Mutant for improving catalytic activity of alpha-agarase and application method thereof

Abstract

The invention discloses a mutant for improving the catalytic activity of alpha-agarase and an application method of the mutant, and belongs to the technical field of gene engineering and enzyme engineering. The invention provides an alpha-agarase mutant which is obtained by mutating amino acid at the 1101st site of agarase with an amino acid sequence as shown in SEQ ID NO.1. The catalytic activity of the alpha-agarase mutant E1101A is improved compared with that of 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 of α-agarase and its application method, belonging to the fields of genetic engineering and enzyme engineering technology. Background Technology

[0002] 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 a variety of biological activities, including antioxidant, moisturizing and whitening, anti-diabetic and anti-obesity, anti-inflammatory, prebiotic, anti-tumor, and neuroprotective activities. They have application potential in food, medicine, agriculture, and other fields, and are high-value-added industrial agar products with broad application prospects. There are two main methods for preparing agar oligosaccharides: chemical degradation and biodegradation. Among these, compared with chemical degradation, biodegradation, represented by enzymatic hydrolysis, produces agar oligosaccharides with good substrate specificity, is environmentally friendly, and produces products with high activity, making it an effective method for preparing agar oligosaccharides. 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, a total of 10 α-agarases have been reported both domestically and internationally, derived from... Alteromonas agarlyticus , Thalassomonas sp.、 Thalas- Somonas sp.、 Catenovulum agarivorans , Catenovulum sediminis and Gilvimarinus agarilyticus The gene pool of α-agarases is still insufficient. Previous studies have achieved secretory expression of these α-agarase genes in *E. coli* and conducted enzymatic property analysis and identification of enzymatic digestion products on the obtained α-agarases. The initial crude enzyme activity of fermentation cultures using heterologous expression technology was consistently below 10 U / mL, which severely hinders the large-scale application of α-agarases. Therefore, it is necessary to enrich the α-agarase gene pool and develop α-agarases with high catalytic activity, good product specificity, high stability, and promising industrial applications, providing key enzyme preparations for the enzymatic production of agar oligosaccharides.

[0003] This invention selects Saccharophagus litoralisSite-directed mutagenesis of Sa-Aga derived from STB26 has resulted in an enzyme with strong substrate specificity, capable of 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, the low enzyme activity of wild-type Sa-Aga significantly limits the practical industrial application of agarase. Therefore, improving the catalytic activity of α-agarase Sa-Aga through genetic engineering and enzyme engineering is of great significance for the targeted molecular modification of α-agarase and the realization of high-efficiency, low-cost agar processing. Summary of the Invention

[0004] To address the shortcomings of the prior art, this invention provides a mutant with enhanced α-agarase catalytic activity and its application method, aiming to solve the problem of low α-agarase activity in the prior art.

[0005] The first technical solution provided by the present invention is an α-agarase mutant, wherein the α-agarase mutant is obtained by mutating glutamic acid at position 1101 of the α-agarase parent with the amino acid sequence as shown in SEQ ID NO.1 to alanine, and is named: E1101A.

[0006] The second technical solution provided by the present invention is a gene encoding the α-agarase mutant described in the first technical solution.

[0007] The third technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution.

[0008] In some embodiments, the recombinant vector is pET-28a(+) as the expression vector.

[0009] 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.

[0010] In some embodiments, the recombinant cells use bacteria or fungi as host cells.

[0011] In some embodiments, the recombinant cells use Escherichia coli BL21(DE3) as the host cell.

[0012] 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.

[0013] 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.

[0014] The sixth technical solution provided by the present invention is a method for improving the catalytic activity of α-agarase, wherein the method involves mutating glutamic acid at position 1101 of the α-agarase parent with the amino acid sequence shown in SEQ ID NO.1 to alanine.

[0015] 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.

[0016] 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 glutamic acid at position 1101 to alanine, resulting in mutant E1101A. Compared with the wild type, the enzyme activity of mutant E1101A is increased to 122.41% of that of the wild type. In the enzymatic hydrolysis products of mutant E1101A, agarotriose is the main product, accounting for about 60%, followed by agarotetraose and agaropentose, while monosaccharides account for the smallest proportion, and the product distribution is highly consistent with that of the wild type. The yield of agarotriose in the E1101A product is 47.1108%, which is about 7% higher than that of the wild type. These conclusions indicate that, compared with the wild type, mutant E1101A improves the production efficiency of continuous production in practical industrial applications and has good prospects for industrial application. Attached Figure Description

[0017] Figure 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 D1001A, D1004A, E1164A, D1252A, E1101A, V1052I and N1357D.

[0018] Figure 2 SDS-PAGE images of wild-type Sa-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 E1101A, D1001A, D1004A, E1164A, D1252A, V1052I and N1357D.

[0019] Figure 3 The relative enzyme activity of wild-type Sa-Aga and its mutants is characterized.

[0020] Figure 4 The degree of polymerization of enzymatic hydrolysates of wild-type Sa-Aga and its mutants was analyzed. Figure A shows the degree of polymerization distribution curves of the enzymatic hydrolysates of wild-type Sa-Aga and its mutants as detected by ion chromatography. Figure B shows the peak areas of the hydrolysates at different degrees of polymerization. Figure C shows the relative peak areas of the hydrolysates at different degrees of polymerization. Figure D shows the yield of the hydrolysates at different degrees of polymerization.

[0021] Figure 5 To detect the mass-to-charge ratio and degree of polymerization of the enzymatic hydrolysis products of wild-type Sa-Aga by mass spectrometry.

