Mutant for improving catalytic activity and stability of alpha-agarase

Abstract

The invention discloses a mutant for improving catalytic activity and stability of alpha-agarase, and belongs to the technical field of gene engineering and enzyme engineering. The invention provides an alpha-agarase mutant, which is obtained by mutating the 1396th amino acid of agarase with an amino acid sequence as shown in SEQ ID NO.1. The catalytic activity and the stability of the alpha-agarase mutant S1396T disclosed by the invention are improved compared with those of a wild type enzyme, and the polymerization degree distribution of a product is highly consistent with that of the wild type enzyme; the production efficiency of continuous production of the enzyme in actual industrial application is improved, and the method has a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to a mutant that enhances the catalytic activity and stability of α-agarase, belonging to the fields of genetic engineering and enzyme engineering technology. Background Technology

[0002] Agar is a widely used and important industrial product. In recent years, the industrial agar market has continued to expand, with low-value-added and low-tech industrial agar products dominating the market. However, high-end, high-quality, and high-value-added industrial agar products are becoming new growth points in the market, leading the industry to a higher level of development. Therefore, it is necessary to find high-quality, high-value-added processing strategies for industrial agar to meet the demands of high-end consumers. Agar oligosaccharides extracted from agar possess various beneficial bioactivities, including anti-inflammatory, antioxidant, probiotic, antibacterial, and whitening / moisturizing properties, and have application potential in food, medicine, and agriculture, making them a feasible approach to achieving high-value-added processing and utilization of agar.

[0003] Agar oligosaccharides are marine oligosaccharides with a degree of polymerization of 2-20 after hydrolysis of agar polysaccharides. They are divided into two categories: agar oligosaccharides and neo-agar oligosaccharides. Compared with neo-agar oligosaccharides, agar oligosaccharides possess various biological activities such as anti-diabetic, anti-obesity, anti-tumor, and neuroprotective activities, making them high-value industrial agar products with broad application prospects. There are two main methods for preparing agar oligosaccharides: chemical degradation and biodegradation. Among these, enzymatic hydrolysis produces agar oligosaccharides with better substrate specificity and is more environmentally friendly, making it an effective method for preparation. α-Agarase is the key enzyme preparation for the enzymatic hydrolysis of agar oligosaccharides, capable of hydrolyzing the α-1,3-glycosidic bonds of agarose. However, the development and application of α-agarase are still far from perfect, and α-agarase generally suffers from problems such as low enzyme activity and poor thermal stability. Currently, ten α-agarases have been reported both domestically and internationally. The initial crude enzyme activity of these α-agarases cultured using heterologous expression fermentation technology is generally below 10 U / mL, and most of them rapidly inactivate above 40℃, severely hindering the large-scale application of α-agarases. Therefore, it is necessary to develop α-agarases with high catalytic activity, good product specificity, high stability, and promising industrial applications to provide key enzyme preparations for the enzymatic production of agar oligosaccharides.

[0004] This invention selects Saccharophagus litoralisSa-Aga, derived from STB26, exhibits strong substrate specificity, hydrolyzing agarose into agarotriose (A3), agarotetraose (A4), and agaropentaose (A5), which is beneficial for producing agar oligosaccharides with different degrees of polymerization and holds great potential for industrial applications. However, agar exhibits decreased flowability and gradual gel formation below 40°C. Furthermore, wild-type Sa-Aga itself suffers from significant performance defects, namely low enzyme activity and poor thermal stability. On the one hand, the lower catalytic efficiency of wild-type Sa-Aga leads to slow reaction rates and low yields, increasing production costs; on the other hand, its poor thermal stability makes it easily inactivated under commonly used industrial medium- and high-temperature conditions, making it difficult to maintain long-term effective catalytic activity. These inherent defects collectively limit the industrial-scale promotion and application of agarase. Therefore, based on the needs of industrial applications, genetic engineering and enzyme engineering technologies were used to perform site-directed mutagenesis and directional modification of α-agarase Sa-Aga, which effectively improved the catalytic activity and thermal stability of Sa-Aga, enabling its performance to break through the limitations of the wild type. This provides a key tool enzyme for the efficient production of agarotriose and is of great significance for realizing the high-value-added processing and utilization of agar. Summary of the Invention

[0005] To address the shortcomings of the prior art, this invention provides a mutant that improves the catalytic activity and stability of α-agarase, aiming to solve the problems of low α-agarase activity or poor stability in the prior art.

