Preparation of double-layer functionalized PS / GO nanofiber membrane and application of double-layer functionalized PS / GO nanofiber membrane in preparation of galactooligosaccharide
Yeast and β-galactosidase were immobilized in a bilayer PS/GO nanofiber membrane prepared by electrospinning, which solved the problems of enzyme stability and purification difficulty in the enzymatic synthesis of galactooligosaccharides and achieved efficient and stable GOS generation and high-purity products.
Patent Information
- Application Number
- CN202510846971.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-24
- Publication Date
- 2025-11-07
AI Technical Summary
In the existing technology, free β-galactosidase has poor stability, is difficult to reuse, has low transglycosylation activity, and is difficult to purify the product, resulting in low GOS yield and difficulty in achieving large-scale production.
A bilayer functionalized PS/GO nanofiber membrane was prepared using electrospinning technology. Yeast and β-galactosidase were immobilized by encapsulation and adsorption methods to construct a co-immobilized catalytic system, which improved the stability and recyclability of the enzyme and utilized yeast to remove monosaccharide impurities.
This method achieves efficient and stable GOS generation, significantly improves product purity and production efficiency, solves the problem of monosaccharide residue in traditional methods, and enhances the yield and purity of GOS.
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Abstract
Description
TECHNICAL FIELD
[0001] The application relates to the fields of material chemistry and food biotechnology, and discloses a preparation of a double-layer functionalized PS / GO nanofiber membrane and a co-immobilization method of yeast and beta-galactosidase, which can realize efficient and low-cost preparation of high-purity GOS. BACKGROUND
[0002] Galactooligosaccharide (GOS) is a functional oligosaccharide formed by one or more galactosyl groups and glucose or galactose through beta (1-3), beta (1-4) or beta (1-6) bond, which has the biological functions of promoting the growth of probiotics, regulating the balance of intestinal flora, the level of sugar and lipid metabolism, promoting the absorption of minerals and improving the immune function. Beta-galactosidase (EC) is a kind of glycosidase with industrial importance, which has the functions of hydrolysis and transglycosylation, can specifically catalyze the glycosidic bond between the terminal non-reducing beta-d-galactoside unit and the glycoside base to produce GOS. At present, the main methods for preparing GOS are enzyme method, acid hydrolysis method and fermentation method. The product yield of the acid hydrolysis method is low, and the obtained product composition is complex. The fermentation method is less studied, and the enzyme method is the most widely used, safest and most efficient method. However, the free beta-galactosidase enzyme method for catalyzing the synthesis of GOS is still limited in industrial production: (1) the dosage of free beta-galactosidase is large in practical application, the cost is high, and it is difficult to put into large-scale industrial production; (2) the stability of free beta-galactosidase is poor in practical application, and it is difficult to be reused and used; (3) improving the yield of GOS is a technical problem to be solved for realizing the large-scale production of galactooligosaccharide by the enzyme method. For the beta-galactosidase-mediated enzyme synthesis route, the yield of galactooligosaccharide is usually 20% to 45% (corresponding to 40% to 60% of the substrate lactose conversion rate), and there is no successful case of greatly improving the yield of galactooligosaccharide by using biological catalysts or process engineering technology optimization; (4) in the final conversion solution for synthesizing galactooligosaccharide by using the enzyme method, there are still unreacted lactose, hydrolysis by-products glucose and galactose, which are difficult to separate and purify, and the cost is high. At present, the mass fraction of most domestic galactooligosaccharide products on the domestic market is less than 57%. In view of the above problems, the application aims to develop a new type of immobilization strategy based on the co-immobilization technology of beta-galactosidase and saccharomyces cerevisiae, and construct a galactooligosaccharide synthesis-purification integrated two-stage process.
