Improved gold-silicon oligosaccharin preparation process based on biotechnology

Abstract

The invention relates to the technical field of biology, and discloses a biotechnology-based gold-silicon oligosaccharin improved preparation process which comprises the following steps: S1, gold-silicon oligosaccharin is generated by catalyzing a gold-silicon raw material through a biological enzyme subjected to directed evolution and modification, and the biological enzyme has a specific affinity tag and is used for subsequent separation and purification; s2, carrying out preliminary separation on a reaction solution subjected to enzyme catalysis by adopting an ultrafiltration membrane system, intercepting a biological enzyme and an unreacted substrate, and enabling the biological enzyme and the unreacted substrate to penetrate through gold-silicon oligosaccharin; and S3, further purifying the penetrating gold-silicon oligosaccharin through an affinity chromatography technology, wherein the chromatography filler is a degradable natural polymer material with an affinity ligand. According to the present invention, by using the directionally evolved bio-enzyme as the catalyst, the yield and the purity of the gold-silicon oligosaccharin are significantly improved, and compared with the traditional chemical synthesis method, the bio-enzyme catalysis reaction can be efficiently performed under the mild condition, the generation of the by-product is reduced, and the purity of the final product achieves more than 95%, which is far higher than the purity of the traditional method.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an improved preparation process of auriosilicon oligosaccharides based on biotechnology. Background Art

[0002] Gold silico-oligosaccharide is a functional oligosaccharide with multiple biological activities. It has been widely studied and applied in the fields of food, health care products, medicine, etc. Its main advantages include enhancing immunity, anti-oxidation, promoting mineral absorption, etc. However, the existing gold silico-oligosaccharide preparation process mainly relies on chemical synthesis methods, and these traditional methods have many significant shortcomings.

[0003] First, traditional chemical synthesis methods usually rely on high temperature, high pressure and strong acid or strong base environment, which not only requires harsh reaction conditions, but also often produces a large number of by-products. These by-products not only reduce the purity of the target product, but also need to be removed through complex separation and purification processes, resulting in increased production costs. In addition, these conditions may also have an adverse effect on the molecular structure and biological activity of gold silico-oligosaccharides, thereby reducing the functionality of the final product.

[0004] Secondly, the use of chemical catalysts often brings environmental problems. Many chemical catalysts will produce harmful waste gas and waste liquid during the reaction process, causing environmental pollution. In addition, the residue of chemical catalysts may also affect the safety of the final product, especially in the field of food and medicine, where catalyst residues may bring potential health risks.

[0005] In addition, traditional methods usually require a long reaction time to achieve a high conversion rate, and the reaction efficiency is low. In order to increase the yield, a large amount of reactants and catalysts are often required, which further increases production costs and resource consumption.

[0006] In order to overcome these shortcomings, biotechnology has gradually become a research hotspot in the field of auriosilicon oligosaccharide preparation. Enzyme-catalyzed reactions have the advantages of high efficiency, strong specificity, mild reaction conditions, and the enzyme-catalyzed process is outstanding in terms of environmental friendliness. However, the catalytic efficiency and stability of natural enzymes may not be sufficient to meet the needs of industrial production, which requires the optimization of enzymes through methods such as directed evolution to improve their industrial application value. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides an improved preparation process of auriosilicon oligosaccharides based on biotechnology, which solves the problems of low yield, low purity, high environmental pollution and harsh reaction conditions caused by the chemical synthesis method in the prior art.

[0008] To achieve the above objectives, the present invention is implemented by the following technical scheme: an improved preparation process of auriosilicon oligosaccharides based on biotechnology, comprising the following steps:

[0009] Step 1: catalyzing the gold-silicon raw material through directed evolution and modified biological enzymes to generate gold-silicon oligosaccharides, wherein the biological enzyme has a specific affinity tag for subsequent separation and purification;

[0010] Step 2: Use an ultrafiltration membrane system to perform preliminary separation on the reaction solution after enzyme catalysis, intercept the biological enzyme and unreacted substrate, and pass through the gold silica oligosaccharide;

[0011] Step 3: further purifying the permeated aurosilicic acid oligosaccharide by affinity chromatography, wherein the chromatography filler is a degradable natural polymer material with affinity ligands;

[0012] Step 4: Use supercritical fluid drying technology to dry the purified auriosilicate oligosaccharide solution to obtain auriosilicate oligosaccharide powder.

[0013] Preferably, the biological enzyme is a glycosyltransferase that has undergone directed evolution, and the directed evolution process comprises the following steps:

[0014] S101, mutating the glycosyltransferase gene by PCR random mutagenesis, saturation mutagenesis and site-directed mutagenesis to construct a mutant library containing at least 10^6 mutants;

[0015] S102, expressing each mutant in the mutant library in an expression system, and screening out the enzyme with the highest catalytic activity under the conditions of 30° C. to 35° C. and pH 6.8 to 7.2 by high-throughput screening technology;

[0016] S103. Further molecular modification is performed on the selected high-quality enzyme mutants, and an affinity tag is introduced at the N-terminus or C-terminus of the enzyme.

