 μF, resistance 200 Ω, and shock time 5-10 mS. The electroporation process was as follows: 80 μL of pre-cooled thawed on ice P. pastoris Add about 10 μg of linearized recombinant plasmid to GS115 competent cells and mix well, put into ice-precooled electroporation cup for 5 minutes, insert into electroporator and electroporate for about 5mS, then add about 1mL of precooled 1mM sorbitol solution and mix quickly to obtain recombinant strain GS115 / pPIC9K-VaAly7.

[0053] 3. Recombinant bacteria expressing alginate lyase VaAly7 1. Inoculate the recombinant bacteria GS115 / pPIC9K-VaAly7 obtained in step 2 above on MD plates (1.34% YNB, 4×10 -5 % biotin, 2% glucose) at 30°C for 3-5 days to obtain His + Transformant.

[0054] 2. After completing step 1, scrape the transformants with sterile water and spread 100 μL 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 culture at 30°C for 3-5 days.

[0055] 3. After completing step 2, take the transformant and inoculate it into BMGY medium (1% yeast extract, 2% tryptone, 100 mM pH 6.0 phosphate buffer solution, 1.34% YNB, 4×10 -5 % biotin, 1% glycerol), and cultured at 30°C, 200 rpm in a shaking incubator for 16-18 h.

[0056] 4. After completing step 3, centrifuge the culture solution at 8000 rpm for 5 min, collect the bacteria, and use BMMY medium (1% yeast extract, 2% tryptone, 100 mM pH 6.0 phosphate buffer solution, 1.34% YNB, 4×10 -5 % biotin, 0.5% methanol) to OD 600 The culture medium was about 1.0 and cultured at 30 °C and 200 rpm. Methanol was added every 24 h to a final concentration of 0.5% (v / v). After 72 h, 1 mL of the culture medium was aspirated and centrifuged at 10,000 rpm for 10 min.

[0057] 5. After completing step 4, take the supernatant for enzyme activity determination.

[0058] 4. High-density fermentation of recombinant Pichia pastoris to produce alginate lyase 1. Seed liquid culture: The recombinant Pichia pastoris strain GS115 / pPIC9K-VaAly7 with the highest enzyme production level obtained in step 3 above was inoculated into 150 mL YPD medium and cultured overnight at 30°C and 200 rpm with shaking until OD 600 8-10, to obtain seed solution.

[0059] 2. Basic culture: Inoculate the seed solution in step 1 into a 5 L fermenter containing 1.35 L of basic culture medium. The basic culture medium formula is: CaSO40.93 g / L, K2SO418.2 g / L, MgSO4·7H2O 14.9 g / L, KOH 4.13g / L, glycerol 40 g / L, 85% H3PO426.7 mL / L, PTM1 salt 4.375 mL / L. The culture medium temperature is 30°C, the pH is 4.0, and the rotation speed is 600 rpm. When the dissolved oxygen rises to more than 80%, indicating that the glycerol is completely consumed, glycerol fed-batch culture is carried out; 3. Glycerol fed-batch culture: feed glycerol with a mass concentration of 50% (w / v), temperature of 30°C, pH of 5.0, maintain dissolved oxygen at 20%-80% by adjusting the flow acceleration, and feed time of 4-8 h; stop feeding glycerol when the wet weight of the bacteria reaches 180-220 g / L; 4. Methanol fed-batch culture: After stopping the feeding of glycerol, starve for 30-60 min, feed 100% methanol for induction, the rotation speed is 800 rpm, the culture temperature is 28-30℃, the pH is 6.0, and the dissolved oxygen is 20%-80%.

[0060] 5. Samples were taken at different time points during the methanol addition process in step 4 to determine the wet weight of the bacteria, protein content and enzyme activity of alginate lyase.

[0061] The results are as follows Figure 1 As shown in A, when the methanol was induced for 168 h, the enzyme activity of alginate lyase reached the highest level, the enzyme activity of the fermentation supernatant was 413 U / mL, the protein concentration was 7.1 mg / mL, and the wet weight of the bacteria was 307.1 g / L.

[0062] The supernatant was tested by electrophoresis. Figure 1 As shown in B, the target band gradually deepened with the extension of fermentation time, and the content of alginate lyase gradually increased.

