Alginate lyase Alg0392, DNA (deoxyribonucleic acid), recombinant plasmid, recombinant bacteria, application and algin splitting preparation

Abstract

The invention discloses algin lyase Alg0392, DNA, recombinant plasmid, recombinant bacteria, application and an algin cracking preparation, and relates to the technical field of microorganisms, the amino acid sequence of the algin lyase Alg0392 is shown as SEQ ID No.1, the algin lyase Alg0392 has endonuclease activity and exonuclease activity, can degrade sodium alginate into monosaccharide, disaccharide and trisaccharide, and the algin lyase Alg0392 can be used for preparing the algin lyase Alg0392. Meanwhile, the method has substrate specificity on polymannuronic acid, and sodium alginate can be well and thoroughly degraded. In addition, dithiothreitol and beta-mercaptoethanol have a promoting effect on the enzyme activity of alginate lyase Alg0392, and can be prepared into an alginate lysing preparation to improve the degradation efficiency of an alginate substrate.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to an alginate lyase Alg0392 and its DNA, recombinant plasmid, recombinant bacteria and application, and an alginate lysate preparation. Background Art

[0002] Brown algae is a multifunctional seaweed with high yield and wide distribution, and is one of the important raw materials for the production of biofuels. Alginate, also known as alginate, is a linear anionic polysaccharide and an important component of the cell wall in brown algae and some red algae. Alginate has been used in bioengineering, medicine, food and biofuel development, but its high degree of polymerization, high molecular weight and high viscosity also limit its potential applications.

[0003] Alginate oligosaccharides (AOS) are degradation products of alginate. Due to their low molecular weight, good water solubility, and ease of absorption by the body, they also have special biological activities that alginate does not have, such as anti-tumor, antioxidant, hypoglycemic, and neuroprotective properties, which expands the application areas of alginate.

[0004] At present, the preparation methods of brown algae oligosaccharides mainly include chemical method and physical degradation method. However, these two preparation methods have many disadvantages such as high cost, poor product recovery rate, and easy generation of by-products. Summary of the invention

[0005] The main purpose of the present invention is to provide an alginate lyase Alg0392, DNA, recombinant plasmid, recombinant bacteria and application, and an alginate lysate preparation, aiming to solve the problem that the current method for preparing brown algae oligosaccharides is low in efficiency and easy to produce by-products.

[0006] To achieve the above object, the present invention provides an alginate lyase Alg0392, the amino acid sequence of the alginate lyase Alg0392 is shown in SEQ ID No.1.

[0007] The present invention provides a DNA molecule, wherein the DNA molecule is used to encode the aforementioned alginate lyase Alg0392.

[0008] In one embodiment, the nucleotide sequence of the DNA molecule is shown as SEQ ID No.2.

[0009] The invention provides a recombinant plasmid, which comprises the DNA molecule.

[0010] The present invention provides a recombinant bacterium, which comprises the nucleotide sequence of the DNA molecule or the recombinant plasmid.

[0011] The present invention provides an application of the alginate lyase Alg0392 in degrading alginate and its derivatives.

[0012] In one embodiment, the derivative comprises polymannuronic acid and / or polyguluronic acid.

[0013] In one embodiment, the temperature at which the alginate lyase Alg0392 degrades alginate and its derivatives is 25°C to 40°C.

[0014] In one embodiment, the pH of the alginate lyase Alg0392 when degrading alginate and its derivatives is 5.0-9.0.

[0015] The present invention also provides an alginate lysing preparation, comprising the alginate lysing enzyme Alg0392 and a solvent, wherein the solvent comprises dithiothreitol and / or β-mercaptoethanol.