[0017] Preferably, the affinity tag is a His tag or a GST tag, and the tag is used to specifically bind to the ligand on the chromatography filler in the affinity chromatography step.

[0018] Preferably, the affinity tag is a His tag or a GST tag, and the tag is used to specifically bind to the ligand on the chromatography filler in the affinity chromatography step.

[0019] Preferably, the operating conditions of the ultrafiltration membrane system include:

[0020] S201, using an ultrafiltration membrane with a pore size of 10 kDa to separate at a temperature of 20 to 25° C., wherein the temperature range is conducive to maintaining the activity of the enzyme;

[0021] S202, during the ultrafiltration process, the membrane flux is controlled at a pressure of 0.5 to 1.0 bar to ensure efficient permeation of the gold silica oligosaccharide while retaining high molecular weight enzymes and unreacted substrates;

[0022] S203, the ultrafiltered enzyme and unreacted substrate are returned to the reactor through a reflux system for further reaction.

[0023] Preferably, the affinity chromatography technique comprises the following steps:

[0024] S301, loading the aurosilicic acid oligosaccharide solution separated by ultrafiltration into an affinity chromatography column, wherein the filler of the chromatography column is chitosan or alginate with high affinity;

[0025] S302, by gradually increasing the pH value of the elution buffer from 4.0 to 7.0, the non-specific binding substances and aurosil oligosaccharides are eluted in sequence;

[0026] S303, the eluted aurosilicic acid oligosaccharide solution is further dialyzed to remove impurities.

[0027] Preferably, the supercritical fluid drying technology comprises the following steps:

[0028] S401, inject the purified gold silica oligosaccharide solution into a supercritical drying system, wherein the system uses supercritical CO 2 As a drying medium, the pressure is controlled between 7.4MPa and 8.5MPa, and the temperature is controlled between 31℃ and 35℃;

[0029] S402, in supercritical state, CO 2 Rapidly mix with water and evaporate, so that the water in the gold silicon oligosaccharide is removed;

[0030] S403, during the drying process, the possible impurity particles are removed by an electrostatic dust removal system to obtain high-purity gold silicon oligosaccharide powder with uniform particle size.

[0031] Preferably, the modification process of the glycosyltransferase includes introducing a His tag at its N-terminus, and performing preliminary purification by affinity chromatography, followed by elution with high concentration of imidazole to obtain a high-purity enzyme for subsequent synthesis of aurosilico-oligosaccharides.

[0032] Preferably, the raw material pretreatment step in step S1 includes the following:

[0033] (a) selecting a gold-silicon compound with a purity of not less than 99%, and treating it with ultrasound at a frequency of 40 kHz at a temperature of 25° C. to 30° C. for 20 to 30 minutes;

[0034] (b) dissolving the ultrasonically treated gold-silicon compound in 50 mM phosphate buffer, and maintaining the pH of the solution between 6.8 and 7.2 by a real-time pH monitoring system;

[0035] (c) The pretreated gold-silicon solution was stored at 4°C.

[0036] Preferably, the filler of the affinity chromatography column is degradable chitosan.

[0037] Preferably, during the supercritical fluid drying process, the CO in the system is gradually reduced. 2 The pressure is gradually reduced from 8.5MPa to atmospheric pressure while maintaining a constant temperature to ultimately obtain a uniform powdered product.

[0038] The present invention provides an improved preparation process of gold silicon oligosaccharide based on biotechnology, which has the following beneficial effects:

[0039] 1. The present invention uses directed evolution bioenzymes as catalysts, which significantly improves the yield and purity of auriosilicon oligosaccharides. Compared with traditional chemical synthesis methods, bioenzyme catalysis reactions can be carried out efficiently under mild conditions, reducing the generation of by-products, and the purity of the final product reaches more than 95%, which is much higher than traditional methods.

[0040] 2. The present invention avoids the destruction of the structure of auriosilicon oligosaccharides by high temperature and strong acidic conditions because the biological enzyme works at a relatively low temperature and mild conditions, thus maintaining the molecular integrity and functionality of the product. The auriosilicon oligosaccharides prepared by the present invention have higher biological activity, including enhancing immunity and antioxidant capacity, which makes the product have higher application value in the fields of health care products and medicine.

[0041] 3. By optimizing the molar ratio of enzyme to substrate and reaction conditions, the process of the present invention can achieve a higher substrate conversion rate in a shorter time. Compared with the traditional chemical synthesis method, the present invention has significant advantages in reaction efficiency and time.

[0042] 4. The present invention adopts green separation and purification technology, avoids the use of organic solvents and strong acids and bases, and reduces environmental pollution. The application of ultrafiltration membrane, affinity chromatography and supercritical fluid drying technology not only improves the separation and purification efficiency, but also significantly reduces the generation of wastewater and waste gas, meets environmental protection requirements, and reduces environmental burden.