[0063] Example 2: Purification and enzymatic properties of recombinant alginate lyase VaAly7 1. Purification of recombinant alginate lyase VaAly7 1. The fermentation supernatant obtained by methanol induction for 168 h in Example 1 was dialyzed for 12 h against a 20 mM PB pH 8.0 buffer solution (buffer solution A), and centrifuged at 4 °C, 10,000 rpm for 10 min, and the supernatant was retained for later use.

[0064] 2. The enzyme solution obtained in step 1 was purified by QSFF strong anion exchange column (1.0 × 10 cm) using the AKTA protein purification system at a flow rate of 0.5 mL / min.

[0065] 3. After completing step 2, set the flow rate to 1 mL / min and elute with buffer solution A until the eluent OD is 280 <0.05.

[0066] 4. After completing step 3, use buffer solution A containing 100-500 mM NaCl to linearly elute to the eluent OD within 90 min. 280 <0.05.

[0067] 5. Collect the eluate obtained in step 4 and check the protein purity by SDS-PAGE.

[0068] The results are as follows Figure 2 As shown in the figure, the recombinant alginate lyase VaAly7 is an electrophoresis grade pure enzyme with a corresponding molecular weight of 44 kDa. The recovery rate of the enzyme is 76%, and the specific enzyme activity of the pure enzyme is 86.1 U / mg.

[0069] 2. Enzymatic properties of recombinant alginate lyase VaAly7 1. Determination of the optimal reaction pH The pure enzyme solution of alginate lyase VaAly7 obtained in step 1 above was used as the test solution, and the enzyme activity was measured in different buffer solution systems at 30 °C, and the relative enzyme activity was calculated with the highest enzyme activity as 100%. The 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), CAPS buffer (pH 10.0-11.0).

[0070] The results are as follows Figure 3 As shown, the optimal pH of alginate lyase VaAly7 is pH 7.0.

[0071] 2. Determination of pH stability The pure VaAly7 enzyme solution was diluted in different buffer solution systems, placed in a 25 ℃ water bath for 30 minutes, quickly placed in ice water for 30 minutes, and the residual enzyme activity was measured at 30 ℃. The 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), CAPS buffer (pH 10.0-11.0). The enzyme activity of the untreated enzyme solution was taken as 100%, and the percentage of residual enzyme activity was calculated.

[0072] The results are as follows Figure 4As shown in the results, VaAly7 has good pH stability, and more than 80% of the enzyme activity remains after being incubated in the pH range of 4.0-9.5 for 30 min.

[0073] 3. Determination of optimum temperature The pure VaAly7 enzyme solution was appropriately diluted with 50 mM Tris-HCl pH 7.0 buffer solution, and the enzyme activity was measured at different temperatures (15-40 °C). The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated.

[0074] The results are as follows Figure 5 As shown, the optimum temperature of VaAly7 is 30 ℃.

[0075] 4. Determination of temperature stability The pure VaAly7 enzyme solution was appropriately diluted with 50 mM Tris-HCl pH 7.0 buffer solution, and then kept at different temperatures (15-40°C) for 30 min, and then quickly placed in ice water for 30 min, and then the residual enzyme activity was measured at 30°C. The percentage of residual enzyme activity was calculated with the enzyme activity of the untreated enzyme solution as 100%.

[0076] The results are as follows Figure 6 As shown in the figure, VaAly7 has good stability below 30 ℃.

[0077] 5. Determination of half-life The enzyme solution was appropriately diluted with 50 mM Tris-HCl pH 7.0 buffer, incubated at 25, 30, and 35 ℃ for 4 h, samples were taken at different time points, and immediately placed in an ice water bath for 30 min. The residual enzyme activity was determined under the 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 taken for the enzyme activity to decrease to 50% was the half-life at the corresponding temperature.

[0078] 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.

[0079] 6. Effect of NaCl on the activity of alginate lyase VaAly7 A 0.3% (w / v) alginate solution (50 mM Tris-HCl pH 7.0) was prepared, and NaCl was added to make the NaCl concentration in the reaction system 0-1 M. The alginate lyase activity at different NaCl concentrations was measured, and the enzyme activity without adding NaCl was taken as 100%, and the relative enzyme activity at different NaCl concentrations was calculated.

[0080] 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.