[0003] Electrospinning shows a large surface area to volume ratio due to its high porosity and nano-to-micron scale structure, which is beneficial for the controlled release of immobilized enzymes and can also improve the catalytic ability of enzymes. (Santos D P J, Zavareze R D E, Dias G R A, Immobilization of xylanase and xylanase-beta-cyclodextrin complex in polyvinyl alcohol via electrospinning improves enzyme activity at a wide pH and temperature range, International Journal of Biological Macromolecules., 118 (2018), 1676-1684.) However, the traditional carrier has the problem of small surface area, which cannot provide enough space, resulting in reduced loading force; there is steric hindrance between the enzyme active site and the substrate after immobilization, which leads to insufficient reaction of the enzyme and the substrate. Nanomaterials are often used to determine the factors that affect the efficiency of nanobiocatalysts by improving key factors, especially improved enzyme loading, large specific surface area, reduced mass transfer resistance, and increased mobility. There are some reports on the immobilization of beta-galactosidase through electrospun nanofiber membranes, and the research team also found in the previous research process that the hydrolysis and transglycosylation activity of beta-galactosidase can be significantly improved by functionalizing graphene oxide nanofiber membranes. However, there is no report on the co-immobilization of beta-galactosidase and yeast to achieve GOS preparation. The present application proposes a novel and practical method for co-immobilizing beta-galactosidase and yeast through electrospun double-layer nanofiber membranes. SUMMARY
[0004] In view of the poor stability, non-reusable, low transglycosylation activity, and high difficulty in product purification of free enzymes used in the current GOS preparation process, the present application provides a novel catalytic system for co-immobilizing beta-galactosidase and yeast, which has better stability and recycling performance, and has more outstanding transglycosylation activity. In addition, the unique properties of yeast can efficiently remove monosaccharide impurities generated during GOS synthesis, which is beneficial for the production of high-purity GOS.
[0005] To achieve the purpose, the present application adopts the following technical solutions.
[0006] A single-layer polystyrene nanofiber membrane, comprising synthesis of functional graphene oxide and preparation of polystyrene nanofiber membrane, the synthesis method of the functional graphene oxide is alkylsiliconization process; the preparation method of the polystyrene nanofiber membrane adopts electrospinning technology. Comprising the following steps: the synthesis method of the functional graphene oxide is alkylsiliconization process; the preparation method of the polystyrene nanofiber membrane adopts electrospinning technology. Comprising the following steps:
[0007] (1) continuously stirring the aqueous solution containing 3-7% (w / v) 3-aminopropyl triethoxysilane (APTES) and acetic acid to adjust the pH to 3.0-5.0, hydrolyzing at room temperature for 5-20 min, then adding 0.5-2% (w / v) graphene oxide, and ultrasonic treatment for 5-20 min. Then, continuously stirring the mixture at 50-80 °C for 0.5-2 h, centrifuging to remove silane residues and other impurities, obtaining precipitated GO-APTES, and washing with ethanol and water for several times, and drying to obtain the functional graphene oxide.
[0008] (2) dissolving 0.3-2.0% (w / v) GO-APTES in 10-50% (w / v) polystyrene (PS) to prepare a polymer carrier PS / GO / APTES ENMS by stirring overnight. The PS / GO-APTES nanocomposite solution is filled into a syringe for electrospinning, with a voltage of 10-50 kV and a temperature of 20-50 °C.
[0009] A nanofiber membrane for fixing yeast by encapsulation method, comprising preparation of functional graphene oxide modified polystyrene electrospun nanofiber membrane for fixing yeast; the preparation method of the polystyrene nanofiber membrane adopts electrospinning technology. Comprising the following steps:
[0010] (1) drying activated Saccharomyces cerevisiae suspensions with different densities at 70 °C for 24 h, and then grinding into Saccharomyces cerevisiae cell powder. The yeast powder is added to the PS / GO / APTES polymer solution to prepare a mixed solution with different SCs mass ratios.
[0011] (2) stirring the mixture at 35 °C for 3 h to fully mix and uniform. Then, the mixture is placed in a 10 mL syringe with a needle diameter of 1.19 mm (16 gauge). Finally, electrospun nanofibers are generated by adjusting the voltage, receiving distance and pushing rate, and collected on an aluminum foil covered receiving plate.
[0012] In the above step (1), preferably, the yeast is Angel Saccharomyces cerevisiae, and the density is 10 4 and 10 5 CFU.
[0013] The electrospinning conditions are preferably a receiving distance of 17 cm, a flow rate of 2.5 mL / h, a voltage of 20-30 kV, a relative humidity of 37%, and a temperature of 26 °C.
[0014] A nanofiber membrane for fixing yeast by adsorption method, comprising the preparation of fixing yeast on a functional graphene oxide modified polystyrene electrospun nanofiber membrane; the preparation method of the polystyrene nanofiber membrane adopts an electrospinning technology. The method comprises the following steps:
[0015] (1) The activated yeast suspension with different densities is added to the YPD culture medium, then 2 g of PS / GO / APTES ENMs are added, and incubation is performed at 30 °C for 24 hours.