[0043] 5. The present invention reduces the amount and cost of catalysts through directed evolution and recycling of enzymes. Compared with traditional chemical catalysts, the use of biological enzymes is not only cheaper, but also can maintain activity in multiple reaction cycles, greatly reducing the overall production cost and improving the economic efficiency of the process.

[0044] In summary, the present invention has the advantages of improving the yield and purity of aurosilicooligosaccharides, enhancing biological activity, shortening reaction time, reducing environmental pollution and production costs, and adapting to the needs of industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1It is a flow chart of the present invention. DETAILED DESCRIPTION

[0046] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] Embodiment 1:

[0048] Please refer to the attached Figure 1 The embodiment of the present invention provides an improved preparation process of auriosilicon oligosaccharides based on biotechnology, comprising the following steps:

[0049] 1. Directed evolution of enzymes

[0050] 1.1 Selection and directed evolution of enzymes

[0051] In this embodiment, the enzyme selection steps are as follows:

[0052] Glycosyltransferase was chosen as the target enzyme because it plays a key role in catalyzing the synthesis of auriosilicon oligosaccharides. The initial form of glycosyltransferase showed certain catalytic activity under mild conditions, but in order to meet the needs of industrial production, it is necessary to improve its stability, catalytic efficiency and adaptability under specific temperature and pH conditions through directed evolution.

[0053] In this embodiment, the directed evolution process includes the following steps:

[0054] First, the mutant library of the glycosyltransferase gene was generated using PCR random mutagenesis. The PCR mutagenesis reaction was set up as follows: 1 μg template DNA, 10 μL 5× PCR buffer, 1 μL 10 mM dNTPs, 1 μL 10 μM forward primer, 1 μL 10 μM reverse primer, and 0.25 μL Taq DNA polymerase were contained in a 50 μL reaction system. The reaction system was denatured at 95°C for 2 minutes, and then amplified for 30 cycles at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute.

[0055] Secondly, the mutant genes were cloned into the pET-28a expression vector and transformed into BL21 (DE3) Escherichia coli host cells. Each mutant was induced to express by IPTG, and the expression conditions were: 30°C for 24 hours, and the final IPTG concentration was 1 mM.

[0056] Then, the catalytic activity of each mutant was detected by high-throughput screening technology. The specific operation was to carry out the reaction at 30°C and pH 7.0, with a reaction system of 50 μL, including 20 mM gold-silicon substrate and 5 μL of expressed mutant enzyme. The reaction product was detected by HPLC to screen out the enzyme mutant with the highest catalytic efficiency.

[0057] Finally, for the selected efficient mutants, a His tag was introduced at its N-terminus through molecular modification. The specific operation is to design a primer containing the His tag sequence, and connect the His tag to the N-terminus of the enzyme through PCR amplification.

[0058] 2. Expression and purification of enzymes

[0059] 2.1 Expression of biological enzymes

[0060] In this embodiment, the expression steps of the biological enzyme are as follows:

[0061] The modified glycosyltransferase gene was cloned into the pET-28a expression vector, and the recombinant vector was transformed into BL21 (DE3) Escherichia coli. After transformation, the monoclonal colony containing the resistance marker was selected and inoculated into LB medium containing 50 μg / mL kanamycin. The culture medium was shaken and cultured at 37°C until the OD600 value reached 0.6, and then IPTG (final concentration of 1 mM) was added to induce expression. The induction expression conditions were: temperature 30°C, culture time 24 hours. After the culture was completed, the bacteria were collected by centrifugation (4°C, 8000rpm, 15 minutes).

[0062] In this embodiment, the preliminary purification steps of the biological enzyme are as follows:

[0063] The collected bacteria were disrupted using an ultrasonic disruptor at 20 kHz for 30 minutes in an ice bath to maintain enzyme activity. The disrupted bacterial suspension was centrifuged (4°C, 12,000 rpm, 30 minutes) and the supernatant was collected as a crude enzyme solution.

[0064] 2.2 Enzyme purification

[0065] In this example, the affinity chromatography purification steps are as follows:

[0066] The crude enzyme solution was loaded onto a Ni-NTA affinity chromatography column pre-equilibrated with an equilibrium buffer (50 mM phosphate buffer, pH 7.0, containing 300 mM NaCl). The column was washed with 10 mM imidazole buffer to remove non-specific binding proteins. Then, the target enzyme was eluted with 50 mM imidazole buffer and the eluate was collected. The eluate was dialyzed using a dialysis bag (molecular weight cut-off 3 kDa) with a 50 mM phosphate buffer, pH 7.0, for 24 hours to remove imidazole and other impurities.

[0067] In this embodiment, the steps of concentrating the enzyme solution are as follows:

[0068] The dialyzed enzyme solution was concentrated using an Amicon ultrafiltration tube (molecular weight cutoff 10 kDa), and the concentrated enzyme solution was stored at 4°C for subsequent aurosilico-oligosaccharide synthesis reaction.