[0006] The first technical solution provided by the present invention is an α-agarase mutant, wherein the α-agarase mutant is obtained by mutating the serine at position 1396 of the α-agarase parent amino acid sequence as shown in SEQ ID NO.1 to tyrosine, and is named: S1396T; Alternatively, the valine at position 1338 of the α-agarase parent with the amino acid sequence shown in SEQ ID NO.1 can be mutated to isoleucine and named: V1338I; Alternatively, the valine at position 1367 of the α-agarase parent with the amino acid sequence shown in SEQ ID NO.1 can be mutated to isoleucine and named: V1367I; Alternatively, the threonine at position 1407 of the α-agarase parent with the amino acid sequence shown in SEQ ID NO.1 can be mutated to serine, and named T1407S.

[0007] In some embodiments, the α-agarase is derived from marine microorganisms. Saccharophagus litoralis The α-agarase of STB26, the nucleotide sequence encoding the α-agarase is shown in SEQ ID NO.2.

[0008] The second technical solution provided by the present invention is a gene encoding the α-agarase mutant described in the first technical solution.

[0009] The third technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution.

[0010] In some embodiments, the recombinant vector is pET-28a(+) as the expression vector.

[0011] The fourth technical solution provided by the present invention is to express the α-agarase mutant described in the first technical solution, or to contain the gene described in the second technical solution, or to transform recombinant cells into the recombinant vector described in the third technical solution.

[0012] In some embodiments, the recombinant cells use bacteria or fungi as host cells.

[0013] In some embodiments, the recombinant cells use Escherichia coli BL21(DE3) as the host cell.

[0014] The fifth technical solution provided by the present invention is a recombinant Escherichia coli, which uses Escherichia coli BL21(DE3) as the host and pET-28a(+) as the expression vector to express the α-agarase mutant described in the first technical solution.

[0015] The present invention also provides a method for preparing the above-mentioned mutant, the method comprising the following steps: (1) Based on the determined mutation sites, design mutation primers for site-directed mutagenesis, and use the vector carrying the α-agarase encoding gene as a template for site-directed mutagenesis; construct a vector containing the gene encoding the mutant. (2) Transform a vector containing a gene encoding a mutant into a microbial cell; (3) Select positive clones for fermentation culture, collect cells by centrifugation, and the cell wall supernatant is the crude enzyme solution of α-agarase mutant.

[0016] The sixth technical solution provided by this invention is a method for improving the catalytic activity and / or thermal stability of α-agarase, wherein the method involves performing any one of the following mutations on the α-agarase parent with the amino acid sequence shown in SEQ ID NO.1: (1) The serine at position 1396 is mutated to tyrosine; (2) The valine at position 1338 is mutated to isoleucine; (3) Valine at position 1367 is mutated to isoleucine; (4) Threonine at position 1407 is mutated to serine.

[0017] The eighth technical solution provided by the present invention is the application of the mutant described in the first technical solution, the gene described in the second technical solution, the recombinant vector described in the third technical solution, the recombinant cell described in the fourth technical solution, or the recombinant Escherichia coli described in the fifth technical solution in the preparation of agar oligosaccharides.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a mutant of α-agarase with the amino acid sequence shown in SEQ ID NO.1, by mutating serine at position 1396 to tyrosine, resulting in mutant S1396T. Compared with the wild type, mutant S1396T exhibits increased enzyme activity and higher thermostability. After incubation at 37°C for 60 min, mutant S1396T retains 57.82% of the original enzyme activity, a 1.27-fold increase compared to the wild type. In the enzymatic hydrolysis products of mutant S1396T, agarotriose is the main product, accounting for approximately 60%, followed by agarotetraose and agaropentose, with monosaccharides accounting for the smallest proportion. The product distribution is highly consistent with that of the wild type. These conclusions indicate that, compared with the wild type, mutant S1396T improves the production efficiency of continuous production in practical industrial applications and has good prospects for industrial application. Attached Figure Description

[0019] Picture 1 Agarose gel electrophoresis images of wild-type Sa-Aga and its mutants; where M: standard molecular weight of DNA, lane 1: wild-type Sa-Aga plasmid, lane 2: [missing information] 8: Mutant plasmids, namely S1396T, V1338I, V1367I, T1407S, E1118A, D1228A and D1272A.

