Alginate lyase mutant Amut8C as well as coding gene and application thereof

Abstract

The invention provides an alginate lyase mutant Amut8C as well as a coding gene and application thereof, belongs to the technical field of gene engineering and enzyme engineering, and can solve the problem that existing alginate lyase cannot meet the requirements of severe reaction conditions such as high temperature and acidity in industrial application. The amino acid sequence of the alginate lyase mutant Amut8C disclosed by the invention is as shown in SEQ ID NO. 1. According to the space structure of the alginate lyase Algt, disulfide bonds in molecules are analyzed, the alginate lyase is mutated, and the obtained alginate lyase mutant Amut8C has heat resistance, acid and alkali resistance and digestive enzyme tolerance, and compared with the alginate lyase Algt, the alginate lyase mutant Amut8C has the advantages that the content of the alginate lyase mutant Amut8C is increased, and the content of the alginate lyase mutant Amut8C is increased. The alginate oligosaccharide with the polymerization degree of 1-4 prepared from the alginate lyase mutant Amut8C under the same enzymolysis condition is higher in yield. The method can be applied to the fields of feeds, foods, detergents 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 Amut8C with improved thermal stability, a coding gene thereof and application. BACKGROUND

[0002] Brown algae is a kind of polysaccharide widely existing in various brown algae, which is the general term of hydrophilic derivatives of alginic acid. Due to the highly complex molecular structure, large molecular weight, high viscosity and high degree of polymerization of brown algae, it faces many challenges such as poor solubility and low bioavailability in practical application, which seriously limits its wide development and utilization in food, medicine and agriculture and other fields. In order to solve the above problems, brown algae is usually converted into brown algae oligosaccharide with simpler structure and smaller molecular weight by degradation method, which not only improves its solubility and bioavailability, but also endows it with various excellent biological activities, such as anti-inflammatory, antioxidant, immune regulation, anti-tumor, promotion of plant root development and other functions, and has good application prospect. At present, the methods for degrading brown algae mainly include physical method, chemical method and enzyme catalysis method, among which the physical method and the chemical method have obvious shortcomings in energy consumption and environmental friendliness, and are easy to produce by-products and increase the subsequent processing cost; compared with the above two methods, the enzyme catalysis method as a kind of efficient, controllable and green biological transformation technology can selectively degrade brown algae under mild conditions, which not only reduces the impact on the environment, but also improves the yield and functional retention of brown algae oligosaccharide, and gradually becomes the main method for degrading brown algae.

[0003] Brown algae lyase is a kind of hydrolytic enzyme that can specifically degrade the α-(1→3) or α-(1→4) glycosidic bond in brown algae molecules, mainly derived from marine microorganisms such as marine bacteria, fungi and part of marine actinomycetes. At present, the brown algae lyase used for industrial production of brown algae oligosaccharide is mainly derived from Bacillus, Pseudomonas and marine Streptomyces, and has shown wide application prospect in the development of functional oligosaccharide, medicinal active substances and agricultural biological stimulants. Brown algae lyase can efficiently and selectively degrade high molecular weight brown algae into low molecular weight brown algae oligosaccharide during preparation, thereby significantly improving the solubility and biological activity of brown algae. In industrial application, in order to meet the demand of harsh reaction conditions such as high temperature and acidity, brown algae lyase with heat resistance, acid and alkali resistance and digestive enzyme resistance needs to be developed to improve its applicability in food, medicine and agriculture and other fields. SUMMARY

[0004] The present application aims at the technical problem that the existing brown algae lyase cannot meet the demand of harsh reaction conditions such as high temperature and acidity in industrial application, and the applicability of brown algae lyase in food, medicine and agriculture and other fields needs to be improved, and proposes a brown algae lyase with heat resistance, acid and alkali resistance and digestive enzyme resistance.

[0005] To achieve the above object, the technical scheme adopted by the present application is as follows: a brown algae pectin lyase mutant Amut8C, the amino acid sequence of the brown algae pectin lyase mutant Amut8C is shown as SEQ ID NO. 1.

