A method for synthesizing galactosylglycerol using beta-galactosidase OUC Sb 4836

Abstract

The application discloses a method for synthesizing galactosyl glycerol by using beta-galactosidase OUC Sb 4836, and the method comprises the following steps: taking glycerol and lactose as substrates, and under the action of beta-galactosidase OUC Sb 4836, galactosyl glycerol is obtained by reaction; the amino acid sequence of the beta-galactosidase OUC Sb 4836 is shown in SEQ ID NO. 1. The application further discloses application of the beta-galactosidase OUC Sb 4836 in synthesis of galactosyl glycerol. The application realizes, for the first time, heterologous expression of beta-galactosidase OUC-Sb-4836, research on enzymatic properties and verification of transglycosylation activity; the enzyme not only has good beta-galactosidase bond hydrolysis capacity, but also has obvious transglycosylation activity, and the conversion rate of galactosyl glycerol can reach 72.38%. The research of the application provides a new feasible way for green biological catalytic preparation of galactosyl glycerol and related glycolipid compounds.

Description

Technical Field

[0001] This invention relates to a method for synthesizing galactosylglycerol using β-galactosidase OUC Sb 4836, belonging to the field of galactosylglycerol synthesis technology. Background Technology

[0002] Galactosylglycerol is a glycoside with the following structural formula: .

[0003] Galactosylglycerol has advantages such as high temperature and high pressure resistance, no hemiacetal hydroxyl group, non-reducing properties, and high stability. It is a key precursor in the biosynthesis and chemical synthesis of galactosylglycerol esters and is the basis for the construction of functional lipids and bioactive molecules. It has broad application prospects in cosmetics, biomedicine, health food and other fields.

[0004] Natural extraction and chemical synthesis of glycerol glycolipids are cumbersome and yield low amounts. Enzymatic synthesis offers advantages such as mild conditions and high selectivity. Therefore, the enzymatic synthesis of the intermediate galactosylglycerol is key to the efficient synthesis of galactosylglycerol esters. Enzymatic synthesis methods for galactosylglycerol mainly include two reaction types: reverse hydrolysis and transglycosylation. Existing research focuses primarily on the reverse hydrolysis pathway. However, reverse hydrolysis is thermodynamically controlled and easily subject to equilibrium limitations, often resulting in low overall conversion rates and difficulty in achieving complete substrate conversion. Transglycosylation, on the other hand, is kinetically controlled, effectively improving substrate utilization and enabling the targeted synthesis of functional glycosides.

[0005] β-Galactosidases are a class of glycosidic hydrolases that catalyze the hydrolysis of β-galactosidic bonds. They are widely found in microorganisms, plants, and animals, and have significant applications in lactose degradation, dairy processing, and the preparation of functional sugars. In addition to their traditional hydrolytic activity, some β-galactosidases also exhibit significant transglycosylation activity. These enzymes can use glycosyl donors as substrates to transfer glycosyl groups to the hydroxyl groups of acceptor molecules, thereby forming new glycosidic bonds and generating galacto-oligosaccharides, galactosyl alcohols, and other galactosyl derivatives. However, the transglycosylation activity of currently reported β-galactosidases is not high, limiting the industrial preparation of galactosylglycerol. Therefore, developing β-galactosidase catalytic systems with high transglycosylation efficiency, low hydrolytic side reactions, and good stability is of great significance for the large-scale synthesis of functional glycosides. Summary of the Invention

[0006] In view of the above-mentioned prior art, in order to efficiently synthesize galactosylglycerol, the present invention provides a method for synthesizing galactosylglycerol using β-galactosidase OUC Sb 4836.

[0007] This invention is achieved through the following technical solution: A method for synthesizing galactosylglycerol using β-galactosidase OUC Sb 4836 is as follows: using glycerol and lactose as substrates, galactosylglycerol is obtained by reaction under the action of β-galactosidase OUC Sb 4836; the amino acid sequence of said β-galactosidase OUC Sb 4836 is shown in SEQ ID NO.1.

[0008] Further, the specific method is as follows: Mix 50-500 μL of glycerol and 140 mg of lactose, add enzyme solution containing β-galactosidase OUC Sb 4836, and react at 37°C and 220 rpm for 3-36 hours.