[0016] In the technical solution of the present invention, the alginate lyase Alg0392 has endo- and exo-enzyme activities, can degrade sodium alginate into monosaccharides, disaccharides and trisaccharides, and has substrate specificity for polymannuronic acid in sodium alginate, and can degrade sodium alginate better and more thoroughly. In addition, dithiothreitol and β-mercaptoethanol promote the enzymatic activity of alginate lyase Alg0392, and can be configured as an alginate lysing preparation to improve the degradation efficiency of alginate substrates. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 This is a PCR verification result diagram of the recombinant plasmid in Example 1 provided by the present invention;

[0019] Figure 2 This is the SDS-PAGE image of the alginate lyase Alg0392 in Example 1 provided by the present invention;

[0020] Figure 3 This is a graph showing the relative enzyme activity results of alginate lyase Alg0392 at different pH values ​​in Example 2 provided by the present invention;

[0021] Figure 4 The pH stability test result of alginate lyase Alg0392 in Example 2 provided by the present invention is shown in FIG.

[0022] Figure 5The relative enzyme activity results of alginate lyase Alg0392 at different temperatures in Example 2 provided by the present invention;

[0023] Figure 6 The temperature stability test result of alginate lyase Alg0392 in Example 2 provided by the present invention;

[0024] Figure 7 The results of the effect of the organic reagent on the alginate lyase Alg0392 in Example 2 provided by the present invention;

[0025] Figure 8 The Michaelis Menten diagram of the alginate lyase Alg0392 in Example 2 provided by the present invention;

[0026] Fig. 9 This is an analysis diagram of the degradation products of alginate lyase Alg0392 in Example 3 provided by the present invention;

[0027] Fig.10 This is another degradation product analysis diagram of the alginate lyase Alg0392 in Example 3 provided by the present invention.

[0028] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0029] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0030] It should be noted that, in the embodiments, those without specifying specific conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. Those without specifying the manufacturer of reagents or instruments used are conventional products that can be purchased commercially. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of the technical schemes is mutually contradictory or cannot be achieved, it should be considered that the combination of such technical schemes does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0031] Brown algae is a multifunctional seaweed with high yield and wide distribution, and is one of the important raw materials for the production of biofuels. Alginate, also known as alginate, is a linear anionic polysaccharide and an important component of the cell wall in brown algae and some red algae. Alginate has been used in bioengineering, medicine, food and biofuel development, but its high degree of polymerization, high molecular weight and high viscosity also limit its potential applications.

[0032] Alginate oligosaccharides (AOS) are degradation products of alginate. Due to their low molecular weight, good water solubility, and ease of absorption by the body, they also have special biological activities that alginate does not have, such as anti-tumor, antioxidant, hypoglycemic, and neuroprotective properties, which expands the application areas of alginate.

[0033] At present, the preparation methods of brown algae oligosaccharides mainly include chemical method and physical degradation method. However, these two preparation methods have many disadvantages such as high cost, poor product recovery rate, and easy generation of by-products.

[0034] In view of this, the present invention provides an alginate lyase Alg0392, the amino acid sequence of the alginate lyase Alg0392 is shown in SEQ ID No.1.

[0035] SEQ ID No.1:

[0036] .

[0037] In the technical solution of the present invention, the alginate lyase Alg0392 has endo- and exo-enzyme activities, can degrade sodium alginate into monosaccharides, disaccharides and trisaccharides, and has substrate specificity for polymannuronic acid in sodium alginate, and can degrade sodium alginate better and more thoroughly. In addition, dithiothreitol and β-mercaptoethanol promote the enzymatic activity of alginate lyase Alg0392, and can be configured as an alginate lysing preparation to improve the degradation efficiency of alginate substrates.

[0038] The present invention provides a DNA molecule, wherein the DNA molecule is used to encode the aforementioned alginate lyase Alg0392. The alginate lyase Alg0392 provided by the present invention is a new alginate lyase.

[0039] In some embodiments of the present invention, the nucleotide sequence of the DNA molecule is shown as SEQ ID No.2.

[0040] SEQ ID No.2:

[0041]

[0042] The present invention provides a recombinant plasmid, the recombinant plasmid comprises the DNA molecule. The vector of the recombinant plasmid comprises a linearized vector pET28a. The recombinant plasmid can achieve high-level gene expression in a host cell, thereby increasing the expression amount of the DNA molecule loaded thereon.