[0022] Figure 6 The mass-charge ratio and degree of polymerization of the enzymatic hydrolysis products of mutant E1101A were determined by mass spectrometry. Detailed Implementation

[0023] 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.

[0024] Test 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.

[0025] 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).

[0026] 2. Detection of the 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.

[0027] 3. Detection of the mass-to-charge ratio of α-agarase products: The mass-to-charge ratio of α-agarase products was determined by ultra-high performance liquid chromatography-electrospray ionization-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The detection conditions were as follows: (1) Column: ACQUITY UPLC BEH Amide column (100 mm × 2.1 mm, 1.7 μm); (2) Mobile phase: A was methanol, B was 0.1% (v / v) formic acid aqueous solution, A:B = 1:99, flow rate 0.3 mL / min; (3) Column temperature 10°C, injection volume 2 μL; (4) Mass spectrometry conditions: Electrospray ionization (ESI) source was used, and mass spectrometry analysis was performed in negative ionization mode. All mass spectrometry results were processed using MassLynx software (version 4.1).

[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-D1001A pET-28a(+) / sa-D1004A pET-28a(+) / sa-E1164A、 pET-28a(+) / sa-D1252ApET-28a(+) / sa-E1101A、 pET-28a(+) / sa-V1052I and pET-28a(+) / sa-N1357D The plasmid extraction verification results are as follows: Figure 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- D1001A pET-28a(+) / sa-D1004A pET-28a(+) / sa-E1164A、 pET-28a(+) / sa-D1252A pET-28a(+) / sa-E1101A、 pET-28a(+) / sa-V1052I and pET-28a(+) / sa-N1357D 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-D1001A ), E. coli BL21(DE3) / (pET-28a(+) / sa-D1004A ), E. coli BL21(DE3) / (pET-28a(+) / sa-E1164A)、E. coli BL21(DE3) / (pET-28a(+) / sa-D1252A)、E. coli BL21(DE3) / (pET-28a(+) / sa-E1101A)、E. coli BL21(DE3) / (pET-28a(+) / sa-V1052I)、 E. coli BL21(DE3) / (pET-28a(+) / sa-N1357D) 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. Figure 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 the wild-type agarase and mutants E1101A, D1001A, D1004A, E1164A, D1252A, V1052I, and N1357D 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: Figure 3 As shown in the figure, the enzyme activity of mutant E1101A was significantly increased compared to wild type (WT), by about 23%; the enzyme activities of mutants V1052I and N1357D were not significantly different from those of wild type, while the enzyme activities of mutants D1001A, D1004A, E1164A, and D1252A decreased significantly.

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

[0042] Example 4: 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 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. Figure 4 As shown.

[0043] 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 relatively stable. Agaric triose was the main product, accounting for approximately 55%, followed by agaric tetraose and agaric pentose, while monosaccharides accounted for the smallest proportion. This indicates that the introduced mutation did not change the degree of polymerization distribution pattern of the enzymatic reaction, and the product distribution of mutant E1101A was highly consistent with the wild type. The yields of monosaccharides, agaric triose, and agaric pentose were quantitatively calculated using standards, and the yields of each product were further compared. Table 4 shows that the yield of agaric triose in E1101A was 47.1108%, an increase of approximately 7% compared to the wild type's 40.3780%, which may be related to the improved enzyme activity and thermal stability of E1101A. Due to low enzyme activity, the peak area of ​​the product in mutant E1164A was small, and the product peak of D1252A was not detected.

[0044] Table 3. Distribution of degree of polymerization of α-agarase products from wild-type and mutant strains.

[0045] Table 4. Product yields of α-agarase wild-type and mutant.

[0046] Example 5: Detection of enzyme product mass-to-charge ratio The mass-to-charge ratios of the products of α-agarase Sa-Aga wild-type and mutant E1101A were detected by ultra-high performance liquid chromatography-electrospray ionization-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) to further verify the degree of polymerization distribution of the products. The molecular weight values ​​of agar oligosaccharides with different degrees of polymerization are shown in Table 5. The specific steps are as follows: The reaction products of Sa-Aga wild-type and mutant E1101A were prepared according to the steps in Example 4, diluted, filtered through a 0.22 μm aqueous membrane, and detected by mass spectrometry. The results are shown in Table 5. Figure 6 As shown.

[0047] Table 5. Molecular weight of agar oligosaccharides with different degrees of polymerization

[0048] The principle of liquid chromatography-mass spectrometry (LC-MS) is to separate oligosaccharides using a BEH Amide column in either positive or negative ion mode, achieving optimal separation according to the degree of polymerization from low to high. In negative ion mode, agar oligosaccharides are separated as quasi-molecular ions [M-H]. - The samples were detected. Based on the molecular weight data of agarose oligosaccharides in Table 5, the degrees of polymerization of P1, P2, P3, and P4 were 1, 3, 4, and 5, respectively. According to the above chromatographic and mass spectrometric results, the products of agarose hydrolysis from Sa-Aga wild-type and mutant E1101A were determined to be A3, A4, A5, and a small amount of monosaccharides. The mass spectrometry data (m / z) of agarose oligosaccharides in negative ion mode are shown in Table 6.

[0049] Table 6. Mass spectrometry data (m / z) of agar oligosaccharides in negative ion mode.

[0050] 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 glutamic acid at position 1101 of the α-agarase parent amino acid sequence as shown in SEQ ID NO. 1 to alanine.

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.

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 of α-agarase, characterized in that, The method involves mutating glutamic acid at position 1101 of the α-agarase parent amino acid sequence shown in SEQ ID NO.1 to alanine.

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.

Images