Alginate lyase mutant, plasmid, recombinant strain and application thereof

Abstract

The invention provides an alginate lyase mutant, a plasmid, a recombinant strain and application thereof, belongs to the technical field of gene engineering and enzyme engineering, and can solve the technical problem that the enzyme activity of the existing alginate lyase cannot meet the efficient, controllable and industrial preparation of alginate oligosaccharide. The alginate lyase mutant disclosed by the invention is an alginate lyase mutant AMDW2, and the amino acid sequence of the alginate lyase mutant AMDW2 is as shown in SEQ ID NO. 1. The alginate lyase mutant AMDW2 is obtained by mutating D amino acid selected around an active center GQIH sequence into W amino acid by original alginate lyase Algt1, and compared with the original alginate lyase Algt1 which is not mutated, the alginate lyase mutant AMDW2 has higher enzyme activity and thermal stability; the alginate oligosaccharide with low polymerization degree can be prepared by using the minimum substrate pentasaccharide, and can be applied to the fields of feeds, foods, cleaning agents or biofuels.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a brown algae lyase mutant, a plasmid, a recombinant strain and application thereof. BACKGROUND

[0002] Brown algae is a natural sulfated polysaccharide widely existing in marine brown algae plants, and has good water solubility and biological activity. Studies have shown that brown algae has wide pharmacological effects in anticoagulation, antioxidant, antiviral, immunomodulation, and antitumor, and thus has important application value in the fields of functional food, drug development, cosmetics, and biological materials. With the promotion of high-value utilization of marine resources, brown algae is becoming one of the hotspots in the research of marine bioactive substances.

[0003] Brown algae is limited in wide application in the fields of food, medicine, and agriculture due to its complex structure, large molecular weight, high viscosity, strong polymerization degree, poor solubility, and low bioavailability. In order to improve the utilization value of brown algae, degradation methods are usually used to convert brown algae into low molecular weight and simple structure brown algae oligosaccharides, which not only improves the solubility and absorption rate of brown algae, but also endows brown algae with multiple biological activities such as anti-inflammatory, antioxidant, immunomodulation, antitumor, and promotion of plant root development. At present, the degradation methods mainly include physical method, chemical method, and enzyme catalysis method. The physical method and the chemical method have problems such as high energy consumption, great environmental pollution risk, and many by-products. In comparison, the enzyme catalysis method has advantages such as high efficiency, mildness, and strong specificity, and can stably produce functional oligosaccharides under green conditions, and gradually becomes the main method for brown algae degradation.

[0004] Brown algae lyase is a kind of hydrolytic enzyme that can specifically degrade the glycosidic bond in brown algae, and widely exists in marine microorganisms such as marine bacteria and fungi. The main function is to decompose high molecular weight brown algae into low molecular weight brown algae oligosaccharides. The enzyme activity of brown algae lyase directly determines the cleavage efficiency and product structure, and has a key influence on the yield, molecular weight distribution, and function of brown algae oligosaccharides. Therefore, obtaining brown algae lyase with high enzyme activity and high stability is of great significance for realizing the efficient, controllable, and industrialized preparation of brown algae oligosaccharides. SUMMARY

[0005] The present application aims at the technical problem that the enzyme activity of the existing brown algae lyase cannot meet the efficient, controllable, and industrialized preparation of brown algae oligosaccharides, and proposes a brown algae lyase mutant AMDW2 with high enzyme activity and high stability. Compared with the original sequence brown algae lyase Algt1, the brown algae lyase mutant AMDW2 has higher enzyme activity and thermal stability, can use the minimum substrate pentasaccharide to prepare oligomerization degree brown algae oligosaccharides, can realize the efficient, controllable, and industrialized preparation of brown algae oligosaccharides, and can be applied in the fields of feed, food, cleaning agent, or biofuel.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: an alginate lyase mutant, namely alginate lyase mutant AMDW2, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0007] FELVDWYLSIPTDNDNGKADSIKENELSGGYENSSYFYTASDGGMVFRCPIAGYKTSTNTSYTRTELREMLRRGDTSIDTQGVNKNNWVFGSAPSSARNAAGGVDGVLRATLAVNHVTTTGDSSQVGRVIIGQIHA NWWEPLRLYYRKLPGNSKGSIYFAHEPEGGSDQWYEMIGSRSSSASDPADGIALNEVFSYEIKVVGNTLTVTISRDGKDDVVKSVDMSDSGYDSSDQYQYFKAGVYNQNNTGDDDDYVQATFYALENSHDGYPY (SEQ ID NO. 1) In one embodiment, the amino acid sequence of the alginate lyase mutant AMDW2 is obtained by mutating the amino acid sequence of alginate lyase Algt1, the amino acid sequence of which is shown in SEQ ID NO.3.

