Alginate lyase Alya1 as well as modification method and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to alginate lyase Alaa1 as well as a modification method and application thereof. The amino acid sequence of the alginate lyase Alya1 is as shown in SEQ ID NO. 1, and the nucleotide sequence of the Alya1 gene for coding the alginate lyase Alya1 is as shown in SEQ ID NO. 2. The 406th glutamic acid of the alginate lyase Alya1 is mutated into histidine or lysine, and the high temperature resistance, the thermal stability, the pH stability and the half-life period of the alginate lyase Alya1 are remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to an alginate lyase Alya1 and a modification method and application thereof. Background Art

[0002] Alginate is a linear anionic polysaccharide polymer formed by connecting β-D-mannuronic acid (M) and its C5 diastereomer α-L-guluronic acid (G) through 1,4-O glycosidic bonds. Due to its high viscosity and gelling properties, it has been used in the food, biomaterials and pharmaceutical industries, but its low solubility limits the further development and application of alginate. After degradation, alginate can obtain brown algae oligosaccharides with low polymerization degree and high solubility. Oligomeric alginate oligosaccharides have multiple physiological activities such as immunomodulation, antibacterial, anti-tumor, anti-inflammatory, anticoagulant, plant growth promotion, improving plant stress resistance, and serving as a fruit and vegetable preservative to extend the shelf life of fruits and vegetables.

[0003] At present, there are three main methods for degrading alginate: physical method, chemical method, and bioenzyme method. The physical degradation method has the disadvantages of low degradation efficiency, high cost, and high product polymerization degree. The chemical degradation process is not easy to control, and the by-products are not conducive to the separation and purification of subsequent products. The bioenzyme method is to specifically degrade alginate by alginate lyase, which has the advantages of mild reaction conditions, easy control, and high product purity. Compared with the physical method and the chemical method, the brown algae oligosaccharides obtained by degrading alginate by bioenzyme have a lower polymerization degree and the non-reducing end of the oligosaccharide contains an unsaturated double bond, which has better biological activity. The wild-type strain that produces alginate lyase contains a variety of alginate lyases, which are essentially isozymes, but the alginate lyases produced by different encoding genes have different substrate specificities. Therefore, it is difficult to controllably produce brown algae oligosaccharides with uniform structure through wild bacteria, and the wild-type strain has a low enzyme production and cannot be industrialized. By specifically expressing an alginate lyase through heterologous expression, we can not only obtain high-yield genetically engineered bacteria but also obtain brown algae oligosaccharides with uniform structure to meet the needs of industrial production. In recent years, molecular modification strategies have been widely used in alginate lyase, which has greatly improved the application performance of alginate lyase. Summary of the invention

[0004] The purpose of the present invention is to provide an alginate lyase Alya1 and a modification method and application thereof.

[0005] To achieve the above object, the present invention adopts the following technical solutions: A alginate lyase Alya1, the amino acid sequence of the alginate lyase Alya1 is shown as SEQ ID NO.1, and the nucleotide sequence of the Alya1 gene encoding the alginate lyase Alya1 is shown as SEQ ID NO.2.

[0006] The preparation method of the alginate lyase Alya1 comprises the following steps: introducing EcoRI and XhoⅠ restriction sites at both ends of the Alya1 gene sequence, and then connecting to the pMD-18T vector to obtain the recombinant vector pMD-18T-Alya1; the recombinant vector pMD-18T-Alya1 and the pET28a vector are both double-digested with restriction endonucleases EcoRI and XhoⅠ, and then connected by T4 DNA ligase to obtain the recombinant vector pET28a-Alya1; the recombinant vector pET28a-Alya1 is transformed into the host bacteria E.coilBL21(DE3) to obtain the recombinant bacteria E.coli BL21(DE3)-pET28a-Alya1; the recombinant bacteria E.coli BL21(DE3)-pET28a-Alya1 is fermented and induced to express and purified to obtain the alginate lyase Alya1.

[0007] The above-mentioned alginate lyase Alya1 is used in degrading sodium alginate, degrading polyguluronic acid, degrading polymannuronic acid, and preparing brown algae oligosaccharides.

[0008] A method for mutating the alginate lyase Alya1, wherein the glutamic acid at position 406 of the alginate lyase Alya1 is mutated to improve the high temperature resistance, thermal stability, pH stability and half-life of the alginate lyase Alya1; Furthermore, the mutation modification is to mutate the 406th glutamic acid of the alginate lyase Alya1 into histidine or lysine.