[0081] 7. Substrate specificity After the pure VaAly7 enzyme solution was appropriately diluted with 50 mM Tris-HCl pH 7.0 buffer, the alginate lyase activity of different substrates was determined. The substrates were poly G (Qingdao Bozhihuili Biotechnology Co., Ltd., MAPG6-8K), poly M (Qingdao Bozhihuili Biotechnology Co., Ltd., MAPM6-8K), and sodium alginate (Sinopharm Chemical Reagent Co., Ltd., 9005-38-3). The concentration of the substrate in the reaction system was 10 mg / mL. The enzyme activity of VaAly7 on sodium alginate was 100%, and the relative enzyme activity and specific enzyme activity of the enzyme on poly G and poly M were calculated.

[0082] The results are shown in Table 1. VaAly7 is a bifunctional enzyme that can degrade sodium alginate, poly M and poly G. The specific enzyme activity for poly G is the highest, which is 162.7 U / mg; followed by sodium alginate, with a specific enzyme activity of 86.1 U / mg; and the specific enzyme activity for poly M is 84.4 U / mg.

[0083] Table 1. Substrate specificity of alginate lyase VaAly7

[0084] 8. Hydrolysis characteristics 1% (w / v) alginate, poly M, poly G and guluronic acid oligosaccharides (G2-G5) were prepared respectively, and 1 U / mL enzyme was added. The samples were hydrolyzed at 30 ℃ for 12 h. Samples were taken at different time points, boiled for 5 min to inactivate the samples, and centrifuged at 10000 rpm for 5 min. The product composition in the supernatant was analyzed by thin layer chromatography. The developing agent was n-butanol:formic acid:water (2:1:1, V / V / V).

[0085] The results are as follows Fig. 9 As shown in the figure, in the initial stage of VaAly7 degradation of alginate, monosaccharides, DP2-4 brown algae oligosaccharides and a series of oligosaccharides with higher polymerization degrees were produced. As the reaction time increased, the oligosaccharides with high polymerization degrees were gradually degraded, and the amount of brown algae oligosaccharides with low polymerization degrees increased ( Fig. 9 A in the middle). Alginate lyase VaAly7 can degrade poly G to generate DP2-4 guluronic acid oligosaccharides, and degrade poly M to mainly generate a series of oligosaccharides with higher polymerization degree ( Fig. 9 VaAly7 cannot hydrolyze G2, G3, and G4, but can degrade G5 to produce guluronate disaccharide ( Fig. 9 Middle C).

[0086] Example 3: Degradation of polyguluronic acid by recombinant alginate lyase VaAly7 to prepare guluronic acid oligosaccharides 1. Separation and preparation of substrate polyguluronic acid Prepare 2% (w / v) alginate solution, stir with an electric stirrer to make it swell, add concentrated hydrochloric acid to make the system HCl concentration 0.5 mol / L, acid hydrolyze at 100 ℃ for 10 h, centrifuge at 4000 rpm for 10 min and discard the supernatant. Dissolve the precipitate with 8% (w / v) NaHCO3, adjust the pH to 2.85 with 0.3 mol / L dilute hydrochloric acid to produce a large amount of white flocculent precipitate, centrifuge at 4000 rpm for 10 min to obtain the precipitate. Dilute the precipitate to 6% with water, adjust the pH to neutral with 10% (w / v) NaOH solution, dissolve the precipitate, filter to remove impurities, adjust the pH to 2.85 with 0.3 mol / L dilute hydrochloric acid to obtain the precipitate, repeat the above operation 2-3 times to obtain a high purity crude polyguluronic acid.

[0087] The J-815 circular dichroism spectrometer was used to measure the circular dichroism spectrum and analyze the M / G ratio of poly G. A 0.1% (w / v) solution was prepared and scanned in the wavelength range of 190 nm-250 nm using a quartz cell with an optical path length of 1 mm, using water as the reference substance and the scanning temperature at 25 °C.

[0088] The results are as follows Fig.10 As shown, mannuronic acid has a peak near 200 nm, and guluronic acid has a trough near 215 nm. According to the ratio of peak height and wave depth, it can be determined that the M / G ratio of the prepared polyguluronic acid is 0.21 and the purity is 82%.

[0089] 2. Efficient preparation of guluronic acid oligosaccharides 10% (w / v) poly G was prepared, and the recombinant alginate lyase VaAly7 was used for enzymatic hydrolysis at 30 ℃ for 6 h, with an enzyme dosage of 100 U / g. The mixture was boiled at 100 ℃ for 5 min to inactivate the enzyme, and the mixture was centrifuged at 4000 rpm for 10 min. The supernatant was taken to obtain guluronic acid oligosaccharides.