[0009] Furthermore, the amount of enzyme solution added is 500 μL, and the enzyme activity of β-galactosidase OUC Sb 4836 in the enzyme solution is 5-15 U / mL.

[0010] Application of β-galactosidase OUC Sb 4836 in the synthesis of galactosylglycerol.

[0011] Furthermore, in specific applications, glycerol and lactose are used as substrates, and galactosylglycerol is obtained by reacting β-galactosidase OUC Sb 4836.

[0012] This invention marks the first successful heterologous expression, enzymatic property study, and transglycosylation activity verification of β-galactosidase OUC-Sb-4836. The results show that this enzyme possesses not only excellent β-galactosidic bond hydrolysis ability but also significant transglycosylation activity. Enzyme activity was determined using the o-nitrophenol-β-D-galactoside (β-oNPG) method. The optimal reaction pH was found to be 8.0, the optimal reaction temperature to be 40℃, and the enzyme retained over 65% of its relative activity after 72 hours of storage at 4℃, indicating good low-temperature stability and promising industrial application potential.

[0013] The method for synthesizing galactosylglycerol according to the present invention achieves a galactosylglycerol conversion rate of 72.38% and a galactosylglycerol yield of 44.56 mg / mL under the conditions of 500 μL glycerol, 140 mg lactose, 500 μL enzyme (15 U / mL), and a reaction time of 37°C for 36 h. Compared with existing methods that mainly use reverse hydrolysis to synthesize galactosylglycerol with a conversion rate of only 55.88%, the present invention significantly improves substrate utilization and target product synthesis efficiency. This research contributes to the construction of a modular, scalable bio-manufacturing route for glycerol glycolipids with galactosylglycerol as the core intermediate, providing a new feasible approach for the green biocatalytic preparation of galactosylglycerol and related glycolipid compounds.

[0014] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0015] Figure 1 : Schematic diagram of SDS-PAGE electrophoresis results, where M is the standard protein marker, lane 1 is the crude enzyme solution, and lane 2 is the pure enzyme solution.

[0016] Figure 2 β-galactosidase OUC Sb Results of determination of relative enzyme activity of 4836 at different reaction temperatures.

[0017] Figure 3 β-galactosidase OUC Sb Results of determination of relative enzyme activity of 4836 at different reaction pH.

[0018] Figure 4 β-galactosidase OUC Sb The results of determining the relative enzyme activity of 4836 after incubation at different temperatures for different times.

[0019] Figure 5 Liquid phase detection results.

[0020] Figure 6 Results of the determination of the conversion rate of transglycosylation reaction at different amounts of glycerol added.

[0021] Figure 7 Results of the conversion rate of transglycosylation reaction under different enzyme dosages and reaction times. Detailed Implementation

[0022] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0023] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0024] Example 1 β-galactosidase OUC Sb Excavation of 4836 To identify β-galactosidases with high transglycosylation activity, the inventors screened genomic data from multiple Streptomyces strains, obtaining several putative β-galactosidases and measuring their transglycosylation activities. Ultimately, a β-galactosidase OUC with high transglycosylation activity was selected. Sb 4836, other hypothetical β-galactosidases showed poor transglycosylation activity.

[0025] β-galactosidase OUC Sb 4836 comes from *Streptomyces bacillus* ( Sb 4836, whose amino acid sequence is shown in SEQ ID NO.1, and whose nucleotide sequence encoding the gene is shown in SEQ ID NO.2. Streptomyces bacillaris (Purchased from Shandong Provincial Engineering Technology Research Center for Marine Microbial Strains Preservation and Application), whole-genome sequencing and amino acid sequence prediction analysis were performed on this strain, revealing a putative β-galactosidase fragment sb4836, named β-galactosidase OUC. Example 2 β-galactosidase OUC Sb Heterologous expression of 4836 (1) Construction of recombinant plasmids The gene fragment shown in SEQ ID NO.2 was artificially synthesized, amplified using conventional methods, and the amplified product was combined with pET using seamless cloning technology. 28a(+) vector ligation; the ligation product was then transformed into Escherichia coli (E. coli). E. coli DH5α competent cells were plated on LB agar plates containing 50 μg / mL kanamycin sulfate and cultured at 37°C for 16 hours. Single clones were picked and cultured in LB liquid medium containing 50 μg / mL kanamycin sulfate at 37°C and 220 rpm for 12 hours. Positive clones were verified by sending clones with the correct band size to a sequencing company for sequencing. After successful sequence alignment, the successfully verified recombinant plasmid was extracted and named pET28a-sb4836.

