Method for preparing agarotriose through double-enzyme synergism

Abstract

The invention discloses a method for preparing agarotriose through double-enzyme synergism, and belongs to the field of agaro-oligosaccharide preparation. According to the method disclosed by the invention, under the condition of the optimal enzymolysis temperature, a method for synergistically hydrolyzing agar by using double enzymes is adopted, so that the specific efficient preparation of the agarotriose is realized. Compared with a method for preparing odd-number agaro-oligosaccharide through an acidolysis method, the method has the advantages that the reaction conditions are mild and easy to control, meanwhile, the influence of system viscosity rising caused by cooling after agar is subjected to high-temperature treatment on subsequent enzymolysis is reduced, the yield of agaro-triose is obviously increased, a certain theoretical basis can be provided for industrial preparation of functional agaro-oligosaccharide, and the method is suitable for industrial production. The method is suitable for food, cosmetic and pharmaceutical fields.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for preparing a triose by two enzymes in cooperation, and belongs to the field of preparation of oligosaccharides. BACKGROUND

[0002] Agar is a colloidal polysaccharide in the cell wall of red algae, which is composed of ionic sulfated agar and neutral agarose. The former is mainly composed of a series of complex polysaccharide chains, and the latter is a linear chain of sugars with alternating β-1, 4- and α-1, 3-glycosidic bonds, which is the main component of agar to form a gel state. Oligosaccharides are derived from agarose, and generally refer to oligosaccharides with a degree of polymerization of 2-10 formed by hydrolysis of agarose polysaccharides. According to the different non-reducing ends, it can be divided into oligosaccharides and neosaccharides. A large number of studies have shown that oligosaccharides have broad application prospects in food, cosmetics, pharmaceutical and other industries due to their anti-inflammatory, liver-protecting, prebiotic, antioxidant and other physiological activities. However, oligosaccharides have not been industrialized so far.

[0003] The preparation methods of oligosaccharides mainly include acid degradation method and biological degradation method. At present, the acid degradation method has the problems of non-uniform product, easy loss of oligosaccharide activity, high energy consumption of high-temperature acidolysis, etc. At the same time, the energy consumption of acidolysis product deacidification is large and not environmentally friendly. The biological degradation method, i.e. enzymatic hydrolysis method, uses agarase to specifically cut the glycosidic bond of agarose polysaccharide, which has the advantages of easy control of reaction conditions, good specificity of product, green process, etc. However, the substrate concentration that can be acted on by a single enzyme is relatively low, and the yield of product is low. In previous laboratory studies, agar was hydrolyzed by acid-enzyme method, and the yield of A3 was increased to 59.11%, which is the highest yield of A3 so far. However, because the hydrolysis conditions contain acid, it is not conducive to large-scale industrial production of triose. Subsequent desalination consumes a large amount of energy and is not environmentally friendly. Therefore, a new method for efficient preparation of triose needs to be found. SUMMARY

[0004] In order to solve the technical problems of non-uniform product, easy loss of activity, high energy consumption of high-temperature acidolysis in the preparation of oligosaccharides by acidolysis method, and low substrate concentration that can be acted on by a single enzyme in the single enzyme hydrolysis method, so that it is difficult to mass-produce functional oligosaccharides in industry, since the α-AGA derived from Catenovulum agarivorans is the highest activity α-agarase in the laboratory at present, the present application first uses α-AGA derived from Catenovulum agarivorans to act on agar, and finds the best conditions by optimizing the process of single enzyme hydrolysis of agar. Under the best single enzyme action process conditions, the other enzyme is selected from α-AGA (Sa-AGA) SA enzyme derived from Saccharophagus litoralis sulfatase derived from Helix pomatia and α-AGA derived from Aspergillus oryzaeβ-galactosidase, which can hydrolyze agar to some extent, to prepare the trisaccharide efficiently by the synergistic effect of the two enzymes. Saccharophagus litoralis The α-AGA (Sa-AGA) SA enzyme derived from Agarivorans salegae has a poor effect on the agar hydrolysis to obtain A3 compared with the CA enzyme. The production method does not contain acid, greatly reduces the deacidification process, and uses the enzymatic process throughout, which is environmentally friendly and produces purer products. The method has great significance for the industrial production of agar oligosaccharides and the expansion of specific agar oligosaccharide preparation technology.

