A method for preparing cross-linked inclusion bodies and its application in hydrolyzing soybean oligosaccharides

Abstract

The present invention relates to a method for preparing a cross-linked inclusion body and its application in hydrolyzing soybean oligosaccharides. The method comprises the following steps: (1) culturing recombinant Escherichia coli containing an α-galactosidase gene and collecting inclusion bodies; (2) adding a cross-linking agent solution to an inclusion body suspension to construct a cross-linking system; stirring and cross-linking for 0.5 to 4 hours, and obtaining cross-linked inclusion bodies after washing. The present invention also provides the application of the cross-linked inclusion bodies prepared by the method in hydrolyzing soybean oligosaccharides in soy milk. The present invention cross-links inclusion bodies containing α-galactosidase catalytic activity for the first time to obtain cross-linked inclusion bodies. There is no need to purify the recombinant α-galactosidase, which effectively reduces the time and cost of enzyme purification preparation, and has the advantages of simple operation, low cost, and no need for high-purity enzyme. At the same time, in the process of enzyme immobilization, no precipitant needs to be added, which can further reduce the cost and workload.

Description

Technical Field

[0001] The invention relates to a preparation method of a cross-linked inclusion body and application thereof in hydrolyzing soybean oligosaccharides, belonging to the technical field of sugar engineering. Background Art

[0002] Soy milk is rich in protein, essential amino acids, fatty acids, vitamins and minerals. Since it does not contain lactose, it provides an alternative source of nutrition for lactose-intolerant people worldwide. Soy milk has rich nutritional value, including reducing the occurrence of cardiovascular diseases, preventing osteoporosis, anti-oxidation, anti-diabetes, anti-cancer and cholesterol-lowering effects. Soy milk contains a high concentration of soluble oligosaccharides, such as raffinose and stachyose, which cannot be hydrolyzed by enzymes in the human digestive tract. People often have problems such as flatulence after eating soy products due to the fermentation of oligosaccharides by intestinal flora. In order to reduce the content of soy oligosaccharides, soaking, steaming, baking, chemical treatment and α-galactosidase treatment are often used in industry. Among them, the use of α-galactosidase to degrade soy oligosaccharides has the advantages of high efficiency, low energy consumption and relatively short processing time. Heterologous recombinant expression of α-galactosidase is an important method for preparing α-galactosidase, but in the preparation process of recombinant α-galactosidase, multiple purification steps are required to obtain high-purity α-galactosidase, and the purified α-galactosidase needs to be immobilized before it can be recycled. Therefore, in industrial applications, enzyme purification and immobilization are still the main cost and technical difficulties.

[0003] Cross-linked enzyme aggregates (CLEAs) are a carrier-free immobilization technology. First, the free enzyme is precipitated by a precipitant such as a water-soluble organic solvent, a neutral salt solution or a non-ionic polymer, and then a cross-linking agent is added to obtain a water-insoluble enzyme aggregate. This process requires the use of a precipitant to precipitate the enzyme, which is a cumbersome process, and the most commonly used cross-linking agent glutaraldehyde (GA) easily binds to the amino acids at the catalytic active site of the enzyme, reducing the activity of the cross-linked enzyme aggregate and affecting the enzyme activity recovery rate. Therefore, it is of great significance to study a cross-linked enzyme aggregate that is cost-effective, simple to operate, has excellent performance and can be mass-produced. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a method for preparing a cross-linked inclusion body and application thereof in hydrolyzing soybean oligosaccharides.

[0005] The technical solution of the present invention is as follows:

[0006] A method for preparing cross-linked inclusion bodies comprises the following steps:

[0007] (1) culturing recombinant Escherichia coli containing an α-galactosidase gene, collecting inclusion body precipitates and suspending them in a potassium phosphate buffer to obtain an inclusion body suspension;

[0008] (2) adding a crosslinking agent solution to the inclusion body suspension to construct a crosslinking system; then placing the crosslinking system in an ice water bath and stirring for crosslinking for 0.5 to 4 hours, removing the supernatant by centrifugation, collecting the precipitate, and washing to obtain the crosslinked inclusion body.