[0009] A mutant of alginate lyase Alya1 is obtained by mutation and transformation using the above method.

[0010] The alginate lyase Alya1 mutant is used for degrading sodium alginate, degrading polyguluronic acid, degrading polymannuronic acid and preparing brown algae oligosaccharides.

[0011] The significant advantages of the present invention are: (1) The alginate lyase Alya1 provided by the present invention is an uncharacterized alginate lyase.

[0012] (2) The present invention successfully achieves heterologous expression of alginate lyase Alya1 by genetic engineering. After purification, the specific activity of alginate lyase Alya1 to sodium alginate is as high as 7749.17 U / mg. Compared with the existing alginate lyase, it has high enzyme activity and high catalytic efficiency, overcoming the disadvantage of low catalytic efficiency of wild bacteria. At the same time, alginate lyase Alya1 can degrade polyG and polyM, so it is a bifunctional alginate lyase.

[0013] (3) The optimal reaction pH of the alginate lyase Alya1 provided by the present invention is 8.5. It is an alkaline enzyme that prefers to degrade sodium alginate. The degradation product is brown algae oligosaccharides with a degree of polymerization of 1 to 4. Existing literature shows that brown algae oligosaccharides have the effects of anti-tumor, anti-inflammatory, lowering blood lipids, and improving plant stress resistance. At the same time, they can also be used in the fields of food. Therefore, the alginate lyase Alya1 has a good application prospect.

[0014] (4) The present invention successfully constructed the alginate lyase Alya1 mutants E406K and E406H, whose specific activities were 9122.50 U / mg and 8359.53 U / mg, respectively, which were 1.18 and 1.08 times that of Alya1, and their half-lives at 35°C were 5.26 and 4.86 times that of the original. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Phylogenetic tree analysis between alginate lyase Alya1 and characterized alginate lyases of PL7 family.

[0016] Figure 2 : SDS-PAGE image of alginate lyase Alya1 after separation and purification, wherein lane M is the marker and lane 4 is the purified alginate lyase Alya1.

[0017] Figure 3 :The effect of temperature on the enzymatic properties of alginate lyase. A, The effect of temperature on the activity of alginate lyase Alya1; B, The effect of temperature on the stability of alginate lyase Alya1.

[0018] Figure 4 :The effect of pH on the activity of alginate lyase Alya1. A, The effect of pH on the activity of alginate lyase Alya1; 4B, The effect of pH on the stability of alginate lyase Alya1.

[0019] Figure 5 :Effects of metal ions and chelating agents on alginate lyase Alya1.

[0020] Figure 6 : Substrate preference of alginate lyase Alya1.

[0021] Figure 7 : Results of the final degradation products of alginate lyase Alya1. A, thin layer chromatography results; B, negative ion electrospray ionization mass spectrometry results.

[0022] Figure 8: SDS-PAGE analysis of alginate lyase Alya1 mutant. Lane M is a marker, lane 1 is E406K unpurified protein, lane 2 is E406H unpurified protein, lane 5 is E406K purified protein, and lane 6 is E406H purified protein.

[0023] Fig. 9 :The effects of pH and temperature on the activity of the alginate lyase Alya1 mutant. A, The effect of temperature on the activity of the alginate lyase Alya1 mutant; B, The effect of pH on the activity of the alginate lyase Alya1 mutant.

[0024] Fig.10 : Thermostability of alginate lyase Alya1 mutants. A, E406K; B, E406H.

[0025] Fig.11 : pH stability of alginate lyase Alya1 mutant.

[0026] Fig.12 :Thermal inactivation kinetic curves of alginate lyase Alya1 mutant. DETAILED DESCRIPTION

[0027] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0028] Example 1: Origin and sequence analysis of alginate lyase Alya1 The alginate lyase Alya1 of the present invention is derived from the genome of Zobellia sp., and its amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the Alya1 gene encoding the alginate lyase Alya1 is shown in SEQ ID NO.2. The homology comparison results show that no sequence similar to SEQ ID NO.1-2 is compared. The phylogenetic tree results obtained based on the amino acid sequence comparison show that the alginate lyase Alya1 belongs to the polysaccharide hydrolase family 7 (PL7) member.