[0026] (2) Construction of recombinant engineered bacteria The extracted plasmid was transformed into Escherichia coli BL21 ΔlacZ competent cells (i.e., Escherichia coli Bl21 with the LacZ gene knocked out), plated on LB medium solid plates containing 50 μg / mL kanamycin sulfate, and single colonies were picked to obtain the recombinant expression strain.

[0027] (3) Cultivation of recombinant engineered bacteria The selected recombinant expression strains were activated in 5 mL LB liquid medium (containing 50 μg / mL kanamycin sulfate), and then inoculated at a 1% inoculum into LB medium containing 50 μg / mL kanamycin sulfate. The cultures were incubated at 37°C and 200 rpm for 4 h using a shaker. The OD of the bacterial culture was measured. 600 When the value was 0.6, isopropyl-β-d-thiogalactoside (IPTG) was added to a concentration of 100 mM, and the expression of β-galactosidase OUC was induced at 20°C for 16 h. Sb 4836.

[0028] (3) β-galactosidase OUC Sb Extraction of 4836 After the above fermentation is completed, the cells are collected by centrifugation, resuspended in ultrapure water, and then sonicated in an ice-water bath for 15 minutes (300 W, sonication on for 3 seconds, off for 3 seconds). The supernatant is collected as crude enzyme solution.

[0029] (4) β-galactosidase OUC Sb Purification of 4836 Based on His-tag fusion proteins, the crude enzyme solution was purified by affinity chromatography using a Ni-NTA column. The column was equilibrated with 10 mM imidazole solution (10 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl), followed by elution of weakly binding contaminating proteins with 20 mM imidazole solution (20 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl), and then eluted with 120 mM imidazole solution (120 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl). The eluent was collected and placed in a 10 kDa ultrafiltration tube for ultrafiltration to remove residual imidazole. The specific ultrafiltration conditions were: centrifugation at 4000 r / min for 20 min at 4°C, followed by rinsing with pure water and centrifugation at 4000 r / min for 40 min to obtain the pure enzyme solution.

[0030] SDS-PAGE analysis of crude enzyme solution and pure enzyme solution; schematic diagram of SDS-PAGE electrophoresis results is shown below. Figure 1 As shown, a pure protein with a molecular weight of approximately 50 kDa was obtained, along with β-galactosidase OUC. Sb 4836 is equivalent.

[0031] Example 3 β-galactosidase OUC Sb Assay of 4836 enzyme activity β-galactosidase activity was rapidly quantified using the β-oNPG hydrolysis method: 50 μL of crude enzyme solution was added to 100 μL of ultrapure water and incubated at 35℃ for 2 min. Then, 50 μL of 10 mM β-oNPG was quickly added, and the reaction was continued at 35℃ for 1 min. Finally, 200 μL of 0.2 M Na2CO3 was added to terminate the reaction, and the absorbance was measured at 410 nm using a microplate reader.

[0032] Enzyme activity is defined as the amount of enzyme that produces 1 μmol of o-nitrophenol within 1 minute under standard conditions.

[0033] Result: The enzyme activity of the crude enzyme solution was 15.6 U / mL.

[0034] Example 4 β-galactosidase OUC Sb Determination of optimal reaction conditions for 4836 (1) Determination of the optimal reaction temperature The β-galactosidase OUC was measured according to the assay method in Example 3. Sb The enzyme activity of 4836 pure enzyme solution at different reaction temperatures (20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 55℃) was determined to identify the optimal reaction temperature.