[0009] Preferably according to the present invention, in step (1), the inclusion bodies are inclusion bodies having α-galactosidase catalytic activity.

[0010] Preferably, according to the present invention, in step (1), the α-galactosidase gene is an α-galactosidase gene that has been publicly annotated in the NCBI database or other gene databases, and can form active inclusion bodies after recombinant expression; the construction of the recombinant Escherichia coli containing the α-galactosidase gene is carried out according to the existing technology.

[0011] Preferably according to the present invention, in step (2), the cross-linking agent is dimethyl pimelimidate dihydrochloride (DMP), dimethyl suberimidate dihydrochloride (DMS), di(sulfosuccinimidyl) suberate (BS3) or pegylated di(sulfosuccinimidyl) suberate [BS(PEG)5].

[0012] Further preferably, when the cross-linking agent is dimethyl pimelimidate dihydrochloride (DMP) and dimethyl suberimidate dihydrochloride (DMS), the stirring cross-linking time is 1 hour; when the cross-linking agent is di(sulfosuccinimidyl) suberate (BS3), the stirring cross-linking time is 2 hours; when the cross-linking agent is polyethylene glycol di(sulfosuccinimidyl) suberate [BS(PEG)5], the stirring cross-linking time is 1.5 hours.

[0013] Preferably, according to the present invention, in step (2), the final concentration of inclusion bodies in the cross-linking system is 5 to 15 mg / mL;

[0014] More preferably, the final concentration of the inclusion bodies is 10 mg / mL.

[0015] Preferably, according to the present invention, in step (2), the final concentration of the cross-linking agent in the cross-linking system is 1 to 10 mM;

[0016] Further preferably, the concentration of the cross-linking agent is 1 mM.

[0017] Preferably, according to the present invention, in step (2), the centrifugation is performed at 10,000 g for 5 min, and the washing is performed by washing the precipitate with potassium phosphate buffer for 3 times.

[0018] The present invention also provides the use of the cross-linked inclusion body prepared by the method in hydrolyzing soybean oligosaccharides in soybean milk.

[0019] The above application comprises the following steps:

[0020] (a) Grinding soybeans into powder with a grinder, adding hexane to defatted and then drying, suspending the defatted soybean powder in distilled water, fully mixing, boiling at 100° C. for 10 minutes, centrifuging at 10000 g for 10 minutes, and collecting the supernatant to obtain a soymilk solution containing soybean oligosaccharides;

[0021] (b) adding cross-linked inclusion bodies to a soymilk stock solution containing soy oligosaccharides to prepare a soy oligosaccharide hydrolysis reaction system; then reacting the hydrolysis reaction system at pH 5-6 and 55-65° C. for 0.5-3 hours, centrifuging at 10000 g for 10 minutes, and collecting the supernatant to obtain soymilk after the soy oligosaccharide hydrolysis.

[0022] Preferably according to the present invention, in step (a), the mass volume ratio of soybean to hexane is 1:1, in g / mL.

[0023] Preferably, according to the present invention, in step (a), the mass volume ratio of the defatted soy flour to distilled water is 1:10, in g / mL.

[0024] Preferably according to the present invention, in step (b), the soybean oligosaccharide hydrolysis reaction system comprises 1 mL soybean milk stock solution and 4-8 U / mL cross-linked inclusion bodies.

[0025] Further preferably, in step (b), the cross-linked inclusion bodies are 6 U / mL and reacted at 65° C. for 1 hour.

[0026] Beneficial Effects

[0027] 1. The present invention cross-links inclusion bodies containing α-galactosidase catalytic activity for the first time to obtain cross-linked inclusion bodies. Compared with the prior art, the present invention does not need to purify recombinant α-galactosidase, and the inclusion body precipitate can be obtained by centrifugation after cell disruption, which effectively reduces the time and cost of enzyme purification preparation, and has the advantages of simple operation, low cost, and no need for high-purity enzyme. At the same time, in the process of enzyme immobilization, inclusion bodies are good materials for the preparation of cross-linked enzyme aggregates. Because the inclusion bodies themselves exist in the form of precipitation, no precipitant needs to be added, which can further reduce costs and workload.