[0005] The present application uses α-agarase, which contains trisaccharide as the final product of enzymatic hydrolysis, uses a mild optimal enzymatic hydrolysis temperature of 35℃ as the reaction temperature, and optimizes the substrate concentration, agar pretreatment temperature, enzymatic hydrolysis time, and enzyme addition amount to effectively improve the yield of trisaccharide. The method has more advantages in simplifying the process, reducing energy consumption, and protecting the environment. In addition, the present application uses high-performance anion exchange chromatography and ultra-performance liquid chromatography-electrospray-quadrupole-time-of-flight mass spectrometry to analyze the product.

[0006] The first technical solution of the present application is a method for efficiently preparing trisaccharide using two enzymes, which comprises the following steps: (1) adding agar in deionized water and mixing uniformly to obtain an agar pretreatment solution; (2) adding Ca-AGA enzyme and auxiliary enzymes to react when the temperature of the pretreatment solution of step (1) is reduced to the enzymatic hydrolysis temperature to obtain a reaction product; (3) obtaining trisaccharide after concentrating and purifying the reaction product of step (2).

[0007] The trisaccharide obtained is analyzed by high-performance anion exchange chromatography and ultra-performance liquid chromatography-electrospray-quadrupole-time-of-flight mass spectrometry.

[0008] In some embodiments, the concentration of agar in the agar pretreatment solution in step (1) is 7.5-15%.

[0009] In some embodiments, the method comprises the following steps: (1) weighing agar and adding it to deionized water, stirring at 40-80℃ and 200-300 rpm for 5-15 min, and mixing uniformly to obtain an agar pretreatment solution; (2) placing the agar pretreatment solution obtained in step (1) in a 35℃, 200-300 rpm incubator for 10 min, then adding Sa-AGA and Ca-AGA for enzymatic hydrolysis for 8-32 h, and boiling for 10-30 min to inactivate the enzymes to obtain an enzymatic hydrolysate containing trisaccharide; (3) obtaining trisaccharide after concentrating and purifying the enzymatic hydrolysate of step (2).

[0010] In some embodiments, the mixing in step (1) is performed at 60°C for 5-15 min with stirring at 200-300 rpm.

[0011] In some embodiments, in step (2), the Ca-AGA enzyme is α-AGA (Ca-AGA) derived from Catenovulum agarivorans and having an amino acid sequence as shown in SEQ ID NO. 3; and the auxiliary enzyme is α-AGA (Sa-AGA) derived from Saccharophagus litoralis and having an amino acid sequence as shown in SEQ ID NO. 4.

[0012] In some embodiments, in step (2), the amount of Ca-AGA enzyme added is 90-150 U / g.

[0013] In some embodiments, in step (2), the amount of Sa-AGA enzyme added is 40-80 U / g.

[0014] In some embodiments, the concentration and purification in step (3) are performed by first lyophilizing and then purifying using a Superdex 30 Increase 10 / 300 GL gel chromatography column.

[0015] In some embodiments, the product analysis in step (3) is performed using a high-performance anion exchange chromatograph, an ultra-high performance liquid chromatograph-electrospray-quadrupole-time-of-flight mass spectrometer.

[0016] In some embodiments, the method comprises the following steps: (1) weighing agar and adding deionized water, stirring at 60°C and 250 rpm for 10 min, and mixing uniformly to obtain a 12.5% (w / w) agar acidolysis solution; (2) placing the agar pretreatment solution obtained in step (1) in a 35°C, 250 rpm incubator for 10 min, then adding 130 U / g Ca-AGA and 70 U / g Sa-AGA, respectively, for enzymolysis for 32 h, and then boiling for 15 min to inactivate the enzymes to obtain an enzymolysis solution containing gulo-triose; (3) concentrating and purifying the enzymolysis solution of step (2) to obtain gulo-triose.

[0017] The second technical solution of the present application is gulo-triose prepared by the method of the first technical solution.

[0018] The third technical solution of the present application is the use of gulo-triose of the second technical solution in the fields of food, cosmetics, and pharmaceuticals.

[0019] The fourth technical solution of the present invention is a method for increasing the yield of agarotriose synthesized by α-agarase catalysis. The method uses agarose as a substrate and utilizes Sa-AGA enzymes with amino acid sequences as shown in SEQ ID NO.4 and Ca-AGA enzymes with amino acid sequences as shown in SEQ ID NO.3 to co-catalyze the synthesis of agarotriose.

[0020] In some implementations, the substrate agarose concentration is 7.5-15%.