[0006] MKRFKATPAMTIGCAAFLSVYMAAANASIQNPGFESDWDNWTDSDPSAISGVAYSCSKSAKITGSGGRFEQQVSVQTGTNYRLTAYVNCDGTVGAVVNGTTYDADVSSSNWQAVNVEFNSGSAQSVTVFGAYNGGEGRFDNFTLEALCDSGSCQSGSNLSVSAAYDDGTNDGHGPANVLDGSLADESRWSSEGVKWITLDLGSVKNVEGVDIAWYKGDQRISYFYIETSTDNSNWTTVLSGGQSSGTSADMEHYDVSNIEARYVRLTCTGNSANSWNSILEFDVIGCGDGSDGGSSSGGGSSSGGGSSSGGSSCCSSSGSGNLDPTLPPSGNFELVDWYLSIPTDNDGNGKADSIKENELSGGYENSSYFYTASDGGMVFRCPIAGYKTSTNTSYTRTELREMLRRGDTSIDTQGVNKNNWVFCSAPSSARNAAGGVDGVLRATLAVNHVTTTGDSSQVGRVIIGQIHANDDEPLRLYYRKLPGNSKGSIYFAHEPEGGSDQWYEMIGSRSSSASDPADGIALNEVFSYEIKVVGNTLTVTISRDCKDDVVKSVDMSDSGYDSSDQYQYFKAGVYNQNNTGDDDDYVQATFYALENSHDGYPY (SEQ ID NO. 1) In one embodiment, the amino acid sequence of the brown algae pectin lyase mutant Amut8C is prepared by mutating the amino acid sequence of brown algae pectin lyase Algt, and the original amino acid sequence of the brown algae pectin lyase Algt is shown as SEQ ID NO. 3.

[0007] MKRFKATPAMTIGCAAFLSVYMAAANASIQNPGFESDWDNWTDSDPSAISGVAYSGSKSAKITGSGGRFEQQVSVQTGTNYRLTAYVNGDGTVGAVVNGTTYDADVSSSNWQAVNVEFNSGSAQSVTVFGAYNGGEGRFDNFTLEALGDSGSCQSGSNLSVSAAYDDGTNDGHGPANVLDGSLADESRWSSEGVKWITLDLGSVKNVEGVDIAWYKGDQRISYFYIETSTDNSNWTTVLSGGQSSGTSADMEHYDVSNIEARYVRLTGTGNSANSWNSILEFDVIGCGDGSDGGSSSGGGSSSGGGSSSGGSSGGSSSGSGNLDPTLPPSGNFELVDWYLSIPTDNDGNGKADSIKENELSGGYENSSYFYTASDGGMVFRCPIAGYKTSTNTSYTRTELREMLRRGDTSIDTQGVNKNNWVFGSAPSSARNAAGGVDGVLRATLAVNHVTTTGDSSQVGRVIIGQIHANDDEPLRLYYRKLPGNSKGSIYFAHEPEGGSDQWYEMIGSRSSSASDPADGIALNEVFSYEIKVVGNTLTVTISRDGKDDVVKSVDMSDSGYDSSDQYQYFKAGVYNQNNTGDDDDYVQATFYALENSHDGYPY (SEQ ID NO. 3) In one embodiment, the amino acid sequence as shown in SEQ ID NO. 3 is mutated to cysteine C at positions 56, 89, 148, 268, 314, 315, 424, 546.

[0008] In one embodiment, the nucleotide sequence of the alginate lyase mutant Amut8C-encoding gene is as shown in SEQ ID NO. 2.

[0009] The application further provides a recombinant expression vector comprising the coding gene of the alginate lyase mutant Amut8C.

[0010] The application further provides a recombinant strain comprising the recombinant expression vector, and the recombinant strain takes Pichia pastoris as a carrier.

[0011] In one embodiment, the Pichia pastoris comprises Pichia pastoris X33.