[0028] 2. The present invention uses DMP, DMS, BS3 and BS (PEG) 5 as cross-linking agents for cross-linking enzyme aggregates for the first time. These four cross-linking agents are mild in nature, have little damage to the protein structure, and can maintain the natural conformation and activity of the enzyme. The enzyme activity recovery rate of the cross-linked inclusion bodies obtained by using these four cross-linking agents reaches 95%, which is higher than the enzyme activity recovery rate of glutaraldehyde cross-linking.

[0029] 3. The cross-linked inclusion bodies prepared by the present invention have good stability and can be reused many times. After 15 batches of cyclic hydrolysis of soybean oligosaccharides, the relative hydrolysis rate of oligosaccharides is maintained at more than 75%, and the residual enzyme activity is more than 50%, which is higher than the relative hydrolysis rate and residual enzyme activity of glutaraldehyde cross-linking. The cross-linked inclusion bodies prepared by the present invention have good stability and reusability, can greatly reduce the cost of large-scale production, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the enzyme activity recovery rate of cross-linked inclusion bodies.

[0031] Figure 2 Infrared spectra of control inclusion bodies and cross-linked inclusion bodies.

[0032] Figure 3 The effect of the ratio of protein to cross-linking agent on the recovery rate of cross-linked inclusion body enzyme activity.

[0033] Figure 4 The effect of cross-linking time on the recovery rate of cross-linked inclusion body enzyme activity.

[0034] Figure 5 The figure shows the effect of temperature on the activity of control inclusion bodies and cross-linked inclusion bodies.

[0035] Figure 6 The effect of temperature on the stability of control inclusion bodies and cross-linked inclusion bodies.

[0036] Figure 7 The effect of pH on the activity of control inclusion bodies and cross-linked inclusion bodies.

[0037] Figure 8 The effect of pH on the stability of control inclusion bodies and cross-linked inclusion bodies.

[0038] Fig. 9 These are the results of thin layer chromatography of inclusion body hydrolyzed soybean oligosaccharides.

[0039] Fig.10 It is the result of hydrolysis of soybean oligosaccharides by cross-linked inclusion body cycle reaction;

[0040] In the figure, Figure A shows the relative hydrolysis rate of soybean oligosaccharides hydrolyzed by the cross-linked inclusion body cyclic reaction; Figure B shows the relative enzyme activity after the cross-linked inclusion body cyclic reaction hydrolyzed soybean oligosaccharides. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto. Anything not clearly defined in the embodiments may be based on the prior art in the art.

[0042] The publicly annotated α-galactosidase gene sequence is obtained from the NCBI database or other gene databases, the gene sequence is artificially synthesized, and the construction of recombinant Escherichia coli containing the gene sequence is carried out according to the existing technology. The α-galactosidase gene can form active inclusion bodies after recombinant expression in Escherichia coli.

[0043] Example 1

[0044] A method for preparing cross-linked inclusion bodies comprises the following steps:

[0045] (1) inoculating a recombinant Escherichia coli containing an α-galactosidase gene into an LB liquid medium, culturing overnight at 37° C. and 200 rpm to obtain a seed solution; inoculating the seed solution into an LB liquid medium at a volume ratio of 1%, and when the cell grows to an OD600 of 0.6, adding IPTG at a final concentration of 1 mM for induction, the induction temperature is 16° C., the induction time is 12 h, centrifuging at 12000 rpm for 5 min, discarding the supernatant, washing the bacterial cells twice with a pH 7.0, 50 mM potassium phosphate buffer, resuspending in a pH 7.0, 50 mM potassium phosphate buffer, using an ultrasonic disruptor to break the cell wall, centrifuging the resulting suspension at 11000 rpm for 30 min, collecting the inclusion body precipitate having α-galactosidase catalytic activity by centrifugation, and suspending the inclusion body precipitate in a potassium phosphate buffer to obtain an inclusion body suspension;

[0046] The gene sequence of α-galactosidase described in this example is shown in No. OP484967 in the NCBI database, and the expression vector in Escherichia coli is pET35b, but is not limited thereto.