[0020] Picture 2 SDS-PAGE images of wild-type Ca-AGA and its mutants; where M: protein standard molecular weight, lane 1: wild-type Sa-Aga plasmid, lane 2: [missing information - likely a typo, should be 2]. 8: Mutants S1396T, V1338I, V1367I, T1407S, E1118A, D1228A and D1272A.

[0021] Picture 3 The relative enzyme activity of wild-type Sa-Aga and its mutants is characterized.

[0022] Picture 4The thermostability of wild-type Sa-Aga and its mutants at 37℃, 40℃, and 45℃ was characterized. Figure A shows the relative enzyme activity of wild-type Sa-Aga and its mutants at 37℃; Figure B shows the relative enzyme activity of wild-type Sa-Aga and its mutants at 40℃; and Figure C shows the relative enzyme activity of wild-type Sa-Aga and its mutants at 45℃.

[0023] Picture 5 The degree of polymerization of enzymatic hydrolysates of wild-type Sa-Aga and its mutants was analyzed. Figure A shows the distribution curve of the degree of polymerization of enzymatic hydrolysates of wild-type Sa-Aga and its mutants as detected by ion chromatography. Figure B shows the peak area of ​​enzymatic hydrolysates with different degrees of polymerization. Figure C shows the relative peak area of ​​enzymatic hydrolysates with different degrees of polymerization. Detailed Implementation

[0024] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0025] Test method: Detection Method 1: Detection of α-agarase activity: The hydrolytic activity of α-agarase was characterized by changes in reducing sugar content determined using the 3,5-dinitrosalicylic acid (DNS) method. A 0.15% (w / w) agarose substrate solution was prepared using Tris-HCl buffer (20 mM, pH 8.0). 0.1 mL of enzyme solution was directly added to 0.9 mL of agarose solution, and the reaction was incubated at 37 °C for 10 min. The reaction was then terminated by adding 1 mL of DNS, boiling for 5 min, and cooling in an ice-water bath. After dilution with 2 mL of deionized water, the absorbance was measured at 540 nm. A standard curve was plotted using inactivated enzyme as a control and D-galactose as a standard.

[0026] Enzyme activity (U / mL) definition: Under the above conditions, the volume of enzyme required to catalyze the production of the equivalent of 1 μmol of reducing sugar (calculated as galactose) per minute is defined as one enzyme activity unit (U).

[0027] Detection Method 2: Degree of Polymerization Distribution of α-Agarase Products: The detection conditions for the high-performance anion exchange chromatography (HPAEC-PAD) were as follows: (1) Column: Dionex CarboPacPA-200 anion exchange column, including an analytical column (3 mm × 250 mm) and a guard column (3 mm × 50 mm); (2) Mobile phase: 96% 100 mM NaOH, 4% 100 mM NaOH & 500 mM NaAc, with a flow rate of 0.5 mL / min; (3) Detection was performed using a four-potential pulse amperometry method with an amperometric detector; (4) Column temperature: 35℃, injection volume: 10 μL. Odd-numbered agar oligosaccharide standards were used as the qualitative basis, and the types of agar oligosaccharides in the sample were determined based on the retention times of the corresponding chromatographic peaks.

[0028] Raw materials used in the examples: 1. LB liquid medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH 7.0.

[0029] 2. LB solid medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH 7.0, 1.5% (w / v) agar.

[0030] 3. Modified TB medium: tryptone 36 g / L, glycerol 15 g / L, KH2PO4 17 mM, K2HPO4 72 mM, pH 7.0.

[0031] 4. pET28a(+) vector Escherichia coli JM109 competent cells, Escherichia coli BL21(DE3) is a commercial plasmid and a commercial strain.