[0021] In some embodiments, the catalytic conditions are as follows: after incubation at 35°C and 200-300 rpm for 10 min, Sa-AGA and Ca-AGA are added for enzymatic hydrolysis for 8-32 h.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes a dual-enzyme hydrolysis method on agar under optimal enzymatic hydrolysis temperature conditions to achieve the specific and efficient preparation of agarotriose. Compared with acid hydrolysis methods for preparing odd-numbered agar oligosaccharides, this invention offers milder and easier-to-control reaction conditions. It also reduces the impact of increased viscosity due to cooling after high-temperature treatment on subsequent enzymatic hydrolysis, resulting in a significant increase in agarotriose yield. This provides a theoretical basis for the industrial preparation of functional agar oligosaccharides and is applicable to the food, cosmetics, and pharmaceutical industries. Attached Figure Description

[0023] Figure 1 The graph shows the yield of A3 prepared by CA single enzyme in Example 1 under different agar pretreatment temperatures.

[0024] Figure 2 The graph shows the yield of CA single enzyme in Example 2 for preparing A3 under different substrate concentrations.

[0025] Figure 3 The graph shows the yield of A3 prepared by CA single enzyme under different enzyme addition conditions in Example 3.

[0026] Figure 4 The graph shows the yield of A3 prepared by CA single enzyme under different enzymatic hydrolysis time conditions in Example 4.

[0027] Figure 5 The image shows the results of HPAEC-PAD preparation of A3 under the optimal process conditions of CA enzyme in Example 6.

[0028] Figure 6 The graph shows the yield of A3 prepared by SA enzyme assisted by CA enzyme under different enzyme addition conditions in Example 6.

[0029] Figure 7The image shows the HPAEC-PAD result of A3 obtained by SA enzyme combined with CA enzyme hydrolysis of agar in Example 8 after purification.

[0030] Figure 8 The image shows the UPLC-Q-TOF-MS result of A3 obtained by enzymatic hydrolysis of agar with SA enzyme combined with Ca-AGA in Example 8 after purification. Detailed Implementation

[0031] 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.

[0032] Test method: 1. DNS method for determining Ca-AGA enzyme activity: The hydrolytic activity of Ca-AGA was characterized by changes in reducing sugar content determined by 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 35 °C for 30 min. The reaction was then terminated by adding 1 mL of DNS, followed by boiling in a water bath for 5 min, followed by rapid cooling. 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 galactose as a standard.

[0033] Enzyme activity definition: Each unit of enzyme activity (U) is defined as the amount of enzyme required to generate 1 μmol of reducing sugar (calculated as galactose) per minute.

[0034] 2. Detection of agarose triose or agarose pentaose After enzymatic hydrolysis of agar, the sample was boiled to inactivate the enzyme for 15 min, centrifuged, diluted, and filtered through a 0.22 μm aqueous membrane. The products were then analyzed using a high-performance anion exchange chromatography system (HPAEC-PAD) and an ultra-high performance liquid chromatography-electrospray ionization-quadrupole-time-of-flight mass spectrometer (UPLC-Q-TOF-MS). The HPAEC-PAD conditions were as follows: Dionex CarboPac PA-200 anion exchange column, column temperature 35°C, mobile phase 100 mM NaOH, 100 mM NaOH & 500 mM NaAc, flow rate 0.5 mL / min. The conditions for UPLC-Q-TOF-MS were as follows: ACQUITY UPLC BEH Amide column, column temperature 40°C, mobile phase A was 0.1% ammonia-acetonitrile solution, mobile phase B was water-methanol (2:1, V / V) solution, flow rate was 0.3 mL / min, and mass spectrometry analysis was performed using an electrospray ionization (ESI) source in negative ion ionization mode.

[0035] 3. Calculation of agar-agar yield and production rate The agar enzyme digestion solution, on a dry basis, has a mass fraction of Q, a total mass of M g, an enzyme dosage of N g, and a sample volume of mg after digestion. After dilution and volume adjustment, the total volume is V mL. The sample is further diluted n times, filtered through a membrane, and then detected by ion chromatography. The concentration of the agarotriose standard is 10 μg / mL, the peak area corresponding to the standard is S1, and the total peak area corresponding to the enzyme-digested sample is S2.

[0036] The mass of agarose contained in the mg sample is Y (g); .

[0037] The total agarotriose content in the agar hydrolysate is Z (g); 。

[0038] Agarose yield in the product (g / g agar): .

[0039] Agarose yield (%) in the product: .

[0040] In summary:

[0041] .