[0047] (2) DMP, DMS, BS3, BS(PEG)5, and GA crosslinker solutions were added to the inclusion body suspension to construct a crosslinking system. The final concentration of the inclusion bodies in the crosslinking system was 10 mg / mL, and the final concentration of the crosslinker was 5 mM. The crosslinking system was then placed in an ice water bath and stirred for crosslinking for 2 hours. The crosslinking was terminated by centrifugation at 10,000 g for 5 min to remove the supernatant. The precipitate was washed 3 times with potassium phosphate buffer to obtain crosslinked inclusion bodies.

[0048] The non-cross-linked inclusion bodies were used as control inclusion bodies. The activity of α-galactosidase in the control inclusion bodies and cross-linked inclusion bodies was determined, and the enzyme activity recovery rate of the cross-linked inclusion bodies was calculated. The results are shown in Figure 1 shown.

[0049] The enzyme activity determination method is as follows: take 30 μL of enzyme solution, add 150 μL of 2 mM p-nitrophenyl-α-D-pyranogalactoside (pNPG) solution, react at 37°C for 10 min, add 1 mL of 0.2 M boric acid buffer to terminate the reaction, and detect the OD400 of the reaction solution.

[0050] The activity unit of the enzyme is defined as: the amount of enzyme that hydrolyzes pNPG to release 1 μmol of p-nitrophenol in 1 min is one enzyme activity unit (U).

[0051] The calculation method of the enzyme activity recovery rate is as follows: the percentage of the enzyme activity of the cross-linked inclusion bodies to the initial enzyme activity of the control inclusion bodies.

[0052] Depend on Figure 1 It can be seen that after cross-linking with the traditional cross-linking agent glutaraldehyde, the enzyme activity recovery rate is greatly lost, only 45%. The inclusion body of the present invention itself is a white precipitate, and the cross-linked inclusion body obtained after glutaraldehyde treatment is pink. The cross-linked inclusion body obtained after treatment with the four cross-linking agents of the present invention does not change in color, and the enzyme activity recovery rate is 52% to 86%, which is higher than the enzyme activity recovery rate of glutaraldehyde.

[0053] Example 2: Infrared spectroscopy characterization of cross-linked inclusion bodies

[0054] The cross-linked and non-cross-linked inclusion bodies were analyzed by infrared spectroscopy. Figure 2 shown.

[0055] The infrared spectrum analysis method is as follows: the dried sample prepared above and KBr are put into a mortar, mixed and ground, and pressed into a transparent sheet. Record at 400-4000cm -1 The spectrometer model is Thermo Fisher Scientific iS50.

[0056] DMP and DMS are imidoester crosslinkers, which react with primary amines to form amidine bonds; BS3 and BS(PEG)5 are NHS ester crosslinkers, which react with primary amines to form amide bonds. Figure 2 It can be seen that in the infrared spectrum of the uncross-linked inclusion bodies as a control, 1236 cm -1 CN stretching vibration (amide III), 3275 cm -1 NH2 stretching vibration, 858cm -1 is the NH2 distortion vibration. In the cross-linked inclusion body, 1236cm -1 The number of CN stretching vibration (amide III) peaks at 3275 cm -1 The NH2 stretching vibration peak at 858cm -1 The torsional vibration peak of the crosslinked inclusion body showed a tip splitting. The above peak shape change indicated that the chemical environment of NH2 and amide bonds changed, proving that the crosslinker reacted with the primary amine. In addition, due to the introduction of CC and COC by the crosslinker, the crosslinked inclusion body -1 Vibration absorption in the vicinity is significantly enhanced.