[0012] The application further provides a preparation method of the alginate lyase mutant Amut8C, comprising the following steps: transforming the recombinant expression vector comprising the alginate lyase mutant coding gene into a host cell to obtain a recombinant strain; culturing the recombinant strain to induce expression of the recombinant alginate lyase mutant Amut8C; recovering and purifying the expressed alginate lyase mutant Amut8C.

[0013] The application further provides an application of the alginate lyase mutant Amut8C in preparation of alginate oligosaccharide.

[0014] The application further provides an application of the alginate lyase mutant Amut8C in feed, food, cleaning agent or biofuel.

[0015] Compared with the prior art, the application has the advantages and positive effects that the application provides the alginate lyase mutant Amut8C with improved thermal stability, which can be applied in feed, food, cleaning agent or biofuel; the application mutates the alginate lyase Algt according to the spatial structure of the alginate lyase Algt by analyzing the intramolecular disulfide bond, and obtains the alginate lyase mutant Amut8C, which has heat resistance, acid and alkali resistance and digestive enzyme resistance, and the enzyme activity and thermal stability of the alginate lyase mutant Amut8C obtained by the application are improved compared with the alginate lyase Algt, and the alginate oligosaccharide with a polymerization degree of 1-4 obtained by the alginate lyase mutant Amut8C under the same enzymolysis condition has a higher yield. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 FIG. 1 is a schematic diagram of comparison of enzyme activity of the alginate lyase mutant Amut8C and the original alginate lyase Algt; Figure 2 FIG. 3 is a schematic diagram of the optimum temperature of the alginate lyase mutant Amut8C; Figure 3 FIG. 5 is a schematic diagram of T of the alginate lyase mutant Amut8C and the original alginate lyase Algt; 50Value comparison schematic diagram; Figure 4 The figure is the yield of polymeric degree 1-4 alginate oligosaccharide and the proportion of each polymeric degree prepared by the original alginate lyase Algt of the application. 1 / 2 Value comparison schematic diagram; Figure 5 The figure is the yield of polymeric degree 1-4 alginate oligosaccharide and the proportion of each polymeric degree prepared by the original alginate lyase Algt of the application. Figure 6 The figure is the yield of polymeric degree 1-4 alginate oligosaccharide and the proportion of each polymeric degree prepared by the alginate lyase mutant Amut8C of the application. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the application.

[0018] The embodiments of the application provide an alginate lyase mutant Amut8C, a coding gene and application thereof. According to the spatial structure of the alginate lyase Algt, the enzyme is mutated by analyzing the intramolecular disulfide bond, so that the alginate lyase mutant Amut8C is obtained, which has heat resistance, acid and alkali resistance and digestion enzyme resistance.

[0019] At present, by cloning, heterologous expression and rational molecular modification of the lyase gene from natural sources, a variety of recombinant enzymes with excellent enzymatic properties have been obtained, which provides strong support for the large-scale application of alginate lyase. The existing alginate lyase Algt has good stability in the pH range of 5.0-9.0, and the optimum temperature is 40℃, and the enzyme activity can be maintained at 80.5% at 45℃ for 30 min. However, as a feed additive, the thermal stability and enzyme activity of the alginate lyase Algt need to be further improved.

[0020] Disulfide bond is a kind of relatively stable covalent bond in protein molecules, which is formed by the oxidation of thiol between two cysteine residues. In proteins, disulfide bond can effectively stabilize the spatial conformation of the peptide chain, and the increase of the number thereof is usually closely related to the improvement of the overall structural stability of the protein. Generally speaking, the more the number of disulfide bonds, the more stable the spatial conformation of the enzyme, and the stronger the stability. Therefore, by reasonably introducing disulfide bond, the resistance of the protein to external environmental factors such as high temperature, acid and alkali can be significantly enhanced, so as to improve its adaptability and application value under industrial conditions. In order to improve the heat resistance of alginate lyase Algt, the spatial structure of the enzyme is analyzed by molecular simulation docking, and the disulfide bond in the structure is predicted. Based on the original amino acid sequence of alginate lyase Algt, the potential number of disulfide bonds of the mutant Amut8C is increased when the 8 mutation sites are mutated to cysteine C. Therefore, the amino acids in the 56th, 89th, 148th, 268th, 314th, 315th, 424th and 546th positions of the predicted amino acid sequence of alginate lyase Algt are all mutated to cysteine C, and the alginate lyase mutant Amut8C is obtained. It is verified by experiment that the enzyme activity of the obtained alginate lyase mutant Amut8C is significantly improved, and the optimum temperature is 40 DEG C; the thermal stability is improved, the T 50 value is 57.7 DEG C, the t 1 / 2 value is 27 min; the yield of alginate oligosaccharide with a polymerization degree of 1-4 is 89%, which is higher than that of alginate oligosaccharide with a polymerization degree of 1-4 prepared by the original sequence alginate lyase Algt under the same enzymolysis conditions.