[0029] Example 2: Construction of recombinant expression engineering bacteria for alginate lyase Alya1 Primers Alya1F and Alya1R were used to add EcoR I and Xho I restriction sites at both ends of the Alya1 gene sequence, respectively.

[0030] Alya1F:5'-CG GAATTC ATGAAAAAAAATGTATTTACGACGCTTCGC-3' (the underlined part is the EcoR I restriction site) Alya1R:5'-CC CTCGAG ATTGTGGGTTACGCTTAGGTTTTTGATAC-3' (the underline is the XhoⅠ restriction site) The PCR reaction was carried out under the following conditions: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 2min, 30 cycles; 72℃ extension for 10min. The PCR product was subjected to 1% agarose nucleic acid electrophoresis and then gel-cutting and recovery to obtain the target gene with restriction sites. The target gene with restriction sites was connected to the pMD-18T vector using the T / A cloning kit purchased from TAKATA. The connection system and conditions were as follows: gel recovery target band 5μL; SolutionⅠ4.3μL; pMD-18T0.7μL, the connection system was placed at 16℃ for connection overnight, and the connection product was transformed into E.coli (DH5α) competent cells the next day, and the positive clones were selected for DNA sequencing. The recombinant vector pMD-18T-Alya1 was extracted from the positive clones with correct sequencing. The pMD-18T-Alya1 and pET28a vectors were double-digested with restriction endonucleases EcoR I and XhoⅠ (purchased from TAKARA), and the digested products were recovered by gel cutting after 1% agarose nucleic acid electrophoresis. The fragments recovered by gel were connected using T4 DNA ligase (purchased from TAKARA), and the connected products were transformed into E. coli (DH5α) competent cells after overnight connection at 16°C, and positive clones were selected for DNA sequencing. The recombinant vector pET28a-Alya1 was extracted from the positive clones with correct sequencing. The recombinant vector pET28a-Alya1 was transformed into the host bacteria E. coli BL21 (DE3) to obtain the recombinant bacteria E. coli BL21 (DE3)-pET28a-Alya1, which is the recombinant expression engineering bacteria of alginate lyase Alya1.

[0031] Example 3: Induced fermentation of alginate lyase Alya1 The recombinant E. coli BL21 (DE3)-pET28a-Alya1 was streaked on a solid LB plate containing 50 μg / mL kanamycin, and a single colony was picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C in a shaking incubator to obtain a seed solution. The seed solution was inoculated into 1 L of LB liquid medium containing 50 μg / mL kanamycin at an inoculum volume of 1 vol%, and cultured until OD 600When the pH value was 0.6-0.8, add isopropylthiogalactoside (IPTG) with a final concentration of 0.4mM, and induce at 15℃ for 16h. Collect the bacteria after IPTG induction by centrifugation at 4℃ and 12000rpm for 15min, resuspend in pre-cooled 10mL Tris-Hcl (pH 8.5), and after ultrasonic disruption for 20min, centrifuge at 4℃ and 10000rpm for 15min to collect the supernatant, which is the crude enzyme solution of alginate lyase Alya1. The enzyme activity was determined by 3,5-dinitrosalicylic acid (DNS) method, as follows: take 120μL crude enzyme solution, add 80μL 0.75wt% sodium alginate, incubate at 35℃ for 1min, then add 100μL DNS reagent, boil in water bath for 10min, add 700μL ultrapure water, centrifuge briefly, and take the supernatant at OD 540 The absorbance value was detected under the conditions of 40 °C, and the enzyme that had been inactivated in advance was used as a blank control. The specific activity of the alginate lyase Alya1 enzyme solution was calculated according to the standard curve formula (1-1). Enzyme activity unit definition: Under the above experimental conditions, the amount of enzyme required to catalyze the substrate to produce 1 μg of reducing sugar per minute is regarded as an enzyme activity unit (U). The enzyme activity calculation formula is:

[0032] In the formula: U is the unit of alginate lyase activity (U / mL); A1 is the absorbance of the experimental group; A2 is the absorbance of the control group; T is the reaction time (min); V is the volume of the enzyme solution (mL).