[0008] FELVDWYLSIPTDNDNGKADSIKENELSGGYENSSYFYTASDGGMVFRCPIAGYKTSTNTSYTRTELREMLRRGDTSIDTQGVNKNNWVFGSAPSSARNAAGGVDGVLRATLAVNHVTTTGDSSQVGRVIIGQIHA NDDEPLRLYYRKLPGNSKGSIYFAHEPEGGSDQWYEMIGSRSSSASDPADGIALNEVFSYEIKVVGNTLTVTISRDGKDDVVKSVDMSDSGYDSSDQYQYFKAGVYNQNNTGDDDDYVQATFYALENSHDGYPY (SEQ ID NO. 3) This invention obtains a recombinant alginate lyase mutant AMDW2 with enhanced enzyme activity by mutating the original alginate lyase Algt1, exhibiting high enzyme activity and high stability.

[0009] In one embodiment, the D amino acids at positions 139 and 140 of the amino acid sequence shown in SEQ ID NO. 3 are mutated to W amino acids.

[0010] The alginate lyase mutant AMDW2 of the present application is obtained by selecting D amino acid mutation to W amino acid around the active center GQIH sequence of the original alginate lyase Algt1, and the recombinant alginate lyase mutant AMDW2 has higher enzyme activity and thermal stability compared with the original sequence alginate lyase Algt1, and can utilize the minimum substrate pentasaccharide to prepare low degree of polymerization alginate oligosaccharide.

[0011] In one embodiment, the nucleotide sequence of the alginate lyase mutant encoding gene is shown in SEQ ID NO. 2.

[0012] TTTGAATTGGTTGATTGGTACTTGTCTATTCCAACTGATAACGATGGTAACGGTAAGGCTGATTCTATTAAGGAAAACGAATTGTCTGGTGGTTACGAAAACTCTTCTTACTTTTACACTGCTTCTGATGGTGGTATGGTTTTTAGATGTCCAATTGCTGGTTACAAGACTTCTACTAACACTTCTTACACTAGAACTGAATTGAGAGAAATGTTGAGAAGAGGTGATACTTCTATTGATACTCAAGGTGTTAACAAGAACAACTGGGTTTTTGGTTCTGCTCCATCTTCTGCTAGAAACGCTGCTGGTGGTGTTGATGGTGTTTTGAGAGCTACTTTGGCTGTTAACCATGTTACTACTACTGGTGATTCTTCTCAAGTTGGTAGAGTTATTATTGGTCAAATTCATGCTAACTGGTGGGAACCATTGAGATTGTACTACAGAAAGTTGCCAGGTAACTCTAAGGGTTCTATTTACTTTGCTCATGAACCAGAAGGTGGTTCTGATCAATGGTACGAAATGATTGGTTCTAGATCTTCTTCTGCTTCTGATCCAGCTGATGGTATTGCTTTGAACGAAGTTTTTTCTTACGAAATTAAGGTTGTTGGTAACACTTTGACTGTTACTATTTCTAGAGATGGTAAGGATGATGTTGTTAAGTCTGTTGATATGTCTGATTCTGGTTACGATTCTTCTGATCAATACCAATACTTTAAGGCTGGTGTTTACAACCAAAACAACACTGGTGATGATGATGATTACGTTCAAGCTACTTTTTACGCTTTGGAAAACTCTCATGATGGTTACCCATAC (SEQ ID NO. 2) The present application also provides a recombinant expression vector comprising the coding gene of the alginate lyase mutant.

[0013] The present application also provides a recombinant strain comprising the recombinant expression vector, and the recombinant strain takes Pichia as a carrier. The present application heterologously expresses the recombinant alginate lyase mutant AMDW2 in Pichia X33 to obtain the expression product of the recombinant alginate lyase mutant.