[0021] In order to more clearly and detailedly introduce the alginate lyase mutant Amut8C and its encoding gene and application provided by the embodiment of the present application, the following will be described in combination with specific examples.

[0022] In the following examples, the molecular biology experimental methods not specifically described are all carried out according to the specific methods listed in the book of Molecular Cloning Experiment Guide, third edition J. Sambrook, or according to the instructions of the kit and product; the reagents and biological materials can be obtained from commercial channels if not specifically described.

[0023] The experimental materials and reagents related to the embodiment of the present application are as follows: 1. Strains and vectors: The expression host Pichia pastoris X33, the expression vector pPICZ alpha A, Amp and Zeocine are purchased from Invitrogen Company.

[0024] 2. Enzymes and reagent kits: PCR enzyme and ligase are purchased from Takara Company, and plasmid extraction kit and gel recovery kit are purchased from Omega Company.

[0025] 3. Culture medium: LB medium: 1% tryptone, 0.5% yeast extract, 1% NaCl; YPD medium: 1% yeast extract, 2% tryptone, 2% glucose; MD medium: 1.34% YNB, 0.4 mg / L biotin, 2% glucose; BMGY medium: 1% yeast extract, 2% tryptone, 1.34% YNB, 0.00004% biotin, 1% glycerol (V / V); BMMY medium: 1% yeast extract, 2% tryptone, 1.34% YNB, 0.00004% biotin, 0.5% methanol (V / V), pH 4.0.

[0026] Example 1 Construction of alginate lyase mutant engineering bacteria Construction of expression vector: The amino acid sequence of alginate lyase published in the DNA sequence database (GenBank) established by the National Center for Biotechnology Information with the sequence number WP_231902423.1 was used as the original amino acid sequence Algt. After site analysis of the original amino acid sequence Algt, the sites at positions 56, 89, 148, 268, 314, 315, 424 and 546 were all mutated to cysteine C by a conventional method to prepare an artificial gene. PCR primers containing the mutant site genes were designed at the corresponding sites of the gene, and PCR amplification was performed using a PCR high-fidelity enzyme. After in vitro recombination and ligation with the pPICZαA vector, the competent cells DH5α of Escherichia coli were heat-shocked and transformed. The transformants were coated on LB+Zeocin plates and incubated at 37°C. After the appearance of the transformants, single colonies were selected for verification. The positive transformants were sequenced, and the transformants with correct sequences were used for large-scale preparation of recombinant plasmids. Expression in Pichia pastoris: The expression plasmid vector DNA was linearized with the restriction enzyme Sac1. 100 uL of yeast competent cells were mixed with 10 uL of linearized vector and then transferred to a pre-cooled electroporation cup for electroporation. The conditions for electroporation were 1.5 kV and 5 msec. After electroporation, 1 mL of sorbitol solution with a concentration of 1 M was added, and the mixture was transferred to a 1.5 mL centrifuge tube and incubated at 30°C for 1 h. Then, the mixture was centrifuged at 5000 rpm for 5 min, and the bacterial cells were collected and coated on YPD selection plates and incubated at 30°C for 2-3 days. The transformants growing on the plates were selected for further expression experiments.