[0016] (2) Then, the Ads-SCs@PS / GO / APTES / ENMs are obtained by centrifugation at 3000 r for 20 minutes. The nanocomposite is washed with a phosphate buffer solution three times to remove residual yeast cells.
[0017] In the step (1), the yeast is preferably Angel yeast, and the density is 10 4 , 10 5 CFU.
[0018] The electrospinning conditions are preferably a receiving distance of 17 cm, a flow rate of 2.5 mL / h, a voltage of 20-30 kV, a relative humidity of 37%, and a temperature of 26 °C.
[0019] Preparation of a double-layer SCs@PS / GO / APTES / / ENMs
[0020] The second layer of nanofiber ( / / ENMs) is prepared on the first layer of yeast-containing nanofiber ( / ENMs), including the encapsulated Enc-SCs@PS / GO / APTES / ENMs and the adsorbed Ads-SCs@PS / GO / APTES / ENMs.
[0021] The above two kinds of nanofibers are placed on the aluminum foil receiving plate of the electrospinning equipment, and the PS / GO / APTES nanofiber layer is electrospun under the same electrospinning conditions (a receiving distance of 17 cm, a flow rate of 2.5 mL / h, a voltage of 20-30 kV, a relative humidity of 37%, and a temperature of 26 °C), and finally the double-layer structure Enc-SCs@PS / GO / APTES / / ENMs and Ads-SCs@PS / GO / APTES / / ENMs are obtained.
[0022] The application provides a preparation method for fixing β-galactosidase by using the above nanofiber membrane.
[0023] Optionally, the application provides a method for immobilizing β-galactosidase using the nanofiber membrane described above, the specific steps are as follows:
[0024] The prepared double-layer structure Enc-SCs@PS / GO / APTES / / ENMs and Ads-SCs@PS / GO / APTES / / ENMs are soaked in a sodium acetate buffer solution (pH 3-6) of β-galactosidase (0-8 mg / mL) at 0-10 °C overnight, and the ENMs are washed with water to remove free β-galactosidase, thereby obtaining immobilized β-galactosidase.
[0025] The application provides the application of the immobilized β-galactosidase described above in the preparation of galactooligosaccharide.
[0026] Optionally, the application provides the application of the immobilized β-galactosidase described above in the preparation of galactooligosaccharide, and the specific steps are as follows:
[0027] (1) Lactose is dissolved in a sodium acetate buffer solution (pH 3-6) to prepare a solution with an optimal initial lactose concentration (100-500 g / L) at 50-70 °C.
[0028] (2) The lactose (100-500 g / L) solution is added to Ads-SCs@PS / GO / APTES / / ENMs @GAL (0-8 mg / mL) and Enc-SCs@PS / GO / APTES / / ENMs @GAL (0-8 mg / mL) respectively, and the mixture is incubated at 30-40 °C for 20 h.
[0029] In the above step, the initial pH value of the lactose solution is preferably 4.5, and the enzyme activity is highest at pH 4.5.
[0030] In the above step, the lactose solution concentration is preferably 200 g / L, and below or above this concentration, the immobilized enzyme galactosidase activity will be inhibited, and at this concentration, the lactose conversion rate is highest and the galactooligosaccharide (GOS) yield is highest.
[0031] In the above step, the enzyme amount of immobilized β-galactosidase is preferably 4 mg / mL, and below this concentration, the GOS generation rate is slow, and above this concentration, the GOS generation rate cannot be significantly improved.
[0032] In the above step, the reaction temperature is preferably 37 °C. Beneficial effects
[0033] 1. The application provides a novel preparation method of a double-layer functionalized PS / GO nanofiber membrane which can be used for co-immobilization of yeast and β-galactosidase.
[0034] 2、The prepared immobilized β-galactosidase exhibits the advantages of high adsorption rate, storage stability and high reusability.
[0035] 3、The catalytic behavior of the prepared immobilized β-galactosidase changes, and the lactose hydrolysis capacity and transglycosylation activity are improved.
[0036] 4、The application successfully constructs a new biological catalytic system integrating GOS biosynthesis and monosaccharide removal: GOS without monosaccharide is obtained in one step with lactose as raw material, which solves the problem of monosaccharide residue in traditional GOS production, and significantly improves the product purity and production efficiency.