[0042] Raw materials used in the examples: LB liquid medium: yeast extract (Sinopharm Group) 5 g / L, tryptone (Shanghai Mairui) 10 g / L, NaCl (Sinopharm Group) 10 g / L, pH 7.0.

[0043] LB solid medium: yeast extract (Sinopharm Group) 5 g / L, tryptone (Shanghai Mairui) 10 g / L, NaCl (Sinopharm Group) 10 g / L, pH 7.0, 1.5% (w / v) agar (Sinopharm Group).

[0044] Fermentation liquid culture medium: yeast powder (Sinopharm Group) 12 g / L, tryptone (Shanghai Mairui) 24 g / L, sucrose (Sinopharm Group) 5 g / L, KH2PO (Sinopharm Group) 17 mM, K2HPO4 (Sinopharm Group) 72 mM, pH 9.0.

[0045] Example 1: Method for preparing agarose using a single enzyme Catenovulum agarivorans Preparation method of α-AGA (CA): (1) Strain construction: Use restriction endonucleases Nco I and Xho I will reorganize Ca-aga The gene (nucleotide sequence shown in SEQ ID NO.1) was cloned into the plasmid pET-28a(+), and the obtained expression vector pET-28a(+) / Ca-aga Transform to E. coli JM109 was plated on a kanamycin-containing plate, and transformants were picked for sequencing and double enzyme digestion verification. The correctly sequenced expression vector was then transformed into competent cells. E. coli BL21(DE3) was cultured to obtain recombinant strains. E. coli BL21(DE3) / pET-28a(+)- Ca-aga .

[0046] (2) Slide the flat board: Use an inoculation loop to pick up the recombinant strain E. coli BL21(DE3) / pET-28a(+)- Ca-aga The bacterial culture was streaked onto LB solid medium containing 100 μg / mL kanamycin and incubated at 37°C for 12 h. (3) Activation: Single colonies were picked and cultured in LB medium containing 100 μg / mL kanamycin in a shake flask at 37℃ and 200 rpm for 8-12 h to prepare seed culture; (4) Fermentation: The seed culture obtained in step (3) was transferred to 50 mL of fermentation medium containing 100 μg / mL kanamycin at an inoculum rate of 4% (v / v). OD 600 When the pH reached 0.6, isopropyl-β-D-thiogalactoside (IPTG, final concentration 0.01 mM) was added, and the cells were cultured in shake flasks at 25 °C and 200 rpm for 20 h. The fermentation broth was collected and centrifuged at 4 °C and 10,000 rpm for 20 min to obtain bacterial cells. An equal volume of lysis buffer (20 mM Tris-HCl, pH 8.0) was added to resuspend the bacterial cells, and the cells were treated with an ultrasonic cell disruptor for 15 min.

[0047] The supernatant was collected by low-temperature high-speed centrifugation to obtain the crude intracellular enzyme solution. The Ca-AGA enzyme activity was measured to be 80.79 U / mL by the DNS method.

[0048] Weigh 5 g of agar and add it to 50 mL of deionized water. Stir at 250 rpm for 10 min at 40℃, 50℃, 60℃, 70℃, and 80℃ respectively until homogeneous, to obtain a 10% substrate concentration agar pretreatment solution. Add 100 U / g of CA enzyme and hydrolyze for 24 h. After boiling for 15 min to inactivate the enzyme, centrifuge the hydrolysate for 10 min, collect the supernatant, and remove the precipitate. The final yield and percentage of agarotriose are shown in Table 1.

[0049] Analysis and calculations were performed using agar oligosaccharide standards. The yield of A3 after enzymatic hydrolysis under different substrate concentrations is shown in the figure. Figure 1 .

[0050] Table 1. Effects of different pretreatment temperatures on the yield and efficiency of A3 production from CA enzyme.

[0051] The results show that, as can be seen from the graph, the yield of A3 obtained by enzymatic hydrolysis first increases and then decreases with the increase of pretreatment temperature; when the pretreatment temperature is 60℃, the final yield of A3 is the highest at 17.77%.

[0052] Subsequent experiments were conducted with agar pretreatment at 60℃.

[0053] Example 2: Method for preparing agarose using CA single enzyme Agar pretreatment solutions with concentrations of 7.5%, 10.0%, 12.5%, and 15.0% (w / w) were prepared by weighing 3.75, 5, 6.25, and 7.5 g of agar, respectively, and adding them to 50 mL of deionized water. The solutions were stirred at 60 °C and 250 rpm for 10 min to ensure homogeneity. The CA enzyme dosage was 100 U / g, and the enzymatic hydrolysis time was 24 h. The final yield and percentage of agarotriose are shown in Table 2.