[0033] Example 4: Isolation and purification of alginate lyase Alya1 The cells induced by IPTG were resuspended in NTA-0 Buffer (20mM Tris-HCl (pH7.4), 0.5M NaCl) twice the dry weight of the cells, and ultrasonically disrupted for 20 minutes. The supernatant was collected by centrifugation at 10000 rpm for 15 minutes at 4°C to obtain the crude enzyme solution of alginate lyase Alya1. The crude enzyme solution was filtered with a 0.22μm filter membrane, and all solutions used for purification were filtered before use. The 20% ethanol in the Ni-NTA agarose purification resin pre-packed column (Shanghai Shenggong Bioengineering Co., Ltd.) was drained off, the column material was washed with 3 to 5 column volumes of UP water, the column material was equilibrated with 3 to 5 column volumes of NTA-0 Buffer, 1 column volume of the filtered crude enzyme solution was loaded and adsorbed for 3 to 5 minutes, the column was washed with 4 to 6 column volumes of NTA-0 Buffer to remove unbound proteins, and 1 to 2 column volumes of NTA-20 Buffer (20 mM Tris-HCl (pH 7.4), 0.5 M NaCl, 20 mM imidazole), NTA-40 Buffer (20 mM Tris-HCl (pH 7.4), 0.5 M NaCl, 40 mM imidazole), NTA-80 Buffer (20 mM Tris-HCl (pH 7.4), 0.5 M NaCl, 80 mM imidazole), NTA-100 Buffer (20 mM Tris-HCl (pH 7.4), 0.5 M NaCl, 100mM imidazole), NTA-200Buffer (20mM Tris-HCl (pH7.4), 0.5M NaCl, 200mM imidazole), NTA-300Buffer (20mM Tris-HCl (pH7.4), 0.5M NaCl, 300mM imidazole), NTA-400Buffer (20mM Tris-HCl pH7.4, 0.5M NaCl, 400mM imidazole) NTA-500Buffer (20mM Tris-HCl (pH7.4), 0.5M NaCl, 500mM imidazole) were sequentially eluted, and the eluate was collected to obtain the purified alginate lyase Alya1 (hereinafter referred to as "pure enzyme Alya1"). Take 50 μL of pure enzyme Alya1, add an equal volume of 2× protein treatment solution (0.8 g SDS, 30 mg bromophenol blue and 8 mL glycerol, 4 mL β-mercaptoethanol, 5 mL Tris-HCl (0.5 mol / L, pH 6.8) and evenly mix, then add 40 mL distilled water), boil at 100°C for 10 min, centrifuge at 12000 rpm for 5 min at room temperature, take the supernatant, and perform SDS-PAGE electrophoresis. Figure 2As shown, the alginate lyase Alya1 is 48 kDa. 1 wt% sodium alginate was prepared as the substrate for the enzymatic reaction using 20 mM PB buffer (pH 8.5) as the solvent, the enzyme activity (unit: U / mL) of the pure enzyme Alya1 was determined by the DNS method, the concentration of the pure enzyme Alya1 was determined by Coomassie Brilliant Blue G-25 (unit: mg / mL), and the specific activity (unit: U / mg) of the pure enzyme Alya1 was calculated. It was calculated that the specific activity of the pure enzyme Alya1 was 7749.17 U / mg.

[0034] Example 5: Enzymatic properties of alginate lyase Alya1 (1) Effect of temperature on the activity of alginate lyase Alya1 Take 120 μL pure enzyme Alya1, add 80 μL 0.75 wt% sodium alginate (solvent: 20 mM PB buffer (pH 8.5)) and place it at 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C for 1 min, and then determine the enzyme activity by DNS method, with the highest enzyme activity as 100%. Figure 3 As shown in A, the optimum temperature of alginate lyase Alya1 is 35℃.

[0035] (2) Effect of temperature on the stability of brown algae lyase Alya1 After the pure enzyme Alya1 was incubated at 20℃, 25℃, 30℃, 35℃, and 40℃ for different time periods, the DNS method was used to determine the remaining enzyme activity at the optimal reaction temperature (35℃), and the enzyme activity without incubation was taken as 100%. Figure 3 As shown in B, alginate lyase Alya1 can remain relatively stable below 35°C.

[0036] (3) Effect of pH on the activity of alginate lyase Alya1 Prepare substrate buffers 0.1M citric acid-sodium citrate buffer (pH 3.0-6.0), 0.2M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH 6.0-8.0), 0.1M Tris-hydrochloric acid buffer (pH 8.0-9.0), and 0.2M glycine-sodium hydroxide buffer (pH 9.0-11.0), respectively, and use substrate buffers of different pH to prepare 0.75wt% sodium alginate. The specific formula of substrate buffer is shown in the table below. Using the DNS method, the reaction system was placed at 35°C for 1 minute and then the enzyme activity was measured, with the highest enzyme activity being 100%. Figure 4 As shown in A, the optimum pH of alginate lyase Alya1 is pH 8.5.