[0037] 5、The immobilized yeast prepared by the encapsulation method and the adsorption method exhibits high adsorption rate, and in the integrated process, the yeast cells can consume more than 90% of the monosaccharide in the GOS synthesis process, while the GOS yield still remains at a high level of 67%. BRIEF DESCRIPTION OF DRAWINGS
[0038] Figure 1 Encapsulation method (Enc) and adsorption method (Ads) of yeast cell immobilization flow chart
[0039] Figure 2 Yeast immobilization rate analysis of encapsulation method (Enc-SCs@PS / GO / APTES / ENMs) and adsorption method (Ads-SCs@PS / GO / APTES / ENMs)
[0040] Figure 3 X-ray diffraction pattern of encapsulation method (Enc-SCs@PS / GO / APTES / ENMs) and adsorption method (Ads-SCs@PS / GO / APTES / ENMs)
[0041] Figure 4 Infrared spectrum of encapsulation method (Enc-SCs@PS / GO / APTES / ENMs) and adsorption method (Ads-SCs@PS / GO / APTES / ENMs)
[0042] Figure 5 Scanning electron microscope image and nanofiber diameter distribution result of encapsulation method (Enc-SCs@PS / GO / APTES / ENMs) and adsorption method (Ads-SCs@PS / GO / APTES / ENMs)
[0043] Figure 6 Confocal laser scanning microscope image of encapsulation method (Enc-SCs@PS / GO / APTES / ENMs) and adsorption method (Ads-SCs@PS / GO / APTES / ENMs)
[0044] Figure 7 Optical microscope results of methylene blue staining of encapsulation method (Enc-SCs@PS / GO / APTES / ENMs) and adsorption method (Ads-SCs@PS / GO / APTES / ENMs)
[0045] Figure 8 Immobilization ratio and enzyme activity analysis of immobilized β-galactosidase
[0046] Figure 9 Optimum pH and optimum temperature analysis of immobilized β-galactosidase
[0047] Figure 10 Storage stability and reusability analysis of immobilized β-galactosidase
[0048] Figure 11 Thin layer chromatography and high performance liquid chromatography results of lactose hydrolysis DETAILED DESCRIPTION
[0049] In order to make the objects, technical solutions and advantages of the present application clearer, the embodiments of the present application will be further described in detail below with reference to the drawings and examples, but the present application is not limited to the embodiments.
[0050] Example 1 Preparation of Enc-SCs@PS / GO / APTES / ENMs by yeast encapsulation method
[0051] (1) The activated Saccharomyces cerevisiae suspension with different cell densities (10¹, 10², 10³, 10 4 and 10 5 CFU) was dried at 70 °C for 24 h, and then ground into Saccharomyces cerevisiae cell powder. The yeast powder was added to the PS / GO / APTES polymer solution to prepare a mixed solution with different SCs mass ratios.
[0052] (2) The mixture was stirred at 35 °C for 3 h to mix evenly. Then the mixture was placed in a syringe with a needle diameter of 1.19 mm (16 gauge). Finally, electrospun nanofibers were produced by adjusting the voltage, receiving distance and propulsion rate, and were collected on an aluminum foil covered receiving plate.
[0053] Example 2 Preparation of Ads-SCs@PS / GO / APTES / ENMs by yeast adsorption method
[0054] (1) The activated Saccharomyces cerevisiae suspension with different cell densities (10¹, 10², 10³, 10 4 and 10 5A suspension of Saccharomyces cerevisiae (CFU) was added to YPD medium, followed by the addition of 2g of PS / GO / APTES ENMs, and incubated at 30 °C for 24 h.
[0055] (2) Ads-SCs@PS / GO / APTES / ENMs were obtained by centrifugation at 3000 r for 20 min. The nanocomposite was washed three times with phosphate buffer solution to remove residual yeast cells.
[0056] Yeast immobilization efficiency can be assessed by the percentage of yeast encapsulated in electrospun fibers; a higher immobilization rate indicates a higher immobilization efficiency. Figure 2 It is clearly demonstrated that the yeast immobilization effect using the adsorption strategy is superior to that using the encapsulation strategy. At different yeast concentrations, the adsorbed samples exhibited the highest immobilization rate, with the highest rate observed at a yeast concentration of 10⁻⁶. 5 At that time, the immobilization rates of the adsorption method and the encapsulation method reached peak values of 99% and 95%, respectively.