[0035] Calculate the relative enzyme activities at different reaction temperatures, taking the highest enzyme activity as 100%. (β-galactosidase OUC) Sb The results of the determination of the relative enzyme activity of 4836 at different reaction temperatures are as follows: Figure 2 As shown, within the temperature range of 20–55℃, the relative enzyme activity first increases and then decreases with temperature. β-galactosidase OUC Sb The optimal reaction temperature for 4836 is 40℃, and its relative enzyme activity can be maintained above 55% within the range of 25~45℃, indicating that β-galactosidase OUC Sb 4836 not only has high activity at low temperatures, but also a wide temperature activity range, which can adapt to temperature fluctuations that may occur in industrial production.

[0036] (2) Determination of the optimal reaction pH The purified enzyme solution was placed in buffer solutions with pH values ​​ranging from 2.0 to 10.0 (the buffer solutions used included: disodium hydrogen phosphate-citric acid buffer solution with pH values ​​ranging from 2.0 to 6.0, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH values ​​ranging from 6.0 to 8.0, and glycine-sodium hydroxide buffer solution with pH values ​​ranging from 8.0 to 10.0), and the β-galactosidase OUC was measured according to the assay method described in Example 3. Sb The enzyme activity of 4836 at 40°C was determined to identify the optimal reaction pH.

[0037] Calculate the relative enzyme activities at different reaction pH values, with the highest enzyme activity defined as 100%. β-galactosidase OUC Sb The results of the determination of the relative enzyme activity of 4836 at different reaction pH are as follows: Figure 3 As shown, the relative enzyme activity reaches its highest level in glycine-sodium hydroxide buffer at pH 8.0, and exhibits good tolerance in the pH range of 6.0–8.0, maintaining a relative enzyme activity above 70%, indicating that β-galactosidase OUC Sb 4836 is a neutral β-galactosidase, which has good application potential under neutral to weakly alkaline conditions.

[0038] (3) Determination of temperature stability The pure enzyme solution was incubated at 4℃, 25℃ (room temperature), and 40℃ for 72 h, respectively, and samples were taken at different time points (0 h, 1 h, 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h). The enzyme activity of each sample was determined at 40℃ and pH 8.0 according to the determination method in Example 3.

[0039] The relative enzyme activities after incubation at different temperatures for different times were calculated, with the highest enzyme activity defined as 100%. β-galactosidase OUC Sb The results of the determination of the relative enzyme activity of 4836 after incubation at different temperatures for different times are as follows: Figure 4 As shown, after incubation at 4℃ for 72 h, more than 65% of the initial enzyme activity was retained, indicating that β-galactosidase OUC Sb 4836 exhibits good storage stability at 4℃. After storage at 25℃ for 72 h, the remaining enzyme activity is only 33.63%, and after storage at 40℃ for 72 h, it is almost completely inactivated.

[0040] Example 5: Utilization of β-galactosidase OUC Sb 4836-catalyzed transglycosylation reaction to synthesize galactosylglycerol Add 500 μL of glycerol to a reaction flask, along with 140 mg of lactose, and then add 500 μL of 5 U / mL enzyme solution (the crude enzyme solution was diluted to 5 U / mL with pure water). Seal the reaction system and incubate at 37℃ and 220 rpm for 24 h. Take a 50 μL sample, dilute it with 950 μL of deionized water, boil for 10 min to inactivate the enzyme, and remove insoluble impurities using a 0.22 μm syringe filter before using it for liquid chromatography analysis.

[0041] The liquid chromatography detection conditions were as follows: Shimadzu high performance liquid chromatograph, differential refractive index detector, SHODEXSUGAR KS801 column, column oven set to 75℃, pure water elution, flow rate of 0.8 mL / min, and analysis time of 12 min.

[0042] Liquid phase detection results as follows Figure 5 As shown, the peak elution times of the substrates glycerol and lactose were 10.466 min and 7.282 min, respectively, while the peak elution time of the product galactosylglycerol was 7.955 min, and the peak elution time of the byproduct glucose was 8.818 min. These results indicate that β-galactosidase OUC can be utilized... Sb 4836 catalyzes the transglycosylation reaction to synthesize galactosylglycerol.