[0037] Table 1 Citric acid-sodium citrate buffer (pH 3.0~6.0) Table 2 PB buffer (pH 6.0~8.0) Table 3 Tris-HCl buffer (pH 8.0~9.0) Table 4 Glycine-sodium hydroxide buffer (pH 9.0~11.0) (4) Effect of pH on the stability of alginate lyase Alya1 After the pure enzyme Alya1 was mixed with equal volumes of the above buffers of different pH values, the mixture was incubated at 4°C for 2 h. The enzyme activity was determined by the DNS method. The pH stability of the enzyme was tested at the optimal temperature (35°C) and the optimal pH (pH 8.5). The untreated enzyme activity was set as 100%. Figure 4 As shown in B, alginate lyase Alya1 has good stability at pH 8.0-9.0.

[0038] (5) Effects of metal ions and chelating agents on alginate lyase Alya1 80 μL of pure enzyme Alya1 was mixed with 120 μL of 0.75 wt% sodium alginate (solvent: 20 mM PB buffer (pH 8.5)), and different metal ions or chelating agents were added at final concentrations of 1 mM, 5 mM, and 10 mM, respectively. After mixing, the enzyme activity was determined by reacting at the optimal temperature (35°C) for 1 min, and the enzyme activity without adding any ions was taken as 100%. Figure 5 As shown, 1.0 mM K + Mg 2+ , 5.0 mM K + , Fe 2+ , 10.0 mM Fe 2+ It promotes the catalytic process of Alya1. + , K + Mg 2+ When the concentration increased to 10.0mM, the enzyme activity of Alya1 was inhibited. Other metal ions and chelating agents inhibited the enzyme activity at the concentration of 1.0mM to 10.0mM.

[0039] (6) Substrate preference of alginate lyase 1wt% sodium alginate, 1wt% polyguluronic acid (polyG), 1wt% polymannuronic acid (polyM), 1wt% carrageenan, 1wt% agar, 1wt% chitosan and 1wt% chitin were prepared using 0.1M Tris-HCl at pH 9.0 as solvent. The DNS method was used to measure the enzyme activity of 80μL pure enzyme Alya1 and 120μL substrate solution at the optimal reaction temperature (35°C). The enzyme activity when sodium alginate was used as the substrate was regarded as 100%, and the relative enzyme activity under the conditions of the other substrate solutions was calculated. Figure 6 As shown, alginate lyase Alya1 has the ability to degrade sodium alginate, polyG and polyM, among which the degradation effect on polyM is the best. It has no degradation ability on carrageenan, agar, chitosan and chitin, indicating that alginate lyase Alya1 may be a bifunctional alginate lyase.

[0040] Example 6: Degradation end products of alginate lyase Alya1 Add 10mL pure enzyme Alya1 to 100mL 4wt% sodium alginate (solvent is 0.1M Tris-HCl (pH 8.5)) substrate solution, and perform enzymatic reaction at 30℃ and 180rpm. Add 10mL pure enzyme Alya1 every 6h for 24h. The reaction product was boiled in 100℃ water bath for 30min to terminate the enzymatic reaction, and the supernatant was collected by centrifugation at 10000×g for 20min. Add 0.2 times the volume of pre-cooled anhydrous ethanol to the supernatant, and precipitate at 4℃ overnight to remove brown algae oligosaccharides with a large degree of polymerization. The next day, collect the supernatant by centrifugation at 10000×g for 20min, concentrate the supernatant to about 10-20mL by rotary evaporation, add 5 times the volume of pre-cooled anhydrous ethanol to the concentrate, and precipitate at 4℃ overnight. The next day, the precipitate was collected by centrifugation at 10,000 × g for 20 min, the precipitate was dissolved in UP water and centrifuged at 10,000 × g for 20 min to collect the supernatant, the supernatant was concentrated again by rotary evaporator, and after concentrating to about 20-30 mL, the concentrate was freeze-dried for 24 hours, and the freeze-dried powder was collected as brown algae oligosaccharide. The freeze-dried brown algae oligosaccharide was desalted using Superdex G-25, and then the degree of polymerization of unsaturated oligosaccharides produced by enzymatic hydrolysis was analyzed using thin layer chromatography (TLC) and quadrupole time-of-flight mass spectrometry (Q-TOF-MS).