[0069] 3. Enzyme-catalyzed synthesis of aurisoligosaccharides

[0070] 3.1 Raw material pretreatment

[0071] In this embodiment, the steps of selecting and processing the gold silicon raw material are as follows:

[0072] Select a gold-silicon compound with a purity of not less than 99%, weigh an appropriate amount of gold-silicon raw material (usually 100g), place it in a glass beaker, and add 200mL of deionized water. Place the beaker in an ultrasonic processor, set the frequency to 40kHz, and the processing time to 20 minutes. The purpose of ultrasonic treatment is to increase the specific surface area of ​​the gold-silicon raw material, expose more active sites, and thus improve the efficiency of subsequent enzyme-catalyzed reactions.

[0073] In this embodiment, the steps of dissolving the gold silicon raw material and adjusting the pH are as follows:

[0074] The gold-silicon raw material after ultrasonic treatment was dissolved in 50 mM phosphate buffer (pH 6.8 to 7.2), and the total amount of the solution was 1 L. The pH of the solution was monitored and adjusted in real time using an automatic titrator to maintain it between 6.8 and 7.2. The dissolved gold-silicon solution was stored at 4°C to prevent premature reaction or degradation.

[0075] 3.2 Enzyme-catalyzed reactions

[0076] In this embodiment, the steps for establishing the enzyme catalytic reaction system are as follows:

[0077] Take 100 mL of the pretreated gold-silicon solution and add it to a 500 mL reactor. Add the modified glycosyltransferase at a molar ratio of 1:100. Keep the temperature in the reactor between 30°C and 35°C and control the temperature by heating in a water bath. Add 2 mM Ca2+ As a cofactor, it enhances the catalytic stability of the enzyme. After the reaction starts, the reactor is stirred at a rate of 200 rpm to ensure that the substrate is fully in contact with the enzyme.

[0078] In this embodiment, the steps of dynamic monitoring of reaction conditions are as follows:

[0079] During the reaction, 10 μL of the reaction solution was collected through the sampling port every 2 hours, and the amount of gold silico-oligosaccharide produced was detected by HPLC. The HPLC analysis conditions are as follows: the mobile phase is water-acetonitrile (30:70), the flow rate is 1.0 mL / min, and the detection wavelength is 254 nm. By monitoring the trend of oligosaccharide production, the reactor temperature and stirring rate are adjusted if necessary to ensure the optimal reaction efficiency. When HPLC shows that the oligosaccharide concentration reaches the maximum value and the substrate conversion rate is close to 100% (usually between 24 and 36 hours), the reaction is terminated.

[0080] 4. Integrated green separation and purification

[0081] 4.1 Preliminary separation

[0082] In this embodiment, the ultrafiltration membrane separation steps are as follows:

[0083] The liquid after the reaction is directly transferred to the ultrafiltration membrane separation device. The pore size of the ultrafiltration membrane used is 10kDa, the operating temperature is maintained at 20 to 25°C, and the membrane permeation pressure is controlled at 0.5 to 1.0 bar. Gold silica oligosaccharides can pass through the ultrafiltration membrane due to their smaller molecular weight, while high molecular weight enzymes and unreacted substrates are retained. The permeated oligosaccharide solution is collected for further purification, and the retained enzymes and substrates are returned to the reactor through the reflux system for the next round of reaction.

[0084] 4.2 Affinity chromatography purification

[0085] In this example, the affinity chromatography purification steps are as follows:

[0086] The ultrafiltered auriosilicon oligosaccharide solution is injected into the affinity chromatography column through a loading pump. The filler of the chromatography column is chitosan or alginate that has been cross-linked. The high affinity of the filler ensures the effective capture of auriosilicon oligosaccharide. The chromatography column is initially washed with a low pH (about 4.0) buffer to remove non-specific binding substances. Subsequently, the pH value of the eluent is gradually increased (from 4.0 to 7.0), and the salt concentration is increased at the same time, so that the auriosilicon oligosaccharide is effectively eluted at high pH and high salt concentration. The eluent is collected and further removed by dialysis to remove salt and other impurities.

[0087] 4.3 Supercritical fluid drying

[0088] In this embodiment, the supercritical fluid drying steps are as follows:

[0089] The purified gold silica oligosaccharide solution is injected into the supercritical fluid drying system. The system uses supercritical CO 2 As the drying medium, the operating conditions of the drying process are: pressure controlled at 7.4 to 8.5 MPa, temperature controlled at 31°C to 35°C. Supercritical CO 2 When the pressure is reduced, the water in the solution can be evaporated quickly, leaving dry gold silicic acid oligosaccharide powder. In order to ensure the uniformity of the product, the impurity particles are removed by the electrostatic dust removal system during the drying process, and finally the gold silicic acid oligosaccharide powder with uniform particle size and high purity is obtained.