[0027] The single colony of the transformed plate with the transformed plate was picked up with the sterilized toothpick, and the plate was placed in the 30 °C incubator for 2 days, until the colony grew. The transformed strain was inoculated in 3 ml BMGY medium and cultured at 30 °C for 48 h, and the bacterial cells were collected by centrifugation. 1 ml of BMMY induction medium containing 1% methanol was added, and the induction culture was continued at 30 °C. After 48 h, the enzyme activity of the supernatant of each strain was detected, and the transformed strain with high glucose oxidase activity was screened.

[0028] Example 2 Fermentation of recombinant alginate lyase mutant Amut8C in Pichia pastoris Fermentation expression of alginate lyase mutant in a shake flask: The transformed strain with high enzyme activity was inoculated in 300 ml BMGY liquid medium and cultured at 30 °C, 200 rpm for 48 h for bacterial enrichment. The bacterial cells were collected by centrifugation at 4000 x g for 5 min, and the supernatant was discarded gently. The bacterial cells were transferred to 100 ml BMMY liquid medium containing 1% methanol and induced at 30 °C, 200 rpm for 72 h. The activity of the original alginate lyase and its mutant was determined. During the induction culture, methanol solution was added every 24 h to maintain the final concentration of methanol at about 1%, and the supernatant was collected by centrifugation at 10000 x g for 10 min.

[0029] Purification of alginate lyase mutant Amut8C: The supernatant of alginate lyase mutant expressed in a shake flask was collected. First, desalting and concentration were performed using a 10 kDa membrane package, and then anion exchange column chromatography was used for purification to collect electrophoretically pure liquid as a sample for enzyme property research.

[0030] Example 3 Enzymatic property analysis of recombinant alginate lyase mutant Amut8C Enzymatic activity of original alginate lyase Algt and its mutant Amut8C: The enzyme activity of alginate lyase mutant and original alginate lyase was determined by DNS method, and the specific activity of the enzyme was calculated according to the protein concentration.

[0031] 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 to an absorbance value between 0.2 and 1.0 when measuring enzyme activity. 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 each test tube to 10 mL with distilled water. Measure the absorbance at 520 nm.

[0032] 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 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 result is as follows: Figure 1 As shown.

[0033] Depend on Figure 1 It can be seen that the specific activity of the alginate lyase mutant Amut8C is 106.8 U / mg, while the enzyme activity of the original alginate lyase Algt is 53.9 U / mg, indicating that the enzyme activity of the alginate lyase mutant Amut8C is significantly improved compared to that of the original alginate lyase Algt.

[0034] The optimal temperature for the alginate lyase mutant Amut8C is: In a buffer system with a pH of 7.0, using 0.8% sodium alginate as a substrate, the enzyme activity of alginate lyase Amut8C was measured at different temperatures (30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, and 55 ℃). The highest enzyme activity was defined as 100%, and the relative enzyme activity at different temperatures was calculated to determine the optimal temperature for alginate lyase Amut8C. The calculation results are shown below. Figure 2 As shown.

[0035] Depend on Figure 2 It can be seen that, under the same pH conditions, the optimal temperature of the alginate lyase mutant Amut8C is 40 °C. Compared with the original alginate lyase Algt, the optimal operating temperature of the alginate lyase mutant Amut8C provided by this invention has not changed significantly.

[0036] The original alginate lyase Algt and its mutant Amut8C T 50 value and t 1 / 2 value: T 50 value and t 1 / 2 The values ​​are two important indicators for measuring heat resistance performance, among which T is... 50 The value refers to the temperature at which enzyme activity decreases to 50% of its maximum activity; a higher temperature indicates better heat resistance. 1 / 2 The value refers to the time required for enzyme activity to decrease to half of its maximum activity when treated at the same temperature for different durations; the longer the time, the better the heat resistance. 50 Value measurement results are as follows Figure 3 As shown.

[0037] Depend on Figure 3 It can be seen that the T of the primitive alginate lyase Algt 50 The value was 53.2 °C, and the T value of the alginate lyase mutant Amut8C was... 50 The value was 57.7 °C, which was 4.2 °C higher than that of the original alginate lyase Algt. This shows that the thermostability of the alginate lyase mutant Amut8C has been improved to a certain extent.