[0041] First, place the prefabricated thin layer chromatography plate in a 90℃ oven to dry for 1 hour. Spot samples 1.5cm from the bottom of the chromatography plate, except for 0.8cm intervals on the same horizontal line. Spot each sample 3 times. After air-drying, develop it in a chromatography cylinder for 1 hour (the developing agent is n-butanol: formic acid: water = 4:6:1 (v / v / v)). After air-drying, spray the color developer (2mL of aniline, 2g of diphenylamine, 10mL of 85% phosphoric acid (v / v), 1mL of hydrochloric acid, mix well and dissolve in 100mL of acetone, mix well again until completely dissolved). Figure 7 As shown in A, alginate lyase Alya1 hydrolyzes sodium alginate to produce brown algal oligosaccharides with a degree of polymerization of 1 to 4.

[0042] The desalted brown algae oligosaccharide was prepared into a 10 mg / mL sample solution for Q-TOF-MS detection. Figure 7 As shown in B, four obvious characteristic peaks were identified in the negative ion mode of the enzymatic hydrolysis product. Among them, the monosaccharide m / z was 175.0217 ([ΔDP1-H] - ), disaccharide m / z is 351.0494 ([ΔDP2-H] - ), trisaccharide m / z is 263.0349 ([ΔDP3-H] 2- )、571.0407([ΔDP3-3H+2Na] - ), tetrasaccharide m / z is 703.1064 ([ΔDP4-H] - ).

[0043] Example 7: Site-directed mutagenesis of alginate lyase Alya1 The amino acid sequence of alginate lyase Alya1 was uploaded to the online website SWISS-model (https: / / swissmodel.expasy.org / ), the protein model of alginate lyase Alya1 was established, and the protein and ligand were molecularly docked using AutoDockTools-1.5.7 software.

[0044] The mutant E406H of alginate lyase Alya1 was a mutation of glutamic acid (E) at position 406 to histidine (H), and the mutant E406K of alginate lyase Alya1 was a mutation of glutamic acid (E) at position 406 to lysine (K). The mutant primers were designed using SnapGene software. The recombinant vector pET28a-AlyA1 was used as a template and the whole plasmid site-directed mutagenesis method was used. The mutant primers were used as specific amplification primers for PCR amplification. The PCR amplification products were ligated to the pET28a vector digested and methylated by DpnⅠ, and then transformed into E.coli (DH5α) competent cells. The positive clones were picked for DNA sequencing to verify whether the E at position 406 of alginate lyase Alya1 was successfully mutated to K or H. The recombinant plasmid with correct sequencing was transformed into the host bacteria E.coli BL21 (DE3) to obtain the recombinant expression engineering bacteria of alginate lyase Alya1 mutant. Refer to Example 3 and Example 4 to carry out induced fermentation and separation and purification of the target protein.

[0045] Mutation primers for the alginate lyase Alya1 mutant E406H: E406H-F: 5'-GAAAATGCACGTTGACGATTCTATCTGAAAAC-3' E406H-R: 5'-TCAACGTGCATTTTCTTTCTAAATACGCTAGAG-3' Mutation primers for the alginate lyase Alya1 mutant E406K: E406K-F: 5'-GAAAATGAAAGTTGACGATCTATCTGAAAAC-3' E406K-R: 5'-TCAACTTTCATTTTCTTTCTAAATACGCTAGAG-3' After the alginate lyase Alya1 mutant was separated and purified, a protein band appeared at the target molecular weight of 48.07 kDa. 20 mM PB buffer (pH 8.5) was used as the solvent to prepare 1 wt% sodium alginate as the substrate for the enzymatic reaction. The DNS method was used to determine the enzyme activity (unit: U / mL) of the purified alginate lyase Alya1 mutant. The concentration of the purified alginate lyase Alya1 mutant was determined by Coomassie Brilliant Blue G-25 (unit: mg / mL), and the specific activity (unit: U / mg) of the purified alginate lyase Alya1 mutant was calculated. It was calculated that the specific activity of mutant E406K was 9122.50 U / mg, and the specific activity of mutant E406H was 8359.53 U / mg, which were 1.18 and 1.08 times higher than Alya1, respectively.