[0090] 5. Product quality control and packaging

[0091] 5.1 Product Testing

[0092] In this embodiment, the product detection steps are as follows:

[0093] The dried auriosilicon oligosaccharide powder was tested for purity by high performance liquid chromatography (HPLC). The HPLC conditions were the same as above to ensure that the product purity was not less than 95%. In addition, the molecular structure of auriosilicon oligosaccharide was verified by mass spectrometry (MS) and nuclear magnetic resonance (NMR) to confirm its structural integrity and biological activity.

[0094] 5.2 Biological activity test

[0095] In this embodiment, the biological activity test steps are as follows:

[0096] A certain amount of gold silico-oligosaccharide powder was taken to test its biological activity in an in vitro cell experiment. The experiment used the MTT method to detect the cell proliferation rate and the changes in the level of ROS (reactive oxygen) in the cells. The results showed that gold silico-oligosaccharide had significant immune enhancement and antioxidant capabilities, which met the expected functions.

[0097] 5.3 Product packaging and storage

[0098] In this embodiment, the product packaging steps are as follows:

[0099] The gold silicon oligosaccharide powder that has passed the quality inspection is packed into composite aluminum foil bags and vacuum packed to prevent the powder from absorbing moisture and deteriorating. The finished product should be stored in a cool and dry place after packaging, with the temperature controlled below 20℃ and the relative humidity not exceeding 50% to ensure its long-term stability.

[0100] 6. Beneficial effects

[0101] In this embodiment, through the above detailed implementation steps, the beneficial effects of the present invention are as follows:

[0102] Improve production efficiency and product quality: Through directed evolution of biological enzymes and optimized enzyme catalytic reaction conditions, the yield of auriosilicon oligosaccharides is significantly improved, the product purity exceeds 95%, while maintaining its high biological activity.

[0103] Reduce production costs and environmental pollution: Through the recycling of enzymes and green separation and purification technology, the production costs are greatly reduced, while avoiding the use of organic solvents and reducing environmental pollution.

[0104] Adapt to industrial needs: The process design is suitable for large-scale production, especially the manufacture of high value-added functional food raw materials, health care product ingredients and pharmaceutical excipients, and has broad application prospects.

[0105] Embodiment 2:

[0106] Example 2 is an improvement on Example 1, aiming to verify the effect of different reaction temperatures on the yield and purity of auriosilicon oligosaccharides. Compared with Example 1, the only variable in this example is that the reaction temperature is set at 25°C to observe the changes in enzyme catalytic efficiency and final product quality under lower temperature conditions.

[0107] The improved preparation process of aurosilicic acid oligosaccharides based on biotechnology disclosed in this embodiment comprises the following steps:

[0108] 1. Directed evolution of enzymes

[0109] In this embodiment, the selection and directed evolution process of the biological enzyme are the same as in Example 1. A highly efficient glycosyltransferase is obtained by PCR random mutagenesis, screening and molecular modification, and the biological enzyme is expressed in BL21 (DE3) Escherichia coli and purified to finally obtain a high-purity glycosyltransferase for subsequent reactions.

[0110] 2. Enzyme-catalyzed synthesis of auris oligosaccharides

[0111] 2.1 Raw material pretreatment

[0112] In this embodiment, the selection and treatment process of the gold silicon raw material are the same as those in Example 1. The specific surface area of ​​the gold silicon raw material is increased by ultrasonic treatment, and it is dissolved in 50 mM phosphate buffer, maintaining the pH between 6.8 and 7.2 to ensure the appropriate reaction activity of the raw material.

[0113] 2.2 Enzyme-catalyzed reactions

[0114] In this example, the enzyme catalytic reaction system was established in the same manner as in Example 1, but the only variable was that the reaction temperature was set at 25°C:

[0115] 100 mL of the pretreated gold-silicon solution and the directed-evolved glycosyltransferase (enzyme to substrate molar ratio 1:100) were added to the reactor, and the reaction temperature was set to 25°C. Other reaction conditions remained unchanged, such as the addition of 2 mM Ca 2+ As a cofactor, the stirring rate was 200 rpm.

[0116] In this example, the dynamic monitoring process of the reaction conditions is the same as that in Example 1. The amount of auriosilicate oligosaccharides produced in the reaction solution is detected every 2 hours by online HPLC, and the effects of temperature, stirring rate and reaction time on the product yield are monitored.

[0117] 3. Integrated green separation and purification

[0118] In this embodiment, the separation and purification process is the same as in Example 1:

[0119] The reaction solution was initially separated using a 10kDa ultrafiltration membrane to separate the oligosaccharides from the enzyme and unreacted substrate. Afterwards, the oligosaccharides were further purified using an affinity chromatography column, and finally the purified oligosaccharide solution was converted into a powdered product using supercritical fluid drying technology.

[0120] 4. Product quality control and packaging

[0121] In this embodiment, the product quality control and packaging process are the same as in the first embodiment:

[0122] The product purity is tested by HPLC, and the integrity of the oligosaccharide structure is verified by mass spectrometry (MS) and nuclear magnetic resonance (NMR). Finally, the purified product is tested for biological activity and packaged and stored in high-barrier packaging.