[0054] Analysis and calculations were performed using agar oligosaccharide standards. The yield of A3 after enzymatic hydrolysis under different substrate concentrations is shown in the figure. Figure 2 .

[0055] Table 2. Effects of different agar substrate concentrations on the yield and efficiency of A3 preparation using CA enzyme.

[0056] The results showed that, as can be seen from the graph, the yield of A3 obtained by enzymatic hydrolysis first increased and then decreased with the increase of substrate concentration; when the substrate concentration was 12.5% ​​(w / w), the final yield of A3 was the highest at 23.29%.

[0057] Subsequent experiments were conducted with agar pretreatment at 60°C and substrate concentration of 12.5% ​​(w / w).

[0058] Example 3: Method for preparing agarose using CA single enzyme Weigh 6.25 g of agar and add it to 50 mL of deionized water. Stir at 60 °C for 10 min to mix evenly. Add different amounts of CA enzyme, namely 90 U / g, 100 U / g, 110 U / g, 120 U / g, 130 U / g, 140 U / g and 150 U / g. The enzymatic hydrolysis time is 24 h. The yield and percentage of agarotriose are shown in Table 3.

[0059] Analysis and calculations were performed using agar oligosaccharide standards. The yield of A3 after enzymatic hydrolysis under different CA enzyme dosages is shown in the figure. Figure 3 .

[0060] Table 3. Effects of different enzyme dosages on the yield and efficiency of A3 preparation from CA.

[0061] As shown in the chart, the yield of A3 obtained by enzymatic hydrolysis first increases and then decreases with the increase of CA enzyme dosage; when the enzyme dosage is 130 U / g, the final yield of A3 is the highest at 31.48%.

[0062] Subsequent experiments were conducted with agar pretreatment at 60℃, substrate concentration of 12.5% ​​(w / w), and enzyme dosage of 130 U / g.

[0063] Example 4: Method for preparing agarose using CA single enzyme Weigh 6.25 g of agar and add it to 50 mL of deionized water. Stir at 60 °C for 10 min to mix evenly. The amount of CA enzyme added is 130 U / g. The enzymatic hydrolysis times are 8 h, 12 h, 16 h, 20 h, 24 h, 30 h and 32 h, respectively. The yield and percentage of agarotriose are shown in Table 4.

[0064] Analysis and calculations were performed using agar oligosaccharide standards. The yield of A3 under different enzymatic hydrolysis times is shown in the figure. Figure 4 .

[0065] Table 4. Effects of different enzymatic hydrolysis times on the yield and efficiency of A3 preparation from CA.

[0066] The results show that, as can be seen from the graph, the yield of A3 obtained by enzymatic hydrolysis first increases and then decreases with the increase of enzymatic hydrolysis time; when the acid hydrolysis time is 30 h, the final yield of A3 is the highest at 36.00%.

[0067] Example 5: Method for preparing agarose using SA single enzyme Saccharophagus litoralis Preparation method of SA enzyme: (1) Strain construction: Specifically as follows: An enzyme with a chemically synthesized nucleotide sequence as shown in SEQ ID NO.2 was ligated with a vector to obtain a recombinant vector. A restriction endonuclease was used. Nco I and Xho I will agaS The clone was inserted into the plasmid pET-28a(+), and the resulting expression vector pET-28a(+)- agaS Transform to E. coli JM109 was plated on a kanamycin-containing plate, and transformants were picked for sequencing and double enzyme digestion verification. The correctly sequenced expression vector was then transformed into competent cells. E. coli BL21(DE3) was cultured to obtain recombinant strains. E. coli BL21(DE3) / pET-28a(+)- agaS .

[0068] (2) Slide the flat board: Use an inoculation loop to pick up the recombinant strain E. coli BL21(DE3) / pET-28a(+)- agaS The bacterial culture was streaked onto LB solid medium containing 100 μg / mL kanamycin and then incubated at 37°C for 12 h.

[0069] (3) Activation: Pick the single colony obtained in step (2) into LB liquid medium containing 100 μg / mL kanamycin, and culture it in a shake flask at 37℃ and 200 rpm for 12 h to prepare the seed culture.