[0057] Example 3: Effect of cross-linking conditions on the recovery rate of cross-linked inclusion body enzyme activity

[0058] 1. Effect of crosslinker concentration

[0059] A cross-linking agent solution (DMP, DMS, BS3, BS(PEG)5) was prepared with 50mM potassium phosphate buffer at pH 8.0, and then the cross-linking agent solution was added to the inclusion body suspension obtained in Example 1 to construct a cross-linking system, so that the final concentration of the cross-linking agent in the cross-linking system was 1mM, 2mM, 3mM, 5mM, and 10mM, respectively, and the final protein concentration of the inclusion body was 10mg / mL (about 0.1mM), that is, the ratio of the amount of substance of the inclusion body to the cross-linking agent was 1:10, 1:20, 1:30, 1:50 and 1:100, respectively. The cross-linking system was stirred in an ice water bath for 2 hours, and then centrifuged at 10000g for 5min to remove the supernatant to terminate the cross-linking, and the precipitate was washed 3 times with potassium phosphate buffer to obtain cross-linked inclusion bodies. The activity of α-galactosidase in the control inclusion body and the cross-linked inclusion body was determined, and the enzyme activity recovery rate of the cross-linked inclusion body was calculated. The results are shown in FIG. Figure 3 shown.

[0060] Depend on Figure 3 It can be seen that when the cross-linking agent concentration is 1 mM, that is, the molar ratio of protein to cross-linking agent is 1:10, the enzyme activity recovery rate of the obtained cross-linked inclusion bodies is the highest, at 76% to 98%. When the cross-linking agent concentration increases, the enzyme activity recovery rate decreases significantly.

[0061] 2. Effect of cross-linking time

[0062] A cross-linking agent solution (DMP, DMS, BS3, BS(PEG)5) was prepared with 50mM potassium phosphate buffer at pH 8.0, and then the cross-linking agent solution was added to the inclusion body suspension obtained in Example 1 to construct a cross-linking system. The concentration of the cross-linking agent in the cross-linking system was 1mM, and the final protein concentration of the inclusion body was 10mg / mL (about 0.1mM). The cross-linking system was stirred in an ice water bath for 0.5 to 4 hours, and then the supernatant was removed by centrifugation at 10000g for 5min to terminate the cross-linking. The precipitate was washed 3 times with potassium phosphate buffer to obtain cross-linked inclusion bodies. The activity of α-galactosidase in the control inclusion bodies and the cross-linked inclusion bodies was determined, and the enzyme activity recovery rate of the cross-linked inclusion bodies was calculated. The results are shown in FIG. Figure 4 shown.

[0063] Depend on Figure 4 It can be seen that when the cross-linking time of DMP and DMS is 1h, the enzyme activity recovery rate is the highest, both are 100%, and there is no loss of enzyme activity; BS3 and BS(PEG)5 reach the highest enzyme activity recovery rate at 2h and 1.5h of cross-linking treatment respectively, both are 95%.

[0064] Example 4: Effect of temperature on the activity and stability of cross-linked inclusion bodies

[0065] 1. Selection of the optimal temperature: Take the control inclusion body and the cross-linked inclusion body, suspend them in 30 μL of pH 5.5, 50 mM sodium acetate buffer, add 150 μL of 2 mM pNPG, vortex and shake, react at 30℃~80℃ for 10 min, then add 1 mL of 0.2 M boric acid buffer to terminate the reaction, centrifuge, take the supernatant and detect OD400 with a spectrophotometer. The results are as follows: Figure 5 shown.

[0066] Depend on Figure 5 It can be seen that the optimum temperature of BS(PEG)5 cross-linked inclusion bodies is 50°C, and the optimum temperature of the other cross-linked inclusion bodies is 60°C, which is consistent with the optimum temperature of the control inclusion bodies.