[0043] The present invention provides a recombinant bacterium, wherein the recombinant bacterium comprises the nucleotide sequence of the DNA molecule or the recombinant plasmid. Preferably, the host cell of the recombinant bacterium is Escherichia coli. More preferably, the host cell of the recombinant bacterium is Escherichia coli BL21 (DE3). The recombinant bacterium can express a large amount of target enzyme relatively quickly.

[0044] The present invention provides an application of the alginate lyase Alg0392 in degrading alginate and its derivatives.

[0045] In the technical solution provided by the present invention, the degradation of alginate and its derivatives by alginate lyase Alg0392 belongs to a biodegradation method. Compared with physical and chemical methods, this method does not introduce other harmful substances. At the same time, alginate lyase Alg0392 has both endo- and exo-enzyme activities, and can degrade alginate and its derivatives more thoroughly. Among them, alginate is also alginate, and alginate includes sodium alginate. Alginate lyase Alg0392 can degrade sodium alginate into monosaccharides, disaccharides and trisaccharides.

[0046] Alginate is polymerized by α-L-guluronic acid (α-L-guluronic acid, G) and β-D-mannuronic acid (β-D-mannuronic acid, M) in an alternating block arrangement, and the two are connected by 1,4-glycosidic bonds. According to the different contents of the two monosaccharides in alginate, it can be divided into three types: polymannuronic acid (polyM), polyguluronic acid (polyG) and mixed (polyMG) alginate. Block type and molecular weight affect the gelling ability and viscosity of alginate. Therefore, alginate has a variety of derivatives. In some embodiments of the present invention, the derivative includes polymannuronic acid and / or polyguluronic acid. The products generated by the degradation of PolyM by alginate lyase Alg0392 are monosaccharides, disaccharides, and trisaccharides, and the degradation activity of PolyG is relatively low.

[0047] In some embodiments of the present invention, the temperature of the alginate lyase Alg0392 when degrading alginate and its derivatives is 25°C to 40°C. The temperature of the alginate lyase Alg0392 when degrading alginate and its derivatives can be 25°C, 30°C or 40°C. When the degradation temperature is within a suitable range, the relative enzyme activity of the alginate lyase Alg0392 is high, and the degradation efficiency of the alginate lyase Alg0392 is high. Preferably, the temperature of the alginate lyase Alg0392 when degrading alginate and its derivatives is 30°C.

[0048] In some embodiments of the present invention, the pH of the alginate lyase Alg0392 when degrading alginate and its derivatives is 5.0 to 9.0. The pH of the alginate lyase Alg0392 when degrading alginate and its derivatives can be 5.0, 7.0 or 9.0. When the degradation pH is within a suitable range, the relative enzyme activity of the alginate lyase Alg0392 is high, and the degradation efficiency of the alginate lyase Alg0392 is high. Preferably, the pH of the alginate lyase Alg0392 when degrading alginate and its derivatives is 6.0.

[0049] The present invention also provides an alginate lysing preparation, comprising the aforementioned alginate lyase Alg0392 and a solvent, wherein the solvent comprises dithiothreitol and / or β-mercaptoethanol. Since dithiothreitol and β-mercaptoethanol can promote the enzymatic activity of alginate lyase Alg0392, they can be configured as an alginate lysing preparation for improving the lysis efficiency of alginate lyase Alg0392.

[0050] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0051] Example 1 Construction of recombinant plasmid and recombinant bacteria, and acquisition of alginate lyase Alg0392

[0052] (1) Construction of recombinant plasmid

[0053] The nucleotide sequence of alginate lyase Alg0392 (as shown in SEQ ID No. 1) was connected to the linearized vector pET28a using the One Step Seamless Cloning kit to obtain a recombinant plasmid. The enzyme ligation reaction system is as follows: 3 μL of target gene, 2 μL of linear vector, and 5 μL of 2×One Step Cloning Mix. After reacting in a 50°C water bath for 30 minutes, the recombinant plasmid was transferred into Escherichia coli BL21 (DE3) competent cells using the heat shock method. After recovery, it was spread on LB solid culture medium containing 50 μg / mL kanamycin and cultured at 37°C overnight. Positive clones were selected for colony PCR verification, and finally the correct positive clones were screened out by sequencing. The results of PCR verification are shown in Figure 1 shown.