[0014] The application also provides the use of alginate lyase mutant AMDW2 in the preparation of alginate oligosaccharides.

[0015] In some embodiments, the minimum unsaturated oligosaccharide substrate of the alginate lyase mutant AMDW2 is a pentasaccharide, and the minimum unsaturated oligosaccharide product is a monosaccharide. The minimum utilization substrate of the alginate lyase mutant AMDW2 of the application is a pentasaccharide, which can be converted into monosaccharides, disaccharides, trisaccharides and tetrasaccharides, indicating that the alginate lyase mutant AMDW2 has the potential application in the preparation of low-polymerization alginate oligosaccharides.

[0016] The application also provides the use of alginate lyase mutant AMDW2 in feed, food, cleaning agent or biofuel.

[0017] Compared with the prior art, the alginate lyase mutant AMDW2 of the application has high enzyme activity and high stability, can realize efficient, controllable and industrialized preparation of alginate oligosaccharides, and can be applied in the fields of feed, food, cleaning agent or biofuel. The alginate lyase mutant AMDW2 of the application is obtained by selecting D amino acids around the active center GQIH sequence of the original alginate lyase Algt1 and mutating them into W amino acids. Compared with the original sequence alginate lyase Algt1, the recombinant alginate lyase mutant AMDW2 has higher enzyme activity and thermal stability, the minimum utilization substrate is a pentasaccharide, which can be converted into monosaccharides, disaccharides, trisaccharides and tetrasaccharides, and can be used for the preparation of low-polymerization alginate oligosaccharides. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a three-dimensional conformational schematic diagram of the alginate lyase mutant AMDW2 of the application; Figure 2 It is a gene amplification nucleic acid electrophoresis schematic diagram of the alginate lyase mutant AMDW2 of the application; Figure 3 It is an enzyme activity schematic diagram of the alginate lyase mutant AMDW2 of the application and the original alginate lyase Algt1; Figure 4 It is an optimum temperature schematic diagram of the alginate lyase mutant AMDW2 of the application; Figure 5 It is a thermal stability schematic diagram of the alginate lyase mutant AMDW2 of the application and the original alginate lyase Algt1; Figure 6 It is a minimum action substrate TLC analysis diagram of the alginate lyase mutant AMDW2 of the application; Figure 7 It is a minimum action substrate FPLC analysis diagram of the alginate lyase mutant AMDW2 of the application. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all of them. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0020] The present application provides a alginate lyase mutant, a plasmid, a recombinant strain and an application thereof, which has a better application potential in the fields of feed, food, cleaning agent or biofuel.

[0021] When sodium alginate is used as a substrate, the enzyme activity of alginate lyase Algt1 is 212.86 U / mg, and the optimum temperature is 40 DEG C. The alginate lyase Algt1 can maintain 80.9% of the enzyme activity after being treated at 40 DEG C for 90 min. However, the enzyme activity of the alginate lyase Algt1 as a feed additive needs to be further improved.

[0022] In order to further improve the enzyme activity of the alginate lyase Algt1, the spatial structure of the enzyme is analyzed by molecular simulation docking analysis, and the enzyme is mutated by analyzing the action center of the enzyme. The enzyme activity of the alginate lyase mutant ADMW2 obtained is 262.95 U / mg, which is greatly improved compared with the enzyme activity of the alginate lyase Algt1. The optimum temperature of the alginate lyase mutant ADMW2 is 40 DEG C. The residual enzyme activity of the alginate lyase mutant ADMW2 is 72.84% after being treated at 40 DEG C for 2 h, which indicates that the alginate lyase mutant described in the present application can still maintain good thermal stability. The minimum substrate of the alginate lyase mutant ADMW2 is pentasaccharide, which can be converted into monosaccharide, disaccharide, trisaccharide and tetrasaccharide, which indicates that the alginate lyase mutant ADMW2 has an application potential in preparing low degree of polymerization alginate oligosaccharide.

[0023] In order to more clearly and specifically introduce the alginate lyase mutant, the plasmid, the recombinant strain and the application thereof provided in the embodiments of the present application, the following will be described in combination with specific embodiments.

[0024] In the following embodiments, the molecular biology experimental methods not specifically described are all carried out according to the specific methods listed in the book of Molecular Cloning: A Laboratory Manual, Third Edition by J. Sambrook, or according to the instructions of the kit and product. The reagents and biological materials can be obtained from commercial channels unless otherwise specified.