[0038] The enzymes were treated at 50 °C for 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min, respectively. The relative enzyme activities of the original alginate lyase Algt and the alginate lyase mutant Amut8C were measured and calculated. The results are shown below. Figure 4 As shown in the figure. The time required for the relative enzyme activity to reach 50% was calculated based on the fitted curve. The results showed that the alginate lyase mutant Amut8C retained 50% of its relative enzyme activity after treatment at 50 ℃ for 27 min, i.e., the time required for the alginate lyase mutant Amut8C to reach 50% of its relative enzyme activity. 1 / 2 The value was 27 min, exhibiting the best heat resistance, while the original alginate lyase Algt had a t value of 27 min. 1 / 2 The value was 19.2 min, and the t value of the alginate lyase mutant Amut8C was... 1 / 2 The value was higher than that of the original alginate lyase Algt. 1 / 2 The value indicates that the heat resistance of the alginate lyase mutant Amut8C is superior to that of the original alginate lyase Algt.

[0039] Example 4: Analysis of the yield and proportion of oligosaccharides with polymerization degrees of 1-4 prepared from alginate lyase and its mutant Amut8C. 100 g of sodium alginate was weighed into 1000 mL of ultrapure water to prepare a substrate stock solution with a concentration of 10%; 50 mL of alginate lyase mutant and original alginate lyase were added to the substrate stock solution, respectively, and stirred at 40°C for 6 h to determine the enzymatic hydrolysate; at the same time, in order to remove impurities, the reaction was centrifuged at 4000 rpm for 10 min, and then purified by freeze-drying.

[0040] The purified product was detected by FPLC: Superdex TM 30Increase10 / 300GL column, the mobile phase was 0.2 M NH4HCO3, the flow rate was 0.3 mL / min, and the variable wavelength detector (VWD) was used to detect at 235 nm, by analyzing the peak area of FPLC and the peak area of oligosaccharides with different degrees of polymerization, the yield of oligosaccharides with degrees of polymerization 1-4 and the proportion of oligosaccharides with different degrees of polymerization were calculated, and the results are shown in Figure 5

[0041] As can be seen from Figure 5 , the yield of alginate oligosaccharides with degrees of polymerization 1-4 prepared by alginate lyase mutant Amut8C is 89%, which is higher than the yield of alginate oligosaccharides prepared by original alginate lyase Algt, which is 82%, indicating that alginate lyase mutant Amut8C is superior to original alginate lyase Algt in preparing alginate oligosaccharides with degrees of polymerization 1-4.

[0042] 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, Amut8C, characterized in that, The amino acid sequence of the alginate lyase mutant Amut8C is shown in SEQ ID NO.

1.

2. The alginate lyase mutant Amut8C according to claim 1, characterized in that, The amino acid sequence of the alginate lyase mutant Amut8C is obtained by mutating the amino acid sequence of alginate lyase Algt, and the amino acid sequence of alginate lyase Algt is shown in SEQ ID NO.

3.

3. The alginate lyase mutant Amut8C according to claim 2, characterized in that, As shown in SEQ ID NO. 3, the amino acid sequence is mutated to cysteine ​​C at positions 56, 89, 148, 268, 314, 315, 424, and 546.

4. The encoding gene of the alginate lyase mutant Amut8C 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 Amut8C 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. A method for preparing an alginate lyase mutant, Amut8C, characterized in that, Includes the following steps: A recombinant expression vector containing the gene encoding an alginate lyase mutant was transformed into host cells to obtain a recombinant strain. The recombinant strain was cultured to induce the expression of the recombinant alginate lyase mutant Amut8C; The expressed alginate lyase mutant Amut8C was recovered and purified.

9. Application of an alginate lyase mutant, Amut8C, in the preparation of alginate oligosaccharides.

10. Application of an alginate lyase mutant, Amut8C, in feed, food, cleaning agents, or biofuels.

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