[0054] Figure 1 In the figure, M represents DNA Marker 15000bp, and lane 2 represents the recombinant plasmid pET28a(+)-alg0392. It was found that the size of the electrophoresis band in lane 2 was consistent with the expected size of gene alg0392, indicating that the recombinant plasmid pET28a-alg0392 was successfully constructed.

[0055] (2) Construction of recombinant bacteria and acquisition of alginate lyase Alg0392

[0056] The successfully verified recombinant bacterial colony was picked and inoculated into 5 mL LB medium, activated at 37°C and 200 rpm overnight, and the bacterial solution was transferred to 500 mL LB medium containing 50 μg / mL kanamycin at a ratio of 1:100, and cultured at 37°C and 200 rpm with shaking until OD 600 =0.4-0.6. Stop shaking, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.1mmol / L, induce expression at 16°C and 200rpm for 16h, centrifuge and discard the supernatant, collect the bacterial precipitate, add 50mmol / L phosphate buffer (PBS) to wash the bacterial precipitate twice, and then resuspend the bacterial precipitate with buffer (50mmol / L PBS, pH=7.4), ultrasonically break for 30min at 400W power, with a working / interval time of 2s / 3s, and collect the supernatant by centrifugation as the crude enzyme solution. The recombinant enzyme in the crude enzyme solution was purified by Ni-NTA agarose affinity chromatography column method, and the eluent was 50mmol / LPBS with 20, 40, 80, and 100mM imidazole, respectively, to obtain the purified alginate lyase Alg0392. The size and purification of alginate lyase Alg0392 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Figure 2 shown.

[0057] Figure 2 In the figure, M indicates protein marker, lane 1 indicates crude enzyme solution, lane 2 indicates flow-through solution, lane 3 indicates 20 mM imidazole eluate, lane 4 indicates 40 mM imidazole eluate, and lane 5 indicates 80 mM imidazole eluate. Figure 2 It can be seen that alginate lyase Alg0392 was eluted at 80 mM imidazole, and the band size was consistent with the expected molecular weight of alginate lyase Alg0392.

[0058] Example 2 Enzymatic property test of alginate lyase Alg0392

[0059] (1) Establishment of enzyme activity determination method

[0060] The 3,5-dinitrosalicylic acid method (DNS) was used to determine the content of reducing sugars generated, and the enzyme activity was determined by the content of reducing sugars. The specific method is: the substrate for determining the enzyme activity is 200mL of 4mg / mL sodium alginate solution (i.e., sodium alginate is dissolved in phosphate / citrate buffer, pH=6.0), 1μL of the crude enzyme solution in Example 1 is added to react in a 30°C water bath for 5min, and then DNS is added to terminate the reaction. After color development in a boiling water bath for 5min, it is quickly placed in ice water and cooled to room temperature. After microcentrifugation, the supernatant is taken to determine its OD value at 540nm. The control group is added with inactivated crude enzyme solution, and three replicates are set for the control group and the experimental group during the enzyme activity determination experiment.

[0061] One enzyme activity (U) is defined as the amount of enzyme required to degrade sodium alginate to produce 1 μmol of reducing sugar per minute at 30°C.

[0062] (2) Optimal reaction pH of alginate lyase Alg0392

[0063] Sodium alginate substrate solutions of different pH concentrations were prepared using 0.1 mol / L citric acid-0.2 mol / L sodium hydrogen phosphate buffer (pH 4.0-8.0) and 50 mmoL / L glycine-sodium hydroxide buffer (pH 8.0-10.0). The enzyme activity of alginate lyase Alg0392 at different pH values ​​was determined according to the enzyme activity determination method in (1). The highest enzyme activity determined was taken as 100%, and the relative enzyme activity of alginate lyase Alg0392 at different pH values ​​was calculated to determine the optimal pH value of the enzyme. The test results are shown in Figure 1. Figure 3 shown.