[0057] The prepared Enc-SCs@PS / GO / APTES / ENMs and Ads-SCs@PS / GO / APTES / ENMs were analyzed by X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy, respectively. X-ray diffraction (… Figure 3 The results showed that the yeast immobilization method had little effect on the crystal structure, and the X-ray diffraction patterns of the samples were basically consistent. Specifically, the encapsulated sample showed enhancement in the two diffraction angle 2θ regions (40.30 and 45.35), with intensity counts of 3790 and 3220, respectively, while the corresponding values for the adsorbed sample were only 1990 and 2020. Fourier transform infrared spectroscopy (FTIR) Figure 4 The results showed that the chemical composition of the encapsulation method and the adsorption method were generally consistent, and their spectra were basically overlapping, differing only at 3024, 1641, and 1602 cm⁻¹. -1 Slight differences were observed in three specific wavenumber regions. The transmittance values of the adsorbed sample at these three wavenumbers (0.097, 0.078, and 0.070) were all lower than those of the encapsulated sample (0.109, 0.099, and 0.096). Scanning electron microscopy results are shown below. Figure 5 As shown, yeast nanofibers were immobilized using an encapsulation method. Figure 5 A and B) have a more regular fibrous structure, with yeast cells encapsulated inside the fibers. Yeast nanofibers immobilized via adsorption (…) Figure 5 The morphology of cells D and E was significantly affected by the immobilization process. Yeast cells mainly adhered to the fiber surface rather than the interior, and their external morphology remained largely unchanged. This indicates that adsorption immobilization has less impact on yeast cells compared to encapsulation immobilization, but may affect the overall immobilization effect. Diameter distribution analysis showed that the average diameter of the encapsulated yeast fibers (500.15 nm) was significantly higher than that of the encapsulated yeast fibers. Figure 5C) less than the average diameter of the adsorbed yeast fibers (573.85 nm, Figure 5 F). This indicates that the embedding of yeast in the electrospinning process affects the properties of the polymer solution, which in turn changes the physical properties of the fibers.
[0058] To further explore the effect of immobilization on the activity of yeast cells, CTC and methylene blue staining were performed on the immobilized yeast, and confocal laser scanning microscopy was used for activity analysis. The results of confocal laser scanning microscopy (CLSM) are shown in Figure 6 As shown, the cells fixed in the fibers by adsorption method showed green fluorescence, while the yeast cells fixed by encapsulation method showed red fluorescence. When fixed by adsorption method, yeast cells adhere to the surface of PS / GO / APTES nanofibers, and this process is mild and generally does not cause significant changes in cell structure or fluorescence properties. Green indicates that the yeast maintains the original structure and emission properties, as adsorption generally does not exert significant chemical or physical stress. The core of the encapsulation method is to encapsulate yeast cells inside the PS / GO / APTES matrix, which enhances mechanical stability and isolates external interference, but may cause greater stress to the cells and trigger chemical and physical changes. Red fluorescence indicates a decrease in cell activity and metabolic function, which may be caused by: (1) the interaction between the encapsulation matrix (PS / GO / APTES) and the green fluorescent marker causing fluorescence quenching or emission spectrum shift; (2) the autofluorescence of PS / GO / APTES matrix itself in the red light band may cover the yeast cell signal; (3) the changed local microenvironment (such as pH value, oxygen concentration, etc.) during the encapsulation process affects the fluorescence properties of the cells. In addition, due to its energy transfer properties, graphene oxide has a fluorescence quenching effect, and in the matrix containing graphene oxide, the green fluorescence may be suppressed, so only red fluorescence is observed. Therefore, the fixation method has a significant impact on the yeast cells. The results of methylene blue staining are shown in Figure 7 A, the yeast cells fixed by encapsulation have reduced activity and cannot metabolize methylene blue, resulting in blue staining; while in the staining image of the yeast cells fixed by adsorption, only a small number of blue cells are detected Figure 7 B). The results show that yeast cells fixed on PS / GO / APTES nanofibers by adsorption method maintain a high activity, while encapsulation method affects the survival state of yeast cells.
[0059] Example 3 Preparation of double-layer SCs@PS / GO / APTES / / ENMs
[0060] A second layer of nanofibers ( / / ENMs) was prepared on the first layer of yeast-containing nanofibers ( / ENMs), including encapsulation type Enc-SCs@PS / GO / APTES / ENMs and adsorption type Ads-SCs@PS / GO / APTES / ENMs.
[0061] The two kinds of nanofibers were placed on the aluminum foil receiving plate of the electrospinning equipment, and the PS / GO / APTES nanofiber layer was electrospun under the same electrospinning conditions (receiving distance 17 cm, flow rate 2.5 mL / h, voltage 20-30 kV, relative humidity 37%, temperature 26 °C), finally obtaining the double-layer structure Enc-SCs@PS / GO / APTES / / ENMs and Ads-SCs@PS / GO / APTES / / ENMs.