[0067] 2. Determination of temperature stability: Take the control inclusion body and the cross-linked inclusion body, suspend them in 30μL of pH 5.5, 50mM sodium acetate buffer, place them at 30℃~80℃ for 2 hours, then add 150μL of 2mM pNPG, vortex and shake, react at 37℃ for 10min, then add 1mL of 0.2M boric acid buffer to terminate the reaction, centrifuge, take the supernatant and detect OD400 with a spectrophotometer. The results are as follows: Figure 6 shown.

[0068] Depend on Figure 6 It can be seen that the stability of the control inclusion bodies and cross-linked inclusion bodies is good around 30°C. When the temperature continues to rise to 40°C-70°C, the temperature stability of DMP cross-linked inclusion bodies, BS3 cross-linked inclusion bodies and BS(PEG)5 cross-linked inclusion bodies are slightly higher than that of the control inclusion bodies.

[0069] Example 5: Effect of pH on the activity and stability of cross-linked inclusion bodies

[0070] 1. Selection of the optimal pH: Take the control inclusion body and the cross-linked inclusion body, suspend them in 30 μL of citric acid-sodium hydrogen phosphate buffer at pH 4.0-8.0, add 150 μL of 2 mM pNPG, vortex and shake, react at 37°C for 10 min, then add 1 mL of 0.2 M boric acid buffer to terminate the reaction, centrifuge, and take the supernatant to detect OD400 with a spectrophotometer. The results are as follows: Figure 7 shown.

[0071] Depend on Figure 7 It can be seen that the optimal pH of DMP cross-linked inclusion bodies and DMS cross-linked inclusion bodies is 5.0, which is consistent with the optimal pH of control inclusion bodies; the optimal pH of BS3 cross-linked inclusion bodies and BS (PEG) 5 cross-linked inclusion bodies is 6.0.

[0072] 2. Determination of pH stability: Take the control inclusion body and cross-linked inclusion body, suspend them in pH 4.0-8.0 citric acid-sodium hydrogen phosphate buffer, place them in an ice water bath for 48 hours, centrifuge them at 10000g for 5 minutes to remove the supernatant, and replace the buffer with pH 5.5, 50mM sodium acetate buffer. Take 30μL of the control inclusion body and cross-linked inclusion body suspension, add 150μL of 2mM pNPG, vortex and shake, react at 37℃ for 10min, then add 1mL of 0.2M boric acid buffer to terminate the reaction, centrifuge, take the supernatant and detect OD400 with a spectrophotometer. The results are as follows: Figure 8 shown.

[0073] Depend on Figure 8 It can be seen that under the conditions of pH 4.0-5.0, the pH stability of the four cross-linked inclusion bodies was higher than that of the control inclusion body. Under the conditions of pH 7.0-8.0, the BS(PEG)5 cross-linked inclusion body also showed higher stability than that of the control inclusion body.

[0074] Example 6: Comparison of kinetic parameters of control inclusion bodies and cross-linked inclusion bodies

[0075] The control inclusion bodies and cross-linked inclusion bodies were suspended in 30 μL of 50 mM sodium acetate buffer at pH 5.5, and 150 μL of 0.05-2 mM pNPG was added respectively. The mixture was vortexed and reacted at 37°C for 10 min. Then 1 mL of 0.2 M boric acid buffer was added to terminate the reaction. The mixture was centrifuged and the supernatant was used to measure OD400 by spectrophotometer. The K of the enzyme was calculated using GraphPad Prism 8.0 software. m and V max The results are shown in Table 1.

[0076] Table 1

[0077]

[0078] As shown in Table 1, the K m The affinity for the substrate pNPG was enhanced; BS3 cross-linked inclusion body K m Increased, the affinity for the substrate pNPG decreased.