[0070] (4) Fermentation: The seed culture obtained in step (3) was transferred to TB liquid medium containing 100 μg / mL kanamycin at an inoculation rate of 4% (v / v). 600 When the pH reached 0.6, isopropyl-β-D-thiogalactopyranoside (IPTG, 10 μL / 50 mL, final concentration 0.005 mM) was added, and the mixture was cultured in shake flasks at 25℃ and 200 rpm for 24 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 manner, 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 1 As shown in lane 1 of the middle swimming pool. The AgaS enzyme activity was measured to be 44.25 U / mL by the DNS method.

[0071] Similar to the method of agar hydrolysis using CA single enzyme, the optimal process conditions for SA enzyme were investigated by adjusting temperature, substrate concentration, enzyme dosage, and reaction time as follows: (1) When the substrate concentration was 10%, the amount of SA added was 50 U / g, and the enzymatic hydrolysis time was 24 h, the yield and efficiency of agarose were finally detected by different pretreatment temperatures, as shown in Table 5.

[0072] Table 5. Effects of different pretreatment temperatures on the yield and efficiency of A3 production from SA enzyme.

[0073] (2) Based on the table above, a pretreatment temperature of 60℃ was selected, and agar pretreatment solutions with concentrations of 7.5%, 10.0%, 12.5%, and 15.0% (w / w) were selected respectively. The amount of SA enzyme added was 50 U / g, and the enzymatic hydrolysis time was 24 h. The final yield and efficiency of agarotriose were shown in Table 6.

[0074] Table 6. Effects of different agar substrate concentrations on the yield and efficiency of A3 preparation using SA enzyme.

[0075] (3) Based on the table above, the pretreatment temperature was 60℃, the substrate concentration was 12.5%, the SA enzyme dosage was 40 U / g, 50 U / g, 60 U / g, 70 U / g and 80 U / g, the enzymatic hydrolysis time was 24 h, and the final yield and efficiency of agarose were shown in Table 7.

[0076] Table 7. Effects of different enzyme dosages on the yield and efficiency of A3 preparation from SA.

[0077] (4) Based on the table above, the pretreatment temperature was selected as 60℃, the substrate concentration as 12.5%, the amount of SA enzyme added as 70 U / g, and the enzymatic hydrolysis time as 8 h, 12 h, 16 h, 20 h, 24 h, 30 h and 32 h respectively. The yield and efficiency of agarose were finally detected as shown in Table 8.

[0078] Table 8. Effects of different enzymatic hydrolysis times on the yield and efficiency of A3 production from SA.

[0079] Based on the optimization of the process conditions for SA single-enzyme hydrolysis of agar, it was found that the highest yield of A3 obtained from SA hydrolysis of agar was 10.45% under the conditions of pretreatment temperature of 60℃, substrate concentration of 12.5%, hydrolysis time of 30 h, and enzyme dosage of 70 U / g, which was significantly lower than that of CA single-enzyme hydrolysis process.

[0080] Example 6: Method for preparing agarose using two enzymes Under the optimal hydrolysis conditions of CA single enzyme, SA enzyme and CA enzyme were used together on agar. Among them, CA enzyme had the highest activity and mainly hydrolyzed agar. The agar pretreatment temperature was 60℃, the substrate concentration was 12.5%, and the CA enzyme dosage was 130 U / g. Then, SA enzyme was added at concentrations of 50 U / g, 60 U / g, 70 U / g, and 80 U / g, respectively, and the enzymes were hydrolyzed together for 32 h. The yield and percentage of agarotriose were finally determined and are shown in Table 9.

[0081] Analysis and calculations were performed using agar oligosaccharide standards. The yield of A3 prepared under two-enzyme conditions with the appropriate amount of SA enzyme is shown in the figure. Figure 5 .

[0082] Table 9. Effects of different SA enzyme dosages on the yield and efficiency of A3 production under two-enzyme conditions.

[0083] As shown in the chart, SA and CA work synergistically with agar. When the enzyme dosage of CA is 130 U / g, the enzyme dosage of SA is 70 U / g, the substrate concentration is 12.5%, the agar pretreatment temperature is 60℃, the enzymatic hydrolysis is carried out for 32 h, and the enzyme is inactivated by boiling. After centrifugation at 10000 rpm for 15 min, the supernatant is obtained. The highest yield of A3 was obtained at 43.70%.

[0084] Take 1 g of the obtained enzymatic hydrolysate and dilute it to 10 mL. After diluting 2000 times, filter it through a 0.22 μm aqueous membrane for HPAEC-PAD detection. The results are as follows: Figure 6 As shown, its main product is agarotriose.