[0123] In this example, the reaction temperature was set at 25°C. Although auriosilicate oligosaccharides were still successfully produced, there were the following differences compared with the effect of Example 1:

[0124] Yield reduction: Due to the low reaction temperature, the catalytic efficiency of glycosyltransferase was reduced, resulting in a 15% reduction in the yield of auriosilicon oligosaccharide compared with Example 1. HPLC detection showed that the amount of the final product was lower than that of Example 1.

[0125] Slightly lower purity: Due to the slow reaction rate under low temperature conditions, incompletely converted substrates and by-products remain in the final product, resulting in a slight decrease in the purity of auriosilicon oligosaccharides compared to Example 1 (about 3% lower in purity).

[0126] Difference in biological activity: The results of the biological activity test showed that due to the low reaction temperature, some biological enzymes may be insufficiently active at low temperatures, and the immune enhancement and antioxidant capacity of the final product are slightly lower than those in Example 1.

[0127] Example 2 is an improvement on Example 1, aiming to verify the effect of different reaction temperatures on the yield and purity of auriosilicon oligosaccharides. Compared with Example 1, the only variable in this example is that the reaction temperature is set at 25°C to observe the changes in enzyme catalytic efficiency and final product quality under lower temperature conditions.

[0128] In summary, although Example 2 can still produce aurosilicooligosaccharides, the reaction efficiency and product quality at 25°C are lower than those of Example 1. Therefore, the process conditions described in Example 1 are more optimal, can achieve higher yield and purity, and have more significant biological activity.

[0129] Embodiment three:

[0130] Example 3 is improved on the basis of Example 1, aiming to verify the effect of different enzyme to substrate molar ratios on the yield and purity of auriosilicon oligosaccharides. Compared with Example 1, the only variable in this example is that the enzyme to substrate molar ratio is set to 1:200, in order to observe the catalytic efficiency and final product quality under the condition of lower enzyme dosage.

[0131] 1. Directed evolution of enzymes

[0132] In this embodiment, the selection and directed evolution process of the enzyme are the same as those in the first embodiment, that is, a highly efficient glycosyltransferase is obtained by PCR random mutation, screening and molecular modification. The enzyme is expressed in BL21 (DE3) Escherichia coli and purified to obtain a high-purity glycosyltransferase for subsequent reactions.

[0133] 2. Enzyme-catalyzed synthesis of auris oligosaccharides

[0134] 2.1 Raw material pretreatment

[0135] In this embodiment, the selection and treatment process of the gold silicon raw material are the same as those in Example 1. The specific surface area of ​​the gold silicon raw material is increased by ultrasonic treatment, and it is dissolved in 50 mM phosphate buffer, maintaining the pH between 6.8 and 7.2 to ensure that the raw material has good reaction activity.

[0136] 2.2 Enzyme-catalyzed reactions

[0137] In this example, the establishment of the enzyme catalytic reaction system is basically the same as in Example 1, but the only variable is that the molar ratio of enzyme to substrate is set to 1:200:

[0138] 100 mL of pretreated gold-silicon solution and directed-evolved glycosyltransferase were added to the reactor, and the molar ratio of enzyme to substrate was set to 1:200. The temperature in the reactor was maintained between 30°C and 35°C, and 2 mM Ca was added to the reaction system. 2+As a cofactor, the stirring rate was 200 rpm to ensure sufficient contact between the substrate and the enzyme.

[0139] In this embodiment, the dynamic monitoring process of the reaction conditions is the same as that in the first embodiment, and the amount of aurosilicic acid oligosaccharide generated in the reaction solution is detected every 2 hours by online HPLC to monitor the progress of the reaction in real time. If necessary, the reaction conditions are adjusted according to the HPLC test results to ensure the highest possible reaction efficiency.

[0140] 3. Integrated green separation and purification

[0141] In this embodiment, the separation and purification process is the same as in Example 1:

[0142] After the reaction, the reaction solution was initially separated using a 10kDa ultrafiltration membrane to retain high molecular weight enzymes and unreacted substrates and pass through the aurosilicon oligosaccharides. The permeated liquid was further purified by affinity chromatography, and finally the purified aurosilicon oligosaccharides solution was converted into a powdered product by supercritical fluid drying technology.

[0143] 4. Product quality control and packaging

[0144] In this embodiment, the product quality control and packaging process are the same as in the first embodiment:

[0145] The purity of the product is tested by HPLC, and the molecular structure of gold silicoligosaccharide is verified by mass spectrometry (MS) and nuclear magnetic resonance (NMR). Products that pass the quality inspection are tested for biological activity and finally vacuum packaged and stored to ensure the stability and safety of the product.

[0146] In summary, although Example 3 can still successfully produce aurosilic oligosaccharides, under the condition that the enzyme to substrate molar ratio is set to 1:200, the reaction efficiency, yield and product quality are inferior to those of Example 1. Therefore, the process conditions of Example 1 achieve better reaction efficiency and product quality at a relatively high enzyme dosage, which is suitable for industrial production.