[0064] Depend on Figure 3 It can be seen that the optimum pH of alginate lyase Alg0392 is 6.0.

[0065] (3) pH stability of alginate lyase Alg0392

[0066] After the crude enzyme solution was incubated with 0.1 mol / L citric acid-0.2 mol / L sodium dihydrogen phosphate buffer (pH 4-8) and 50 mmoL / L glycine-sodium hydroxide buffer (pH 8.0-10.0) at 4°C for 1 hour, the residual enzyme activity was determined. The enzyme activity of the untreated crude enzyme solution was taken as 100%, and the relative enzyme activity of alginate lyase Alg0392 treated with different pH buffers was calculated to determine the pH stability of alginate lyase Alg0392. The test results are shown in Figure 2. Figure 4 shown.

[0067] Depend on Figure 4 It can be seen that after incubation at different pH values ​​for 1 hour, the enzymatic activity of alginate lyase Alg0392 can be maintained above 50% in the range of pH 5.0 to 9.0, and the enzyme has good pH stability.

[0068] (4) Optimal reaction temperature of alginate lyase Alg0392

[0069] Using sodium alginate solution prepared with citric acid-sodium hydrogen phosphate buffer (pH=6.0) as substrate, the enzyme activity of alginate lyase Alg0392 in the temperature range of 0-90°C was determined. The highest enzyme activity determined was 100%, and the relative enzyme activity of alginate lyase Alg0392 at different temperatures was calculated to determine the optimal temperature of the enzyme. Figure 5 shown.

[0070] Depend on Figure 5 It can be seen that the optimum temperature of alginate lyase Alg0392 is 30°C.

[0071] (5) Temperature stability of alginate lyase Alg0392

[0072] The crude enzyme solution was incubated at different temperatures for 30 minutes and then its residual enzyme activity was measured. The enzyme activity of the untreated enzyme solution was taken as 100%, and the relative enzyme activity of alginate lyase Alg0392 at different temperatures was calculated to determine the temperature stability of alginate lyase Alg0392. The test results are shown in Figure 6 shown.

[0073] Depend on Figure 6 It can be seen that after incubation at different temperatures for 30 minutes, the relative enzyme activity of alginate lyase Alg0392 can be maintained at more than 90% in the temperature range of 5-30°C, and at 30-40°C, alginate lyase Alg0392 still has 80% relative enzyme activity. When the temperature is raised to 45°C, the enzyme activity decreases significantly, and after exceeding 50°C, alginate lyase Alg0392 is completely inactivated.

[0074] (6) Effects of organic reagents on alginate lyase Alg0392

[0075] Low concentration (1 mmol / L or 10 mg / mL) and high concentration (5 mmol / L or 50 mg / mL) organic reagents were added to the enzyme activity assay reaction system in (1), wherein in the experiment of adding low concentration organic solvents, the concentrations of methanol, ethanol, isopropanol, and n-butanol were 10 mg / mL, and the concentrations of the other reagents including sodium dodecyl sulfate (SDS), dithiothreitol (DTT), guanidine hydrochloride, acetone, β-mercaptoethanol (β-Met), dimethyl sulfoxide (DMSO), Triton-100, and EDTA were 1 mmol / L; high concentrations of In the organic reagent experiment, the concentration of methanol, ethanol, isopropanol, and n-butanol was 50 mg / mL, and the concentration of other reagents including SDS, DTT, guanidine hydrochloride, acetone, β-Met, DMSO, Triton-100, and EDTA was 5 mmol / L. The enzyme activity was measured after 5 minutes of reaction under the optimal reaction conditions; an equal volume of citric acid-disodium hydrogen phosphate buffer (pH = 6.0) was added to the blank control group. The enzyme activity of the blank control group was taken as 100%, and the relative enzyme activity of alginate lyase Alg0392 after adding different organic reagents was calculated to analyze the effect of organic reagents on enzyme activity. The test results are as follows: Figure 7 shown.