[0085] The literature (Jiang, Liu, Sun, et al. A Novel Route for Agarooligosaccharide Production with the Neoagarooligosaccharide-Producing β-Agarase as Catalyst[J]. Catalysts, 2020, 10(2):214.) reported that after 15% (w / v) agarose was initially liquefied with 2.5% (w / v) citric acid at 90℃ and 0.5 MPa for 50 min, the yield of agarotriose was 14% after being catalyzed by β-agarooligosaccharide DagA (a new agarohexasaccharide-producing enzyme). It can be seen that the method for preparing agarotriose in Example 8 is better than the existing preparation technology, and the dual-enzyme action environment is acid-free, which is green and environmentally friendly.

[0086] Example 7: Preparation and purification of agarose The specific steps are as follows: (1) Weigh 6.25 g of agar and add it to 50 mL of deionized water. Stir at 60 °C and 250 rpm for 10 min until well mixed to obtain an agar pretreatment solution with a concentration of 12.5% ​​(w / w). (2) At the optimal enzymatic hydrolysis temperature of 35℃, 70 U / g SA enzyme and 130 U / g CA enzyme were added to the agar pretreatment solution obtained in step (1) and enzymatically hydrolyzed for 32 h, and then boiled for 15 min to inactivate the enzyme.

[0087] (3) Centrifuge the reaction system obtained in step (2), collect the supernatant, freeze-dry and concentrate, and weigh. After reconstitution, separate the sample by size exclusion chromatography (SEC) using a Superdex 30 Increase 10 / 300 GL column. Elute with Milli-Q water at a flow rate of 0.1 mL / min, collect in separate tubes, and verify the purity of agarotriose by analysis using HPAEC-PAD and UPLC-Q-TOF-MS.

[0088] Example 8: Characterization of agarose products (1) The ion chromatography results of the product obtained in Example 7 are as follows: Figure 7 As shown, the purified agarose is free of other sugars and has a high purity of up to 98.3%. The patent (Mao Xiangchao, Wang Qidong, Sun Jian'an, et al. An immobilized enzyme producing 3,6-endoether-L-galactose: 201810572993[P][2023-08-10].) provides an immobilized enzyme producing 3,6-endoether-L-galactose for degrading agarose to prepare agarose. The purified agarose has a purity higher than 96.0%. It can be seen that the method for preparing agarose in Example 8 is superior to existing preparation techniques.

[0089] (2) The liquid chromatography-mass spectrometry results of the product obtained in Example 7 are as follows: Figure 8 As shown, the component analysis results are as follows: Agarotriose [MH] - In the mass spectrometry, the doubly charged ion (m / z 242.0021) appeared at half the mass of the single-charged ion (m / z 485.0080) of the same mass unit, indicating that the two peaks belong to the same substance. The mass spectrometry analysis further confirms that high-purity agarotriose was obtained after separation and purification.

[0090] Comparative Example 1: A method for preparing agarose using two enzymes Preparation method of sulfatase-co-CA enzyme: Source Helix pomatiaSulfatetaase was purchased commercially from Shanghai Titan Company. Under the optimal hydrolysis conditions for CA single enzyme, sulfatetaase and CA enzyme were co-treated on agar. CA enzyme had the highest activity and mainly hydrolyzed agar. The agar pretreatment temperature was 60℃, the substrate concentration was 12.5%, and the CA enzyme dosage was 130 U / g. Then, 200 U, 300 U, and 400 U of sulfatetaase were added respectively, and the co-hydrolysis was carried out for 32 h. The final yield and percentage of agarotriose are shown in Table 10. The highest agarotriose yield (26.92%) was achieved when the sulfatetaase dosage was 300 U / g.

[0091] Table 10 Effect of different sulfatase dosages on the yield and productivity of A3 production under two-enzyme conditions.

[0092] Comparative Example 2: A method for preparing agarose using two enzymes Preparation method of galactosidase synergistic CA enzyme: Source Aspergillus oryzae The β-galactosidase was commercially purchased from Shanghai Titan Company. Under the optimal hydrolysis conditions of CA single enzyme, β-galactosidase and CA enzyme were co-treated on agar. CA enzyme had the highest activity and mainly hydrolyzed agar. The agar pretreatment temperature was 60℃, the substrate concentration was 12.5%, and the CA enzyme dosage was 130 U / g. Then, 300 U, 400 U, and 500 U of β-galactosidase were added respectively, and co-hydrolyzed for 32 h. The yield and percentage of agarotriose were finally determined and are shown in Table 11. The highest agarotriose yield (8.38%) was achieved when the β-galactosidase dosage was 400 U / g.