[0062] Example 4 Preparation of immobilized β-galactosidase The prepared nanofiber membranes (including encapsulated Enc-SCs@PS / GO / APTES / / ENMs and adsorbed Ads-SCs@PS / GO / APTES / / ENMs) were soaked in a sodium acetate buffer solution (0.1 M, pH 4.5) containing β-galactosidase (0-8 mg / mL) at 4 °C overnight. The enzyme-yeast-containing composite double-layer nanofiber structures were prepared: encapsulated Enc-SCs@PS / GO / APTES / / ENMs@GAL and adsorbed Ads-SCs@PS / GO / APTES / / ENMs@GAL. Finally, the ENMs were thoroughly rinsed with water to remove free β-galactosidase. The supernatant and washing liquid were collected and the concentration of unabsorbed protein was determined using the Bradford method.
[0063] Figure 8 A shows that when the concentration of β-galactosidase is 4 mg / ml, the maximum adsorption yield of the adsorbed yeast nanofiber membrane is 92%, the encapsulated yeast nanofiber membrane is slightly lower than the adsorbed method, with a maximum adsorption yield of 88%, and the blank nanofiber membrane without immobilized yeast has the lowest adsorption yield of only 81%.
[0064] Example 5 Stability analysis of immobilized β-galactosidase
[0065] (1) Enzyme activity determination: ONPG (20 mM, pH 4.5) was used as the substrate, and free β-galactosidase was used as the control to analyze the change in enzyme activity of the immobilized β-galactosidase. The specific operation was as follows: the prepared nanofiber membranes (0.2 g) were added to 0.1 M sodium acetate buffer solution, reacted with ONPG at 37 °C for 15 min, and the reaction was terminated with 2.0 mL of sodium carbonate solution (1 M). The absorbance at 405 nm was measured. The results are shown in Figure 8 B), the activity of the adsorbed yeast nanofiber membrane combined with the enzyme reached 97%, reaching the maximum activity level. The encapsulated yeast nanofiber membrane combined with the enzyme reached a maximum of 95%, and the blank nanofiber membrane combined with the enzyme reached a maximum of 87%, all of which were higher than the free enzyme of 79%.
[0066] (2) Optimum temperature analysis: The enzyme activity of immobilized β-galactosidase was determined in 0.1 M sodium acetate buffer (pH 4) at different temperatures (20 °C-70 °C), with the activity recorded at 50 °C as the control (100%) for the percentage of residual activity. As shown in Figure 9 As shown in FIG. 2A, compared with the immobilized enzyme, the free enzyme exhibited the lowest stability at each temperature. The activity of the free enzyme reached a peak at 40 °C, but the activity of the β-galactosidase combined with the different nanofiber membranes reached a maximum at 50 °C, which further expanded the applicable range of the β-galactosidase in the production process. Among them, the thermal stability of the enzyme combined with the adsorbed yeast nanofiber membrane was optimal, followed by the encapsulated yeast nanofiber membrane, and the worst was the nanofiber membrane without yeast.
[0067] (3) Optimum pH analysis: The β-galactosidase was reacted with ONPG as the substrate in a buffer (0.1 M) at pH 3.0-8.0 at 50 °C for 15 min. Among them, the buffer used was glycine-HCl (pH 3.0), sodium acetate (pH 4.0-5.0), sodium phosphate (pH 6.0-7.0), and Tris-HCl (pH 8.0). The results are shown in Figure 9 As shown in FIG. 2B, the free enzyme had the highest activity at pH 6.5, while the immobilized enzyme reached the maximum activity at pH 4.5. By comparing the different nanofiber membrane carriers, the enzyme stability of the nanofiber membrane without yeast was the lowest, which was significantly lower than that of the nanofiber membrane fixed with yeast cells by the encapsulation method or the adsorption method.
[0068] (4) Storage stability: The storage stability of the immobilized β-galactosidase was analyzed and determined by storing at 4 °C for two months, and the results are shown in Figure 10 As shown in FIG. 2A, the activity of the free enzyme decreased significantly, while the nanofiber membranes of various types had a protective effect on the enzyme (with less difference in the protection effect). Among them, the adsorbed yeast nanofiber membrane had the best protection effect on the enzyme, followed by the encapsulated yeast nanofiber membrane, and finally the nanofiber membrane without yeast. This indicates that the double-layer nanofiber membrane containing yeast cells has a positive regulatory effect on the operational stability of the enzyme, which can effectively reduce the operating cost in actual application.