[0076] Figure 7 In the figure, 1 is the blank control group, 2 is SDS, 3 is DTT, 4 is guanidine hydrochloride, 5 is acetone, 6 is β-Met, 7 is DMSO, 8 is Triton-100, 9 is EDTA, 10 is methanol, 11 is ethanol, 12 is isopropanol, and 13 is n-butanol. Figure 7It can be seen that at low and high concentrations, DTT and β-Met both have a significant promoting effect on the activity of alginate lyase Alg0392, and ultimately increase the activity of alginate lyase Alg0392 by nearly 10%. It is speculated that the active center of alginate lyase Alg0392 has a -SH bond, and DTT and β-Met as reducing agents can maintain its active state, thereby maintaining the active conformation of the enzyme, and finally promoting the activity of the enzyme. Under the action of SDS, alginate lyase Alg0392 is completely inactivated. Under the low concentration condition of 1mmol / L or 10mg / mL, dimethyl sulfoxide (DMSO), TritonX-100, EDTA, methanol, ethanol, isopropanol and n-butanol had little effect on the enzyme activity of alginate lyase Alg0392, and its relative enzyme activity was basically maintained at more than 80%. Guanidine hydrochloride and acetone had little inhibitory effect on alginate lyase Alg0392, and the enzyme activity could be maintained at more than 70%. Alginate lyase Alg0392 had good tolerance to TritonX-100 and isopropanol. When the concentration of organic reagents was increased to 5mmol / L or 50mg / mL, the relative enzyme activity of alginate lyase Alg0392 could still be maintained at more than 80%. Under the action of EDTA, guanidine hydrochloride, acetone, methanol and ethanol, the relative enzyme activity of alginate lyase Alg0392 could also be maintained at more than 60%, but high concentration of n-butanol would basically inactivate alginate lyase Alg0392.

[0077] (7) Substrate specificity of alginate lyase Alg0392

[0078] Sodium alginate solution, polyguluronic acid (PolyG) solution and polymannuronic acid (PolyM) solution with a concentration of 4 mg / mL were prepared using citric acid-disodium hydrogen phosphate buffer (pH=6.0), and the crude enzyme solution was added to react for 5 min under the optimal reaction conditions. The optimal reaction conditions were: pH 6.0 and temperature 30°C. The enzyme activity measured when sodium alginate was used as the substrate was defined as 100%, and the preference of alginate lyase Alg0392 for different substrates was analyzed.

[0079] The results showed that alginate lyase Alg0392 had good degradation activity for sodium alginate and PolyM, with the highest degradation activity for PolyM, with a specific activity of 48.15U / mg; the degradation activity for sodium alginate was second, with a specific activity of 30.56U / mg; the degradation activity for PolyG was lower, with a specific activity of only 11.90U / mg. Therefore, it is inferred that the enzyme is a polymannuronate lyase that is specific for PolyM.

[0080] (8) Kinetic parameters of alginate lyase Alg0392

[0081] Sodium alginate solutions of different mass concentrations (0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5 mg / mL) were prepared using citric acid-disodium hydrogen phosphate buffer (pH = 6.0), and the enzyme activity of alginate lyase Alg0392 at different substrate concentrations was determined according to the enzyme activity determination method in (1).

[0082] The reaction rate V is taken as the ordinate and the substrate concentration S is taken as the abscissa, and the MichaelisMenten model is used for fitting (eg Figure 8 The maximum reaction rate (Vmax), Michaelis constant (Km), catalytic constant (Kcat) and Kcat / Km of alginate lyase Alg0392 were calculated.