[0090] The sample was filtered through a 0.22 μm filter membrane and the composition of guluronic acid oligosaccharides was determined by ESI-MS. The detection was in negative ion mode, the ionization source was ESI, the spray voltage was 4 kV, the capillary temperature was 350 °C, the tube lens was 120 V, the sheath gas pressure was 45 AU, and the molecular weight screening range was 150-1500 m / z.

[0091] 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: TSK-gelG3000PWXL column, mobile phase 0.1 M NaNO3, column temperature 30 ℃, detector temperature 30 ℃, flow rate 1 mL / min.

[0092] The results are as follows Fig.11 As shown, the composition of the hydrolysate was analyzed by ESI-MS, which mainly included monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and pentasaccharides ( Fig.11 The results of HPGPC analysis showed that the molecular weight of alginate was 2327 kDa, the molecular weight of prepared polyguluronic acid was 11.5 kDa, and the molecular weight of guluronic acid oligosaccharide was 2.1 kDa ( Fig.11 Middle B).

[0093] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A protein, which is any of the following: 1) The protein whose amino acid sequence is sequence 1 in the sequence list; 2) The amino acid sequence of the protein is from position 22 to position 453 of sequence 1 in the sequence listing; 3) Proteins obtained by replacing and / or deleting and / or adding one or more amino acid residues of the proteins in 1) or 2) and having the same function; 4) A fusion protein is obtained by connecting a protein tag to the N-terminus or / and the C-terminus of 1) or 2) or 3).

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid molecule is the DNA molecule described in 1) or 2) or 3) or 4) below: 1) The nucleotide sequence is the DNA molecule of sequence 2 in the sequence list; 2) The nucleotide sequence is the DNA molecule from position 64 to 1362 of sequence 2 in the sequence list; 3) A DNA molecule that has 85% or more identity with the nucleotide sequence defined in 1) and encodes the protein of claim 1; 4) A DNA molecule that hybridizes with the nucleotide sequence defined in 1) or 2) under stringent conditions and encodes the protein of claim 1.

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

5. The biomaterial according to claim 4, characterized in that The recombinant vector is a recombinant vector obtained by inserting the nucleotide sequence from positions 64 to 1362 of sequence 2 into the multiple cloning site of vector pPIC9K; the recombinant bacteria is a recombinant Pichia pastoris obtained by introducing the recombinant vector into yeast; and the yeast is Pichia pastoris.

6. The method for preparing the protein according to claim 1, characterized in that: The method comprises introducing a DNA molecule capable of expressing the protein of claim 1 into a target microorganism to obtain a recombinant microorganism, and culturing the recombinant microorganism to obtain the protein.

7. The preparation method according to claim 6, characterized in that: Cultivating the recombinant microorganism comprises the following steps: 1) inoculating the recombinant microorganism into a fermentation medium for cultivation, and feeding glycerol after the glycerol is completely consumed to ensure that the dissolved oxygen content in the fermentation system is 20%-80%; 2) stopping the glycerol feeding when the wet weight of the microorganism reaches 180-220 g / L; 3) starving the microorganism for another 30-60 minutes, and starting to feed 100% methanol by volume for induction culture to obtain the protein.

8. The preparation method according to claim 6 or 7, characterized in that: The recombinant microorganism is a recombinant Pichia pastoris, and the recombinant Pichia pastoris contains a recombinant vector capable of expressing the protein according to claim 1.

9. A method for preparing guluronic acid oligosaccharide, characterized in that: Polyguluronic acid is used as a substrate, and the protein according to claim 1 is used to perform an enzymatic hydrolysis reaction on the substrate to obtain the guluronic acid oligosaccharide.

10. Use of the protein according to any one of claims 1, and / or the biomaterial according to claim 4 or 5, and / or the method according to any one of claims 6 to 8 in any of the following: 1) Application of preparing alginate lyase; 2) Application in the preparation of products containing alginate lyase activity; 3) Application in the preparation of brown algae oligosaccharides; 4) Application in degradation of polyguluronic acid; 5) Application in the preparation of guluronic acid oligosaccharides.

Images