[0069] (5) Reusability: The same immobilized enzyme was repeatedly used for 20 times in the ONPG hydrolysis process, among which the retention activity of the enzyme combined with the adsorbed yeast nanofiber membrane was the highest, which could reach 69%, the retention activity of the enzyme combined with the encapsulated yeast nanofiber membrane could reach 63%, and the retention rate of the enzyme activity of the nanofiber membrane without yeast was only 54%. As shown in Figure 10 B.
[0070] Example 6 Synthesis of galactooligosaccharides
[0071] (1) Lactose (200 g / L) was dissolved in sodium acetate buffer solution (0.1 M, pH 4.5) at 60 °C, and the solution was cooled to 37 °C before the encapsulated yeast cell membrane-bound enzyme (4 mg / mL) and the adsorbed yeast cell membrane-bound enzyme (4 mg / mL) were added, respectively. The mixture was incubated at 37 °C with 200 rpm shaking, and samples were taken at different times and inactivated by heating in boiling water for 10 min. The samples were filtered using 0.45 μm nylon filters and diluted 40 times.
[0072] (2) Thin layer chromatography (TLC) analysis: 2 μL of the diluted sample was applied to a silica gel 60 pre-coated aluminum plate (20 x 20 cm) with a chloroform / acetic acid / water (6:7:1, v / v / v) mixture as the mobile phase. Sulfuric acid (95:5, v / v) was sprayed onto the plate, which was then baked at 110 °C for 10 min. The plate was developed in iodine vapor and the results were observed by the appearance of a dark spot on a light background. Figure 11 It can be seen that the TLC plate shows that both types of cell membrane-bound enzyme exhibit high transglycosylation activity. Compared with Enc-SCs@PS / GO / APTES / / ENMs @GAL (A), Ads-SCs@PS / GO / APTES / / ENMs @GAL (B) shows higher and faster monosaccharide removal capacity, with a significant decrease in monosaccharides from the 4th hour of the catalytic reaction. This indicates that the galactosylation conversion process can be accelerated by using the biocatalyst double layer PS / GO / APTES / / ENMs @GAL. By co-immobilizing yeast with β-galactosidase, the monosaccharide byproduct generated during the catalytic reaction can be efficiently and rapidly removed. Figure 11 A) and Ads-SCs@PS / GO / APTES / / ENMs @GAL (B) show higher and faster monosaccharide removal capacity, with a significant decrease in monosaccharides from the 4th hour of the catalytic reaction. This indicates that the galactosylation conversion process can be accelerated by using the biocatalyst double layer PS / GO / APTES / / ENMs @GAL. By co-immobilizing yeast with β-galactosidase, the monosaccharide byproduct generated during the catalytic reaction can be efficiently and rapidly removed. Figure 11 B) show higher and faster monosaccharide removal capacity, with a significant decrease in monosaccharides from the 4th hour of the catalytic reaction. This indicates that the galactosylation conversion process can be accelerated by using the biocatalyst double layer PS / GO / APTES / / ENMs @GAL. By co-immobilizing yeast with β-galactosidase, the monosaccharide byproduct generated during the catalytic reaction can be efficiently and rapidly removed.
[0073] (3) High performance liquid chromatography (HPLC) analysis: Aminex HPX-87H chromatographic column (300 x 7.8 mm) was used for quantitative analysis of the synthesized carbohydrates using a high performance liquid chromatograph (SHIMADZU) equipped with a differential refractive index detector (RID-10A) with ultrapure water as the mobile phase at a flow rate of 0.5 mL / min. Standard solutions of lactose, glucose, galactose, and galactooligosaccharides with known concentrations were used for quantitative analysis.
[0074] Figure 11 The time course of GOS synthesis by immobilized enzymes during a 20 h reaction time at 37 °C is shown. The results are shown in Table 1. Figure 11 Table 1: Time course of GOS synthesis by immobilized enzymes during a 20 h reaction time at 37 °C.
[0075] The results showed that the adsorbed yeast nanofiber membrane combined with enzyme had high GOS synthesis capacity (56%) and excellent monosaccharide degradation capacity.