[0032] 5. Raw material sources: Tryptone and yeast extract were purchased from Oxoid, UK; sodium chloride, glycerol, KH2PO4, and K2HPO4 were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0033] Example 1: Preparation of mutants The specific steps are as follows: (1) The α-agarase Sa-Aga, with a nucleotide sequence as shown in SEQ ID NO.2, was chemically synthesized, and an expression vector for α-agarase Sa-Aga was constructed using PCR and homologous recombination. Based on the α-agarase gene, upstream primers were designed as follows: 5'-ATGGATACAATTCAACTGCAAGCAGAAG-3'; downstream primer: 5'-GTGGTGGTGCTCGAGATGTGCCAGTTCCAGAATGCCC-3'. Based on the pET28a(+) vector sequence, upstream primers were designed as follows: 5'-GACGGGCTTGTCTGCTCC-3'; downstream primer: 5'-TTGAATTGTATCCATGGCCATGGTATATCT-3'; upstream primer: 5'-CTCGAGCACCACCACCAC-3'; downstream primer: 5'-GCAGACAAGCCCGTCAGG-3'. The target gene SEQ ID NO.2 and the pET28a(+) vector containing the signal peptide sequence were cloned. The PCR system for the α-agarase gene was as follows: 25 µL of 2×phanta Max Master Mix (Dye plus), 2 µL of forward primer (20 µM), 2 µL of reverse primer (20 µM), 1 µL of template DNA, and double-distilled water to a final volume of 50 µL. The PCR amplification conditions were: 95℃ pre-denaturation for 3 min; followed by 30 cycles (95℃ for 15 s, 60℃ for 15 s, 72℃ for 4.5 min); and finally, incubation at 72℃ for 5 min. The PCR system for the pET28a(+) vector was as follows: 25 µL of 2×phanta Max Master Mix (Dye plus), 2 µL of forward primer (20 µM), 2 µL of reverse primer (20 µM), 1 µL of template DNA, and double-distilled water to a final volume of 50 µL. The PCR amplification conditions were as follows: pre-denaturation at 95℃ for 3 min; followed by 30 cycles (95℃ for 15 s, 60℃ for 15 s, 72℃ for 3 min); and finally incubation at 72℃ for 5 min.

[0034] The PCR products of the agarase gene and the pET28a(+) vector were subjected to nucleic acid electrophoresis and then recovered by gel extraction. Homologous recombination was used to ligate the agarase gene and the pET28a(+) vector. The ligation system consisted of: 2 μL of purified agarase PCR fragment (50 ng / μL), 1 μL of purified pET28a(+) vector PCR fragment (180 ng / μL), 4 μL of 5× CE II Buffer, 2 μL of Exnase II, and 10 μL of ddH2O. Homologous recombination was performed at 37℃ for 30 min, followed by transformation into *E. coli* JM109, plating on LB agar plates containing 20 μg / mL kanamycin, and selecting transformants for sequencing and nucleic acid electrophoresis verification to obtain the expression vector pET-28a(+) / containing the α-agarase gene. sa-wt .

[0035] (2) The recombinant plasmid pET-28a(+) / prepared in step (1) sa-wt Using DNA as a template, complementary primer chains were designed for the experiment (see Table 1). Primers were synthesized by Genewiz Biotechnology Co., Ltd. One-step PCR was used for site-directed mutagenesis. The PCR system for the α-agarase mutant was: 25 µL 2×phanta Max Master Mix (Dye plus), 2 µL forward primer (20 µM), 2 µL reverse primer (20 µM), 1 µL template DNA, and double-distilled water to a final volume of 50 µL. PCR amplification conditions were: 95℃ pre-denaturation for 3 min; followed by 30 cycles (95℃ for 15 s, 58℃ for 15 s, 72℃ for 4.5 min); and finally, incubation at 72℃ for 5 min. PCR products were digested with DpnI enzyme. The DpnI digestion system was: 50 µL PCR product, 10 µL 10×Buffer Y, 5 µL DpnI enzyme, and double-distilled water to a final volume of 100 µL. The DpnI digestion conditions were: 37℃ constant temperature water bath for 2-3 hours. The processed PCR products were then... E. coli The JM109 competent state conversion method was transferred to... E. coli Transformants were prepared from JM109 and spread on LB solid medium containing kanamycin (20 μg / mL). The medium was incubated at 37°C for 12 h in an inverted position. Positive clones were picked and transferred to LB liquid medium containing kanamycin (20 μg / mL), and incubated at 37°C and 200 rpm for 10–12 h. Plasmids were extracted and sent to the company for sequencing. Recombinant plasmids containing the mutant gene were obtained: pET-28a(+) / sa-S1396T pET-28a(+) / sa-V1338I pET-28a(+) / sa-V1367I, pET-28a(+) / sa-T1407SpET-28a(+) / sa- E1118A, pET-28a(+) / sa-D1228A and pET-28a(+) / sa-D1272A The plasmid extraction verification results are as follows: Picture 1 As shown.