[0147] Table 1 Comparison table of experimental data of embodiment

[0148]

[0149] Example 1: Under the conditions of 30-35°C and an enzyme to substrate molar ratio of 1:100, the yield, purity and biological activity of auriosilicon oligosaccharides all reached the optimal state, the reaction time was relatively short, the by-product content was the lowest, and the overall performance was excellent.

[0150] Example 2: The reaction temperature was lowered to 25°C. Although auriosilicate oligosaccharides were still produced, the yield, purity and biological activity were lower than those in Example 1. In addition, the reaction time was prolonged, the content of by-products increased, and the reaction efficiency decreased.

[0151] Example 3: While maintaining the temperature at 30-35°C, the enzyme-to-substrate molar ratio was reduced to 1:200. Although the amount of enzyme used was reduced, the reaction time was prolonged, the yield and purity decreased, the biological activity was also affected to a certain extent, the by-product content increased, and the reaction efficiency was the lowest.

[0152] Comparative Example 1:

[0153] This comparative example discloses a traditional improved preparation process of auriosilicon oligosaccharides, comprising the following steps:

[0154] Chemical synthesis process

[0155] 1. Raw material selection

[0156] In this comparative example, the raw materials are selected as follows:

[0157] The same gold-silicon compound is selected as the reaction substrate, and the purity is not less than 99%. During the chemical synthesis process, the gold-silicon raw material does not need to be ultrasonically treated and is directly dissolved in the reaction solution at high temperature.

[0158] 1.2 Reaction conditions

[0159] In this comparative example, the chemical catalytic synthesis reaction steps are as follows:

[0160] 100 g of gold silicon raw material was added to a 500 mL reactor and concentrated sulfuric acid (H 2 SO 4 ) as a catalyst, the amount of the catalyst is 10% of the substrate mass. The temperature in the reactor is heated by an electric heating mantle and set to 120°C. The entire reaction system is stirred and reacted at high temperature for 4 hours. After the reaction is completed, the crude product of gold silicon oligosaccharide is obtained by cooling, filtering and neutralization.

[0161] 2. Separation and purification

[0162] In this comparative example, the conventional separation and purification steps are as follows:

[0163] The crude product after the reaction is dissolved in a large amount of water, and an organic solvent (such as ether) is added for liquid-liquid extraction to separate the auriosilicon oligosaccharide from the by-products. The extraction process needs to be performed multiple times to improve the purity of the product. Subsequently, the solid product of auriosilicon oligosaccharide is obtained by evaporating the organic solvent. Finally, the product is further purified by recrystallization, using ethanol as a solvent for recrystallization.

[0164] 3. Product testing and packaging

[0165] In this comparative example, the product testing steps are as follows:

[0166] The purity of the final product was tested by HPLC, and the testing conditions were the same as those in Example 1. At the same time, the molecular structure of the gold silico-oligosaccharide was verified by mass spectrometry (MS) and nuclear magnetic resonance (NMR). Since a large amount of acid and organic solvent are used in the chemical synthesis process, there are many by-products, and neutralization treatment is required after the reaction, and acidic substances may remain.

[0167] Table 2 Comparison table of comparative experimental data

[0168] parameter Embodiment 1 Comparative Example 1 Reaction temperature (℃) 30-35 120 Catalyst dosage The molar ratio of enzyme to substrate was 1:100 10% of substrate mass Response time (hours) 24-36 4 Yield of auris oligosaccharides (g / L) 95 60 Purity of Gold Silico-oligosaccharide (%) 95 80 By-product content (%) 2 15 Biological activity (relative value) 100 60 HPLC test results (peak area) 8500 5000 Reaction efficiency (%) 90 50 Substrate conversion rate (%) 95 50

[0169] Yield and purity: The chemical synthesis method of the comparative example is carried out under high temperature conditions. Although the reaction time is short, due to the use of a more aggressive chemical catalyst, more reaction by-products are produced, resulting in the purity and yield of auriosilicon oligosaccharides being significantly lower than those in Example 1.

[0170] Biological activity: Due to the high temperature and harsh chemical conditions, the biological activity of the final product is significantly lower than that of the gold silicon oligosaccharide prepared by the bio-enzyme catalysis method, especially in terms of immune enhancement and anti-oxidation, the effect is not as good as that of Example 1.

[0171] In summary, the preparation process of gold silico-oligosaccharides based on bio-enzyme catalysis in Example 1 of the present invention has obvious advantages over traditional chemical synthesis methods. It can obtain products with higher purity and higher biological activity under milder conditions, while greatly reducing environmental pollution. It is a more superior industrial production method.