[0025] Example 1 Three-dimensional structure analysis of original alginate lyase and mutation site design As shown in Figure 1 The original alginate lyase Algt1 three-dimensional conformation is mainly β-pleated sheet, and the active center of the original alginate lyase Algt1 contains a GQIH conserved active sequence, which plays a key role in the lyase activity of the enzyme, and the charge of the amino acids around it also has an important influence on the activity of the enzyme. Generally speaking, the stronger the hydrophobicity of the amino acid residues around the active center, the more hydrophobic interactions can be formed with the substrate, and thus the stronger the binding affinity of the enzyme and the substrate. In this embodiment, by analyzing the three-dimensional structure and the conserved site region of the original alginate lyase Algt1, the mutation site and sequence of the alginate lyase mutant are designed, the D amino acid around the GQIH sequence in the active center of the original alginate lyase Algt1 is mutated to W amino acid, i.e., the D amino acids at positions 139 and 140 are both mutated to W amino acid, to obtain the alginate lyase mutant AMDW2, and the hydrophobicity thereof is enhanced.

[0026] Example 2 Gene amplification of alginate lyase mutant AMDW2 DNA amplification enzyme: Phanta ® Super-Fidelity DNA Polymerase The reaction system refers to the enzyme: Buffer 10 μL, dNTP 1 μL, Phanta 1 μL, forward primer 2 μL, reverse primer 2 μL, synthetic template 1 μL, and ddH2O to make up the system to 50 μL The amino acid sequence of the alginate lyase published in the sequence number WP_231902423.1 in the DNA sequence database (GenBank) established by the National Center for Biotechnology Information is used as the amino acid sequence of the wild-type enzyme, and the structure of the wild-type enzyme contains three domains, CBM_4_9, F5_F8_type_C, and Alginate lyase2. The amino acid sequence of the wild-type enzyme is truncated to only retain the Alginate lyase2 domain, to obtain the original alginate lyase Algt1. The gene sequence of the original alginate lyase Algt1 is used as a template for gene mutation PCR amplification of the alginate lyase mutant, wherein the DNA amplification enzyme Phanta® Super-Fidelity DNA Polymerase is used in the reaction. The reaction system refers to the enzyme instruction manual, specifically: Buffer 10 μL, dNTP 1 μL, Phanta 1 μL, forward primer 2 μL, reverse primer 2 μL, synthetic template 1 μL, and ddH2O to make up the system to 50 μL. The annealing temperature in the reaction condition is set to 56 ℃, and the extension time at 72 ℃ is 6 min. Other reaction conditions are performed according to the enzyme instruction manual.

[0027] After the reaction is completed, the PCR reaction product is tested by agarose nucleic acid gel electrophoresis. The results of the nucleic acid electrophoresis test of the gene amplification of the alginate lyase mutant AMDW2 are shown in Figure 2 The DNA amplification band with a molecular weight of 813 bp is obtained. The tested PCR product is further subjected to Dpnl enzyme digestion and Cycle Pure Kit PCR purification kit purification. The specific operation steps are carried out according to the instructions of Dpnl enzyme and Cycle Pure Kit PCR purification kit. The recovered DNA fragment after purification is the PCR product of the alginate lyase mutant.

[0028] Example 3 Construction of recombinant expression vector of alginate lyase mutant AMDW2 2 microliters of Not I and EcoR I enzymes are added to the PCR product, 5 microliters of enzyme reaction buffer are added, and the reaction is carried out at 37°C for 6h. The pichia pastoris X33 expression vector is linearized by enzyme cutting using Not I and EcoR I enzymes. The PCR product of the alginate lyase mutant is connected to the linearized expression vector by in vitro homologous recombination. The heat shock method is used to transfer the linearized recombinant expression vector into E. coli DH5a, and after 12-16h of culture at 37°C, single colony picking is carried out. After verification of the positive transformant clones, the construction of the recombinant expression vector of the alginate lyase mutant is completed.