[0036] Table 1. Introduction of α-agarase mutation sites

[0037] 1 The underlined bases correspond to the mutated amino acids. Example 2: Construction of genetically engineered bacteria and expression of mutants The specific steps are as follows: (1) The recombinant plasmid pET-28a(+) / obtained in Example 1 was respectively... sa-wt pET-28a(+) / sa- S1396T pET-28a(+) / sa-V1338I pET-28a(+) / sa-V1367I pET-28a(+) / sa-T1407S pET-28a(+) / sa-E1118A pET-28a(+) / sa-D1228A and pET-28a(+) / sa-D1272A Transfer to E. coli In BL21(DE3), respectively prepared the following: E. coli BL21(DE3) / (pET-28a(+) / sa-wt ), E. coli BL21(DE3) / (pET-28a(+) / sa-S1396T ), E. coli BL21(DE3) / (pET-28a(+) / sa-V1338I ), E. coli BL21(DE3) / (pET-28a(+) / sa-V1367I), E. coli BL21(DE3) / (pET-28a(+) / sa- T1407S), E. coli BL21(DE3) / (pET-28a(+) / sa-E1118A), E. coli BL21(DE3) / (pET-28a(+) / sa-D1228A), E. coli BL21(DE3) / (pET-28a(+) / sa-D1272A) Recombinant strain.

[0038] (2) The recombinant strains were streaked on LB solid medium containing kanamycin (20 μg / mL) and cultured in a constant temperature incubator at 37°C for 12 h. Positive single clones were picked and cultured in LB liquid medium containing kanamycin (20 μg / mL) at 37°C and 200 rpm for 8-10 h to prepare seed liquid.

[0039] (3) The seed culture was inoculated at a rate of 2% (v / v) into fermentation broth containing kanamycin (20 μg / mL) in shake flasks for fermentation and cultured at 37°C until OD reached. 600 When the pH reached 0.6-0.8, isopropyl-β-D-thiogalactoside (IPTG, 100 μL / 50 mL, final concentration 0.05 mM) was added, and the mixture was cultured in shake flasks at 20℃ and 200 rpm for 12-16 h. The resulting fermentation broth was centrifuged at 4℃ and 10000 rpm for 20 min, and the cells were collected. The cells were resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0) in a proportional ratio, sonicated, and the supernatant was collected by high-speed centrifugation to obtain the crude intracellular enzyme solution. The SDS-PAGE gel electrophoresis image is shown below. Picture 2 As shown in the figure, the results indicate that both the original enzyme and the mutant enzyme were expressed.

[0040] Example 3: Enzyme activity assay The specific steps are as follows: The enzyme activities of wild-type agarase and mutant enzymes V1338I, V1376I, S1396T, T1407S, E1118A, D1228A, and D1272A obtained in Example 2 were determined. The wild-type enzyme activity was set as 100%, and the relative enzyme activities of the mutant enzymes were detected and calculated. The results are as follows: Picture 3 As shown in the figure, mutants V1338I, V1367I, S1396T, and T1407S exhibited significantly increased enzyme activities compared to the wild-type (WT), with an average increase of approximately 10%; while mutants E1118A, D1228A, and D1272A showed a substantial decrease in enzyme activity. After excluding mutants E1118A, D1228A, and D1272A, which initially showed significantly reduced enzyme activity, mutants V1338I, S1396T, T1407S, and V1367I, whose enzyme activities were higher than those of the wild-type, were selected for subsequent experiments.

[0041] Table 2. Average enzyme activity of α-agarase wild-type and mutant.

[0042] Example 4: Determination of the thermal stability of enzymes The specific steps are as follows: The crude enzyme solution obtained in Example 2 was incubated at 37℃, 40℃, and 45℃ for 1 min, 2 min, 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min, respectively, before the incubation was terminated. The enzyme activity of the enzyme solution before and after incubation was measured. The inactivated enzyme was used as a blank control, and the enzyme without incubation was used as 100% to calculate the relative enzyme activity. The results are as follows: Picture 4 As shown.