[0172] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An improved preparation process of gold silicon oligosaccharide based on biotechnology, characterized in that: The following steps are involved: S1. The gold-silicon raw material is catalyzed by directed evolution and modified biological enzymes to generate gold-silicon oligosaccharides, wherein the biological enzymes have specific affinity tags for subsequent separation and purification; S2, using an ultrafiltration membrane system to perform preliminary separation on the reaction solution after enzyme catalysis, intercepting the biological enzyme and unreacted substrate, and passing through the gold silica oligosaccharide; S3, further purifying the permeated aurosilico-oligosaccharide by affinity chromatography, wherein the chromatography filler is a degradable natural polymer material having an affinity ligand; S4. Use supercritical fluid drying technology to dry the purified auriosilicate oligosaccharide solution to obtain auriosilicate oligosaccharide powder.

2. The improved preparation process of gold silicon oligosaccharides based on biotechnology according to claim 1, characterized in that: The biological enzyme is a glycosyltransferase that has undergone directed evolution, and the directed evolution process comprises the following steps: S101, mutating the glycosyltransferase gene by PCR random mutagenesis, saturation mutagenesis and site-directed mutagenesis to construct a mutant library containing at least 10^6 mutants; S102, expressing each mutant in the mutant library in an expression system, and screening out the enzyme with the highest catalytic activity under the conditions of 30° C. to 35° C. and pH 6.8 to 7.2 by high-throughput screening technology; S103. Further molecular modification is performed on the selected high-quality enzyme mutants, and an affinity tag is introduced at the N-terminus or C-terminus of the enzyme.

3. The improved preparation process of gold silicon oligosaccharides based on biotechnology according to claim 1, characterized in that: The affinity tag is a His tag or a GST tag, and the tag is used to specifically bind to the ligand on the chromatography filler in the affinity chromatography step.

4. The improved preparation process of gold silicon oligosaccharides based on biotechnology according to claim 1, characterized in that: The operating conditions of the ultrafiltration membrane system include: S201, using an ultrafiltration membrane with a pore size of 10 kDa to separate at a temperature of 20 to 25° C., wherein the temperature range is conducive to maintaining the activity of the enzyme; S202, during the ultrafiltration process, the membrane flux is controlled at a pressure of 0.5 to 1.0 bar to ensure efficient permeation of the auriosilicon oligosaccharides while retaining high molecular weight enzymes and unreacted substrates; S203, the ultrafiltered enzyme and unreacted substrate are returned to the reactor through a reflux system for further reaction.

5. The improved preparation process of gold silicon oligosaccharides based on biotechnology according to claim 1, characterized in that: The affinity chromatography technique comprises the following steps: S301, loading the aurosilicic acid oligosaccharide solution separated by ultrafiltration into an affinity chromatography column, wherein the filler of the chromatography column is chitosan or alginate with high affinity; S302, by gradually increasing the pH value of the elution buffer from 4.0 to 7.0, the non-specific binding substances and aurosil oligosaccharides are eluted in sequence; S303, the eluted aurosilicic acid oligosaccharide solution is further dialyzed to remove impurities.

6. The improved preparation process of gold silico-oligosaccharides based on biotechnology according to claim 1, characterized in that: The supercritical fluid drying technology comprises the following steps: S401, injecting the purified aurosilicic acid oligosaccharide solution into a supercritical drying system, wherein the system uses supercritical CO2 as a drying medium, the pressure is controlled at 7.4 MPa to 8.5 MPa, and the temperature is controlled at 31° C. to 35° C.; S402, in a supercritical state, CO2 and water are quickly mixed and evaporated, so that the water in the gold silicon oligosaccharide is removed; S403, during the drying process, the possible impurity particles are removed by an electrostatic dust removal system to obtain high-purity gold silicon oligosaccharide powder with uniform particle size.

7. The improved preparation process of gold silico-oligosaccharides based on biotechnology according to claim 1, characterized in that: The modification process of glycosyltransferase includes the introduction of a His tag at its N-terminus and preliminary purification by affinity chromatography, followed by elution with high concentration of imidazole to obtain a high-purity enzyme for subsequent synthesis of aurosilico-oligosaccharides.

8. The improved preparation process of gold silico-oligosaccharides based on biotechnology according to claim 1, characterized in that: The pretreatment step of the raw material in step S1 includes the following contents: (a) selecting a gold-silicon compound with a purity of not less than 99%, and treating it with ultrasound at a frequency of 40 kHz at a temperature of 25° C. to 30° C. for 20 to 30 minutes; (b) dissolving the ultrasonically treated gold-silicon compound in 50 mM phosphate buffer, and maintaining the pH of the solution between 6.8 and 7.2 by a real-time pH monitoring system; (c) The pretreated gold-silicon solution was stored at 4°C.

9. The improved preparation process of gold silico-oligosaccharides based on biotechnology according to claim 1, characterized in that: The filler of the affinity chromatography column is degradable chitosan.

10. The improved preparation process of gold silico-oligosaccharides based on biotechnology according to claim 1, characterized in that: During the supercritical fluid drying process, the CO2 pressure in the system is gradually reduced from 8.5 MPa to atmospheric pressure while maintaining a constant temperature, ultimately obtaining a uniform powdered product.

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