[0093] Table 11 Effects of different β-galactosidase dosages on the yield and efficiency of A3 production under two-enzyme conditions.

[0094] Although both Comparative Examples 1 and 2 are enzymes catalyzed by a double displacement mechanism, sulfatase theoretically acts on the sulfate bonds in agar, enabling desulfation of agar; β-galactosidase, as a glycosidic bond hydrolase, forms an "endo-exo" synergistic effect with agarase to achieve efficient hydrolysis of agar. However, in practice, the efficiency of these two enzymes in assisting the hydrolysis of agar is relatively low. SA enzyme has a higher efficiency in assisting the hydrolysis than these two enzymes, with the highest yield of agarotriose reaching 43.70%.

[0095] 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. A method for preparing agarose, characterized in that, Includes the following steps: (1) Add agar to deionized water and mix well to obtain an agar pretreatment solution; (2) When the temperature of the pretreatment solution in step (1) drops to the enzymatic hydrolysis temperature, add Ca-AGA enzyme and coenzyme to react and obtain the reaction product. (3) After concentrating and purifying the reaction product of step (2), agarose is obtained; Among them, Ca-AGA enzyme is derived from Catenovulum agarivorans The α-agarase, whose amino acid sequence is shown in SEQ ID NO.3; the coenzyme is derived from... Saccharophagus litoralis The α-agarase has the amino acid sequence shown in SEQ ID NO.

4.

2. The method according to claim 1, characterized in that, The method includes the following steps: (1) Add agar to water and stir for 5 to 15 minutes at 40 to 80°C and 200 to 300 rpm until well mixed to obtain an agar pretreatment solution; (2) After the agar pretreatment solution obtained in step (1) is kept at 35℃ and 200~300 rpm for 10 min, Ca-AGA enzyme and coenzyme are added to it for enzymatic hydrolysis for 8~32 h. After boiling for 10~30 min to inactivate the enzyme, an enzymatic hydrolysate containing agarose is obtained. (3) After concentrating and purifying the enzymatic hydrolysate from step (2), agarose is obtained.

3. The method according to claim 1 or 2, characterized in that, In step (1), the mixing is carried out at 60°C and 200-300 rpm for 5-15 min; the concentration of agar substrate in the agar pretreatment solution is 7.5-15%.

4. The method according to claim 1 or 2, characterized in that, In step (2), the amount of Ca-AGA added is 90~150 U / g, and the amount of the coenzyme added is 40~80 U / g.

5. The method according to claim 1 or 2, characterized in that, In step (3), the concentration and purification are carried out by first lyophilizing and then purifying using a Superdex 30 Increase 10 / 300 GL gel chromatography column; the product analysis is carried out using a high performance anion exchange chromatograph and an ultra-high performance liquid chromatography-electrospray ionization-quadrupole-time-of-flight mass spectrometer.

6. The method according to any one of claims 1 to 5, characterized in that, The method includes the following steps: (1) Weigh agar, add it to deionized water, stir at 60℃ and 250 rpm for 10 min, mix well, and obtain a 12.5% ​​(w / w) agar acid hydrolysis solution; (2) After the agar pretreatment solution obtained in step (1) was kept at 35°C and 250 rpm for 10 min, 130 U / g Ca-AGA and 70 U / g coenzyme were added to it for enzymatic hydrolysis for 32 h. After boiling for 15 min to inactivate the enzyme, an enzymatic hydrolysate containing agarose was obtained. (3) After concentrating and purifying the enzymatic hydrolysate from step (2), agarose is obtained.

7. Agarose prepared by the method according to any one of claims 1 to 6.

8. The use of the agarose according to claim 7 in the food, cosmetic and pharmaceutical fields.

9. A method for increasing the yield of agarotriose synthesized by α-agarase, characterized in that, The method involves using agar as a substrate and utilizing the Sa-AGA enzyme (as shown in SEQ ID NO.4) and the Ca-AGA enzyme (as shown in SEQ ID NO.3) to co-catalyze the synthesis of agarotriose.

10. The method according to claim 9, characterized in that, The substrate agarose concentration was 7.5-15%; The catalytic conditions are as follows: after incubation at 35℃ and 200~300 rpm for 10 min, Sa-AGA and Ca-AGA are added for enzymatic hydrolysis for 8~32 h.

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