Claims
1. A nanofiber membrane comprising yeast, characterized in that Preparation of yeast immobilized on functional graphene oxide modified polystyrene electrospun nanofiber membrane by encapsulation and adsorption method; the preparation method of the polystyrene nanofiber membrane adopts electrospinning technology. It comprises the following steps: (1) continuously stirring the aqueous solution containing 3-7% (w / v) 3-aminopropyl triethoxysilane (APTES) and acetic acid to adjust the pH to 3.0-5.0, and then adding 0.5-2% (w / v) graphene oxide after hydrolysis at room temperature for 5-20 min, and ultrasonic treatment for 5-20 min. Then, the mixture is continuously stirred at 50-80 °C for 0.5-2 h, and the silane residue and other impurities are removed by centrifugation to obtain a precipitate GO-APTES, which is washed with ethanol and water several times and dried to obtain functional graphene oxide. (2) Dissolve 0.3-2.0% (w / v) GO-APTES in 10-50% (w / v) polystyrene (PS) and stir overnight to prepare the polymer carrier PS / GO / APTES ENMS. Fill the PS / GO-APTES nanocomposite solution into a syringe for electrospinning, with a voltage of 10-50 kV and a temperature of 20-50 °C.
2. The preparation of encapsulated SCs@PS / GO / APTES / ENMs according to claim 1, characterized in that, It comprises the following steps: (1) The activated S. cerevisiae suspensions with different cell densities (10 4 and 10 5 CFU) were dried at 70 °C for 24 h, and then ground into yeast powder. The yeast powder was added into the PS / GO / APTES polymer solution to prepare a mixed solution with different SCs mass ratios. (2) The mixture was stirred at 35 °C for 3 h to allow it to mix thoroughly. The mixture was placed in a 10 mL syringe with a needle diameter of 1.19 mm (16 gauge). Finally, the nanofibers were electrospun by adjusting the voltage, receiving distance, and propulsion rate, and were collected on an aluminum foil-covered receiving plate.
3. The preparation of adsorptive nanofiber membrane (Ads-SCs@PS / GO / APTES / ENMs) according to claim 1, characterized in that, It comprises the following steps: (1) The activated suspension of S. cerevisiae with different densities (10 4 and 10 5 CFU was added to the YPD medium, followed by the addition of 2 g of PS / GO / APTES ENMs and incubation at 30 °C for 24 h. (2) The Ads-SCs@PS / GO / APTES / ENMs were obtained by centrifugation at 3000 r for 20 min. The composite was washed three times with phosphate buffer solution to remove residual yeast cells.
4. A process for the preparation of a double layer functionalized PS / GO nanofiber membrane for co-immobilization of β-galactosidase with yeast, characterized by, It comprises the following steps: Place the successfully synthesized Enc-SCs@PS / GO / APTES / ENMs and Ads-SCs@PS / GO / APTES / ENMs on the aluminum foil receiving plate of the electrospinning equipment, and electrospin the PS / GO / APTES nanofiber layer under the same electrospinning conditions (receiving distance 17 cm, flow rate 2.5 mL / h, voltage 20-30 kV, relative humidity 37%, temperature 26 °C) to obtain a double-layer structure Enc-SCs@PS / GO / APTES / / ENMs and Ads-SCs@PS / GO / APTES / / ENMs.
5. A method for the preparation of an immobilized β-galactosidase, characterized by The nanofiber membrane according to claim 4 is made.
6. The immobilized β-galactosidase of claim 5, wherein, It comprises the following steps: soaking the prepared double-layer composite carrier in a sodium acetate buffer solution (pH 3-8) containing β-galactosidase (0-8 mg / mL) at 0-10 °C overnight, and then thoroughly rinsing the ENMs with water to remove free β-galactosidase. At this time, the adsorption yield and enzyme activity of β-galactosidase are the highest.
7. The use of the immobilized enzyme according to claim 6 in the preparation of galactooligosaccharides (GOS).
8. Use according to claim 7, characterized in that, It comprises the following steps: (1) Dissolve lactose in a sodium acetate buffer solution (pH 3-6) at 50-70 °C to prepare a solution with an optimal initial lactose concentration (100-500 g / L). (2) Add the prepared Ads-SCs@PS / GO / APTES / / ENMs @GAL (0-8 mg / mL) and Enc-SCs@PS / GO / APTES / / ENMs @GAL (0-8 mg / mL) to the lactose (100-500 g / L) solution, respectively, and incubate the mixture at 30-40 °C for 20 h.
9. Use according to claim 8, wherein the immobilized β-galactosidase is repeatedly used, with good performance.