[0043] The thermal stability of V1338I, S1396T, T1407S and V1367 was tested after incubation at 40℃. The results showed that the thermal stability of V1338I was similar to that of the wild type, while the thermal stability of S1396T, T1407S and V1367 was improved compared with the wild type. After 10 min at 40℃, the residual enzyme activity of wild-type and V1338I was about 20% of the original, while the residual enzyme activity of S1396T, T1407S and V1367 was about 40%-50% of the original. This indicates that the thermal inactivation rate of the three mutants was slower than that of the wild-type. Further incubation at 37℃ and 45℃ and detection of the thermostability of S1396T, T1407S and V1367 showed that after 30 min of incubation, the wild-type retained only 45.42% of the original enzyme activity; while the mutant S1396T retained 57.82% of the original enzyme activity, which was 1.27 times higher than that of the wild-type; the mutant V1367I retained 20.14% of the original enzyme activity, which was 44.34% lower than that of the wild-type; and the mutant T1407S retained 45.05% of the original enzyme activity, which was basically the same as that of the wild-type. In summary, the thermal stability of mutant S1396T was improved compared to the wild type, while mutants V1367I and T1407S showed only average performance.

[0044] Example 5: Degree of Polymerization Analysis of Enzyme Products The specific steps are as follows: Using 0.5% (w / v) agarose solution as substrate, an appropriate amount of Sa-Aga mutant prepared in Example 2 and wild-type crude enzyme solution (substrate to enzyme solution volume ratio 10:1) were added. The mixture was placed in a 35℃ constant temperature shaking water bath for 24 h, and then samples were taken. The enzyme was inactivated by boiling for 10 min, and the supernatant was collected by centrifugation. The supernatant was then diluted, filtered through a 0.22 μm aqueous filter, and analyzed using HPAEC-PAD. Using agarotriose (A3), agaropentose (A5), and agaroheptaose (A7) standards as quantitative and qualitative standards, the monosaccharides, disaccharides, and oligosaccharides in the product were detected using HPAEC-PAD. The results are shown in Table 3. Picture 5 As shown.

[0045] The absolute yield of oligosaccharides at different degrees of polymerization was characterized by peak area, and the proportion of oligosaccharides was characterized by relative peak area. The results showed that although there were some differences in product yield among different mutants, the relative distribution pattern of products at different degrees of polymerization remained basically stable. Agaric triose was the main product, accounting for about 60%, followed by agaric tetraose and agaric pentose, while monosaccharides accounted for the smallest proportion. This indicates that the introduced mutation did not change the distribution pattern of the degree of polymerization in the enzymatic reaction, and the product distribution of the mutants was highly consistent with that of the wild type.

[0046] Table 3. Distribution of degree of polymerization of α-agarase products from wild-type and mutant strains.

[0047] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An α-agarase mutant, characterized in that, The α-agarase mutant is formed by mutating serine at position 1396 of the α-agarase parent amino acid sequence as shown in SEQ ID NO.1 to tyrosine; or mutating valine at position 1338 of the α-agarase parent amino acid sequence as shown in SEQ ID NO.1 to isoleucine; or mutating valine at position 1367 of the α-agarase parent amino acid sequence as shown in SEQ ID NO.1 to isoleucine; or mutating threonine at position 1407 of the α-agarase parent amino acid sequence as shown in SEQ ID NO.1 to serine.

2. The gene encoding the α-agarase mutant of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector is pET-28a(+) as the expression vector.

5. Recombinant cells expressing the α-agarase mutant of claim 1, or containing the gene of claim 2, or transformed with the recombinant vector of claim 3 or 4.

6. The recombinant cell according to claim 5, characterized in that, The recombinant cells use bacteria or fungi as host cells.

7. The recombinant cell according to claim 6, characterized in that, The recombinant cells used Escherichia coli BL21(DE3) as the host cell.

8. A recombinant Escherichia coli, characterized in that, The α-agarase mutant of claim 1 was expressed using Escherichia coli BL21(DE3) as the host and pET-28a(+) as the expression vector.

9. A method for improving the catalytic activity and / or thermal stability of α-agarase, characterized in that, The method involves performing any of the following mutations on the α-agarase parent with the amino acid sequence shown in SEQ ID NO.1: (1) The serine at position 1396 is mutated to tyrosine; (2) The valine at position 1338 is mutated to isoleucine; (3) Valine at position 1367 is mutated to isoleucine; (4) Threonine at position 1407 is mutated to serine.

10. The use of the mutant of claim 1, the gene of claim 2, the recombinant vector of claim 3 or 4, the recombinant cell of any one of claims 5 to 7, or the recombinant Escherichia coli of claim 8 in the preparation of agar oligosaccharides.

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