[0043] Example 6 Optimization of transglycosylation reaction conditions (1) Optimization of glycerol addition amount Based on the reaction system of Example 5, the amount of glycerol added was adjusted to 50 μL, 100 μL, 200 μL, 300 μL, 400 μL, and 500 μL. The conversion rates of galactosylglycerol and byproducts (digalactosylglycerol and galactose) were calculated by liquid chromatography (using peak area normalization).

[0044] The results of the conversion rate of the transglycosylation reaction at different glycerol addition amounts are as follows: Figure 6 As shown, when the amount of glycerol added increases to 300 μL, the conversion rate of galactosylglycerol continuously increases due to the increasing substrate ratio; further increasing the amount of glycerol added to 500 μL leads to a decreasing trend in conversion rate. Although the conversion rate of the target product is not highest under the 500 μL glycerol condition, the amount of byproducts (digalactosylglycerol and galactose) generated is significantly reduced, which is beneficial for subsequent product separation and purification. Therefore, considering both product conversion rate and byproduct control, a glycerol addition amount of 500 μL was selected for subsequent reaction optimization. Under this condition, the conversion rate of galactosylglycerol reached 52.15%.

[0045] (2) Optimization of enzyme dosage and reaction time Based on the reaction system of Example 5, the enzyme dosage was adjusted to 500 μL of enzyme solution at concentrations of 5 U / mL, 10 U / mL, and 15 U / mL (pure enzyme solution was diluted with pure water to 5 U / mL, 10 U / mL, and 15 U / mL, respectively), and the reaction time was adjusted to 3 h, 6 h, 12 h, 24 h, and 36 h. The conversion rates of galactosylglycerol and byproducts (digalactosylglycerol and galactose) were calculated by liquid chromatography.

[0046] The results of the transglycosylation conversion rate determination under different enzyme dosages and reaction times are as follows: Figure 7 As shown in the figure. The results indicate that with the increase of enzyme dosage and reaction time, the galactosylglycerol conversion rate increased from 20.59% to 72.38%, indicating that β-galactosidase OUC Sb 4836 exhibits excellent transglycosylation ability and target product synthesis efficiency. However, as the reaction time increases, the amount of byproducts in the reaction system tends to increase. Therefore, considering both the conversion rate of the target product and the control of byproducts, 15 U / ml and 36 h were selected as the optimal enzyme dosage and reaction time, at which point the highest galactosylglycerol yield (72.38%) was obtained.

[0047] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A method for synthesizing galactosylglycerol using β-galactosidase OUC Sb 4836, characterized in that: Using glycerol and lactose as substrates, galactosylglycerol is obtained by reaction under the action of β-galactosidase OUC Sb 4836; the amino acid sequence of the β-galactosidase OUC Sb 4836 is shown in SEQ ID NO.

1.

2. The method for synthesizing galactosylglycerol using β-galactosidase OUC Sb 4836 according to claim 1, characterized in that: Mix 50–500 μL of glycerol and 140 mg of lactose, add an enzyme solution containing β-galactosidase OUC Sb 4836, and react at 37°C and 220 rpm for 3–36 hours.

3. The method for synthesizing galactosylglycerol using β-galactosidase OUC Sb 4836 according to claim 2, characterized in that: The amount of enzyme solution added was 500 μL, and the enzyme activity of β-galactosidase OUC Sb 4836 in the enzyme solution was 5-15 U / mL.

4. The method for synthesizing galactosylglycerol using β-galactosidase OUC Sb 4836 according to claim 3, characterized in that: The enzyme activity of β-galactosidase OUC Sb 4836 in the enzyme solution was 15 U / mL.

5. The method for synthesizing galactosylglycerol using β-galactosidase OUC Sb 4836 according to claim 2, characterized in that: The amount of glycerol added is 500 μL.

6. The method for synthesizing galactosylglycerol using β-galactosidase OUC Sb 4836 according to claim 2, characterized in that: The reaction time is 36 hours.

7. Application of β-galactosidase OUC Sb 4836 in the synthesis of galactosylglycerol, wherein the amino acid sequence of β-galactosidase OUC Sb 4836 is shown in SEQ ID NO.

1.

8. The application according to claim 7, characterized in that: In practical applications, glycerol and lactose are used as substrates, and galactosylglycerol is obtained by reacting β-galactosidase OUC Sb 4836.

Images