[0029] Example 4 Expression of alginate lyase mutant AMDW2 in pichia pastoris 2 microliters of Sac I are added to the extracted recombinant expression vector, 5 microliters of enzyme reaction buffer are added, and the reaction is carried out at 37°C for 6h. The constructed recombinant expression vector is linearized by enzyme cutting using Sac I. The linearized recombinant expression vector is transformed into pichia pastoris X33 competent cells by electroporation. The specific transformation method is as follows: the linearized recombinant expression vector is transferred into the competent cells, which are incubated in an ice bath for 30 min, and then subjected to electric shock transformation using an electric transformer. 1 mL of pre-cooled 1 M sorbitol solution is added, and the bacteria are incubated in a 30°C incubator for 1 h. The bacteria are spread on LB plates containing Zeocin antibiotic and cultured at 30°C for 2-3 days.

[0030] The grown yeast transformants are subjected to positive transformant screening. The screened yeast transformants are inoculated into BMGY liquid medium and subjected to shake flask fermentation. The fermentation conditions are as follows: 30°C, 200 rpm, addition of methanol solution once every 24 h to make the final concentration of methanol 1%, induction, and a total of 3 times of addition of methanol solution. After the expression is completed, the bacteria are centrifuged, and the supernatant is collected, which is the prepared alginate lyase mutant AMDW2.

[0031] Example 5 Enzyme activity determination of alginate lyase mutant AMDW2 The heterologous expression mutant alginate lyase AMDW2 and the original alginate lyase Algt1 were purified by Ni+ affinity chromatography, and the protein concentration of the purified enzyme samples was determined using a BCA kit. Enzyme activity was determined using the DNS method, and the specific activity of the enzyme was calculated based on the protein concentration.

[0032] The enzyme activity assay includes the following steps: Add 900 μL of 0.8% sodium alginate solution and 100 μL of appropriately diluted enzyme solution to a 10 mL test tube as the experimental group. The enzyme solution should be diluted until the absorbance value at the time of enzyme activity assay falls between 0.2 and 1.0. Add 900 μL of 0.8% sodium alginate solution and 100 μL of inactivated enzyme solution to a 10 mL test tube as the control group. Vortex the experimental and control group test tubes and react them in a 40 ℃ water bath for 30 min. After the water bath, add 1 mL of DNS solution to the experimental and control group test tubes and boil them in a water bath for 10 min. Cool immediately and bring the volume of the experimental and control groups to 10 mL with distilled water. Measure the absorbance at 520 nm.

[0033] Subtracting the absorbance of the control group from the absorbance of the experimental group gives the absorbance of the reducing sugar produced by enzymatic hydrolysis. The enzyme activity unit of alginate lyase is defined as the amount of enzyme required to produce 1 μmol of reducing sugar per minute. The enzyme activity calculation formula is: Enzyme activity = (Content of reducing sugar produced by enzymatic hydrolysis × Enzyme solution dilution factor) / (Reaction time × Amount of diluted enzyme solution added × M), where the content of reducing sugar produced by enzymatic hydrolysis is in μg, the reaction time is in min, the amount of diluted enzyme solution added is in mL, and M refers to the molar mass of mannuronic acid and guluronic acid, with a value of 180.16. The enzyme activity calculation results of the alginate lyase mutant AMDW2 and the original alginate lyase Algt1 are shown below. Figure 3 As shown.

[0034] Depend on Figure 3 It can be seen that the specific activity of the original alginate lyase Algt1 is 212.86 U / mg, and the specific activity of the alginate lyase mutant AMDW2 is 262.95 U / mg. The specific activity of the alginate lyase mutant AMDW2 is significantly improved compared with that of the original alginate lyase Algt1, indicating that the original alginate lyase Algt1 was mutated at a site to obtain the alginate lyase mutant AMDW2 with improved enzyme activity. Example 6 Optimal temperature of alginate lyase mutant AMDW2 In a buffer system with a pH of 7.0, using 0.8% sodium alginate as a substrate, the enzyme activity of the recombinant alginate lyase mutant AMDW2 was measured at different temperatures (30℃, 35℃, 40℃, 45℃, 50℃, and 55℃). With the highest enzyme activity defined as 100%, the relative enzyme activity at different temperatures was calculated to determine the optimal temperature for the recombinant alginate lyase mutant AMDW2. The calculated results of the relative enzyme activity of the alginate lyase mutant AMDW2 at different temperatures are shown below. Figure 4 As shown.