[0083] Depend on Figure 8 It can be seen that the maximum reaction rate Vmax of alginate lyase Alg0392 is 0.102 μmol / min, the Michaelis constant Km is 3.772 mg / mL, and the Kcat is 87.775 s －1 , Kcat / Km is 23.271 mL / mg·s.

[0084] Example 3 Analysis of hydrolysis products of alginate lyase Alg0392

[0085] Thin layer chromatography (TLC) was used to analyze the degradation product components of each substrate by alginate lyase Alg0392. 5 μL of crude enzyme solution was added to 200 μL of 4 mg / mL sodium alginate, PolyG and PolyM substrate solutions respectively. After the reaction was carried out under the optimal conditions, the supernatant was centrifuged and spotted. The optimal reaction conditions were: pH 6.0 and temperature 30°C. Two groups were set up for comparison, one group reacted for 30 minutes and the other group reacted overnight. After the reaction, the reaction product was obtained and analyzed by TLC. The specific analysis method was as follows: the sample volume was 6 μL, the developing agent was n-butanol, the developing agent was n-butanol: formic acid: water = 4:6:1, after the development was completed, the silica gel plate was dried, 10% sulfuric acid ethanol was evenly sprayed on the silica gel plate, and the color was developed at 100°C for 30 minutes. The test results are as follows Fig. 9 and Fig.10 shown.

[0086] Fig. 9 and Fig.10In the table, M is alginate oligosaccharide standard, 1 and 5 are sodium alginate, 2 is the degradation product of sodium alginate catalyzed by alginate lyase Alg0392 for 30 min, 3 and 7 are PolyM, 4 is the degradation product of PolyM catalyzed by alginate lyase Alg0392 for 30 min, 6 is the degradation product of sodium alginate catalyzed by alginate lyase Alg0392 overnight, and 8 is the degradation product of PolyM catalyzed by alginate lyase Alg0392 overnight. When the reaction time was 30min, the products generated by the degradation of sodium alginate by alginate lyase Alg0392 were mainly disaccharides and trisaccharides, and the products generated by the degradation of PolyM were monosaccharides, disaccharides, and trisaccharides; when alginate lyase Alg0392 reacted with each substrate overnight, the products generated by the degradation of sodium alginate by alginate lyase Alg0392 were mainly monosaccharides, disaccharides, and trisaccharides, and the products generated by the degradation of PolyM were all converted into monosaccharides; in the experimental group with PolyG as the substrate, no obvious substrate generation was observed, which was speculated to be due to the low degradation activity of the enzyme for PolyG. Therefore, it can be seen that alginate lyase Alg0392 is an alginate lyase with a preference for PolyM and both endo- and exo-cutting properties.

[0087] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. An alginate lyase Alg0392, characterized in that The amino acid sequence of the alginate lyase Alg0392 is shown in SEQ ID No.

1.

2. A DNA molecule, characterized in that The DNA molecule is used to encode the alginate lyase Alg0392 as claimed in claim 1.

3. The DNA molecule according to claim 2, characterized in that The nucleotide sequence of the DNA molecule is shown as SEQ ID No.

2.

4. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the DNA molecule as claimed in claim 3.

5. A recombinant bacterium, characterized in that: The recombinant bacteria comprises the nucleotide sequence of the DNA molecule as claimed in claim 3 or the recombinant plasmid as claimed in claim 4.

6. Use of the alginate lyase Alg0392 as claimed in claim 1 in degrading alginate and its derivatives.

7. The use according to claim 6, characterized in that The derivatives include polymannuronic acid and / or polyguluronic acid.

8. The use according to claim 6, wherein the temperature at which the alginate lyase Alg0392 degrades alginate and its derivatives is 25°C to 40°C.

9. The use according to claim 6, wherein the pH of the alginate lyase Alg0392 when degrading alginate and its derivatives is 5.0 to 9.

0.

10. An alginate lysate preparation, characterized in that: The method comprises the alginate lyase Alg0392 as claimed in claim 1 and a solvent, wherein the solvent comprises dithiothreitol and / or β-mercaptoethanol.

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