[0046] The enzymatic properties (optimal reaction temperature, optimal reaction pH, thermal stability, pH stability, and half-life) of the purified alginate lyase Alya1 mutant were studied with reference to Example 5: like Fig. 9 As shown in A, the optimal reaction temperature of mutants E406K and E406H is 25°C; when the reaction temperature reaches 55°C or above, the relative enzyme activity of the two mutants is always higher than that of Alya1. When the temperature reaches 75°C, Alya1 has lost its activity, while mutant E406K still has about 33% relative enzyme activity, indicating that the mutants have a higher tolerance to high temperatures. Fig. 9 As shown in B, the optimal reaction pH of mutants E406K and E406H were 8.0 and 9.0, respectively; compared with the optimal pH of Alya1, the optimal reaction pH of the mutants did not change significantly.

[0047] like Fig.10 As shown, after incubation at 25℃, 30℃ and 35℃ for 1h, the remaining relative enzyme activities of mutant E406K were 110.47%, 99.01% and 84.45%, respectively. After incubation at 25℃, 30℃ and 35℃ for 1h, the remaining relative enzyme activities of mutant E406H were 93.3%, 108.54% and 90.75%, respectively. However, the remaining relative enzyme activities of Alya1 under the same incubation conditions were only 70.46%, 78.07% and 70.75%, respectively. Therefore, the thermal stability of mutants E406K and E406H is significantly improved compared with the non-mutated parent. Fig.11 As shown, the residual relative enzyme activity of the mutants in pH 6.0 citrate buffer and pH 8.0-9.0 Tris-HCL buffer was greatly improved; the relative enzyme activities of E406K and E406H reached the maximum at pH 9.0, which were 138.72% and 171.18%, respectively, indicating that the pH stability of the mutants E406K and E406H was significantly improved compared with the unmutated parent.

[0048] like Fig.12 As shown, the half-life of the mutant at 35°C was t 1 / 2 The half-life of Alya1 at 35°C is t 1 / 2 The half-life of mutant E406K at 35°C was 70.04 min. 1 / 2 The half-life t1 / 2 of mutant E406H at 35°C was 340.11 min, which was 4.86 times that of Alya1.

[0049] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. An alginate lyase Alya1, characterized in that: The amino acid sequence of the alginate lyase Alya1 is shown in SEQ ID NO.1, and the amino acid sequence encoding the alginate lyase Alya1 is shown in SEQ ID NO.

1. Alya1 The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

2. The method for preparing the alginate lyase Alya1 according to claim 1, characterized in that: exist Alya1 The two ends of the gene sequence were introduced EcoR I and XOt I restriction enzyme site, and then connected to the pMD-18T vector to obtain the recombinant vector pMD-18T-Alya1; both the recombinant vector pMD-18T-Alya1 and the pET28a vector were treated with restriction endonucleases EcoR I and XOt Ⅰ double enzyme digestion, and then connected with T4 DNA ligase to obtain the recombinant vector pET28a- Alya1 ; The recombinant vector pET28a- Alya1 Transformed host bacteria E.coil BL21(DE3), obtained recombinant bacteria E. coli BL21(DE3)-pET28a- Alya1 ; Recombinant bacteria E. coli BL21(DE3)-pET28a- Alya1 Fermentation-induced expression and purification were performed to obtain the alginate lyase Alya1.

3. Use of the alginate lyase Alya1 as claimed in claim 1 in degrading sodium alginate, degrading polyguluronic acid, degrading polymannuronic acid, and preparing brown algal oligosaccharides.

4. A method for mutating the alginate lyase Alya1 according to claim 1, characterized in that: The glutamic acid at position 406 of the alginate lyase Alya1 was mutated to improve the high temperature resistance, thermal stability, pH stability and half-life of the alginate lyase Alya1.

5. The method according to claim 4, characterized in that: The mutation modification is to mutate the 406th glutamic acid of the alginate lyase Alya1 into histidine or lysine.

6. An alginate lyase Alya1 mutant, characterized in that: Obtained by mutation and modification according to any one of claims 4 to 5.

7. Use of the alginate lyase Alya1 mutant according to claim 6 in degrading sodium alginate, degrading polyguluronic acid, degrading polymannuronic acid, and preparing brown algal oligosaccharides.

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