[0035] Depend on Figure 4 It can be seen that, under neutral buffered pH conditions, the optimal temperature of the alginate lyase mutant AMDW2 is 40℃, which is not significantly different from the optimal temperature of the original alginate lyase Algt1.

[0036] Example 7 Temperature stability of the alginate lyase mutant AMDW2 A 0.8% sodium alginate solution was prepared using a pH 7.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer as a substrate. The original alginate lyase Algt1 and the recombinant alginate lyase mutant AMDW2 were treated at 40 °C for 2 h. Samples were taken every 0.5 h, and enzyme activity was determined using the DNS method. The activity of the untreated enzyme solution was considered 100%. The residual enzyme activities of the original alginate lyase Algt1 and the alginate lyase mutant AMDW2 after treatment at the same temperature for different times were calculated. The thermostability results of the alginate lyase mutant AMDW2 and the original alginate lyase Algt1 are shown below. Figure 5 As shown.

[0037] Depend on Figure 5 It can be seen that after treatment at 40 ℃ for 2 h, the residual enzyme activity of the alginate lyase mutant AMDW2 was 72.84%, while the residual enzyme activity of the original alginate lyase after treatment at 40 ℃ for 2 h was 70.86%, indicating that the alginate lyase mutant described in this invention can still maintain good thermal stability. The alginate lyase mutant AMDW2 can still maintain high enzyme activity after long-term treatment at 40 ℃, indicating that the alginate lyase mutant can be active at 40 ℃ to prepare alginate oligosaccharides. Moreover, the comparison shows that the thermal stability of the alginate lyase mutant AMDW2 is improved compared with that of the original alginate lyase Algt1.

[0038] Example 8 Minimum Utilization Substrate of Alginate Lyase Mutant AMDW2 To determine the minimum utilization substrate of alginate lyase mutant AMDW2, 100 μL of mannuronic acid with a concentration of 1 mg / mL and a degree of polymerization of 2-6 was used as the substrate, and then 100 μL of purified and diluted alginate lyase mutant AMDW2 enzyme solution with a concentration of 10 U was added, and incubated at 40 ℃ for 12 h. The determination was performed by using thin layer chromatography and FPLC, respectively. The determination result of thin layer chromatography is shown in Figure 6 The determination result of FPLC is shown in Figure 7 .

[0039] As shown in Figure 6 and Figure 7 , the minimum utilization substrate of alginate lyase mutant AMDW2 is pentasaccharide, which can be converted into monosaccharide, disaccharide, trisaccharide and tetrasaccharide, indicating that alginate lyase mutant AMDW2 has application potential for preparing oligosaccharide of low degree of polymerization alginate.

[0040] The above-described embodiments only describe the preferred embodiments of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications, modifications, evolutions and improvements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. An alginate lyase mutant, characterized in that, The alginate lyase mutant is alginate lyase mutant AMDW2, and the amino acid sequence of the alginate lyase mutant AMDW2 is shown in SEQ ID NO.

1.

2. The alginate lyase mutant according to claim 1, characterized in that, The amino acid sequence of the alginate lyase mutant AMDW2 was obtained by mutating the amino acid sequence of alginate lyase Algt, as shown in SEQ ID NO.

3.

3. The alginate lyase mutant according to claim 2, characterized in that, As shown in SEQ ID NO. 3, the D amino acid at positions 139 and 140 is mutated to the W amino acid.

4. The encoding gene of the alginate lyase mutant according to claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

2.

5. A recombinant expression vector, characterized in that, Includes the encoding gene of the alginate lyase mutant as described in claim 4.

6. A recombinant bacterial strain, characterized in that, The recombinant expression vector of claim 5 is included, wherein the recombinant strain uses Pichia pastoris as the vector.

7. The recombinant strain according to claim 6, characterized in that, The Pichia pastoris includes Pichia pastoris X33.

8. Application of the alginate lyase mutant AMDW2 in the preparation of alginate oligosaccharides.

9. The application of the alginate lyase mutant AMDW2 according to claim 8 in the preparation of alginate oligosaccharides, characterized in that, The smallest unsaturated oligosaccharide substrate of the alginate lyase mutant AMDW2 is a pentasaccharide, and the smallest unsaturated oligosaccharide product is a monosaccharide.

10. Application of the alginate lyase mutant AMDW2 in feed, food, cleaning agents or biofuels.

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