[0079] Example 7: Hydrolysis of soy oligosaccharides in soy milk by cross-linking inclusion bodies

[0080] A method for hydrolyzing soybean oligosaccharides in soybean milk by using cross-linked inclusion bodies comprises the following steps:

[0081] (1) Grind soybeans into powder using a grinder, add hexane (1:1, w / v) to defatted and then dry, suspend the defatted soybean powder in distilled water (1:1, w / v), mix thoroughly, boil at 100°C for 10 min, centrifuge at 10000 g for 10 min to remove protein, and the supernatant is the soymilk solution rich in soybean oligosaccharides;

[0082] (2) Three types of soy oligosaccharide hydrolysis reaction systems were prepared, including 1 mL of soy milk stock solution, 4, 6, and 8 U / mL of cross-linked inclusion bodies, and then reacted at pH 5.5 and 55-65°C for 0.5-3 hours, boiled at 100°C for 10 min to terminate the reaction, centrifuged at 12000 rpm for 10 min, and the supernatant was analyzed by thin layer chromatography and high performance liquid chromatography. The results are as follows: Fig. 9 shown.

[0083] HPLC quantitative analysis results show that each gram of soybean contains 16.9 mg of raffinose and 43.2 mg of stachyose. Fig. 9 It can be seen that after the four cross-linked aggregates were reacted with 4U / mL of α-galactosidase for 1.5 hours or 6U / mL for 1 hour, raffinose and stachyose in soybean oligosaccharides were almost completely hydrolyzed into galactose and sucrose, and the hydrolysis rate was nearly 100%, indicating that the cross-linked inclusion bodies prepared by this patent can efficiently hydrolyze soybean oligosaccharides.

[0084] The analysis method of the thin layer chromatography plate is as follows: after spotting, the sample is developed in a developing agent, a color developer is sprayed, and then baked at 100°C for 3 minutes to color the sugar spot; the developing agent is prepared by mixing propanol, acetic acid and water in a volume ratio of 10:15:1; the color developer is a 3,5-dihydroxytoluene solution with a mass concentration of 0.5% prepared by a sulfuric acid solution with a volume percentage of 20%. The thin plate is scanned and quantitatively analyzed by using the software ImageJ v1.51k (http: / / rsb.info.nih.gov / ij / ).

[0085] The equipment and conditions used for the high performance liquid chromatography analysis are as follows: SHIMADZU LC-20AD liquid chromatograph; SHIMADZU RID-20A differential refractometer; Waters BEH amide analytical column (250 mm×4.6 mm); mobile phase acetonitrile / water (7:3, v / v); flow rate 1.0 mL / min, column temperature 40° C. After the reaction solution was boiled and centrifuged, the supernatant was filtered with a 0.22 μm filter membrane and then injected for analysis.

[0086] Example 8: Cross-linked inclusion body cyclic batch hydrolysis of soybean oligosaccharides in soybean milk

[0087] According to the reaction system and conditions of Example 7, the cross-linked inclusion bodies were mixed with soybean milk stock solution to carry out the first batch of soybean oligosaccharide hydrolysis reaction. The enzyme amount was 6 U / mL. After 1 hour of reaction, the cross-linked inclusion bodies were resuspended in fresh soybean milk stock solution. The second batch reaction was carried out according to Example 7, and the cyclic batch hydrolysis reaction was carried out by analogy.

[0088] The supernatants of all the above batches were subjected to thin layer chromatography and the hydrolysis efficiency was quantitatively analyzed. The reusability of the immobilization method was evaluated based on the hydrolysis efficiency of soybean oligosaccharides after batch reaction and the enzyme activity of cross-linked inclusion bodies. Fig.10 shown.

[0089] Depend on Fig.10 It can be seen that, calculated by relative hydrolysis rate, that is, the efficiency of hydrolyzing soybean oligosaccharides in the first reaction batch is 100%, and the relative hydrolysis rate of DMP cross-linked inclusion bodies and DMS cross-linked inclusion bodies after 10 times of recycling and hydrolyzing soybean oligosaccharides is almost still 100%. After 15 times of recycling and hydrolyzing soybean oligosaccharides, the relative hydrolysis rates of DMP cross-linked inclusion bodies, DMS cross-linked inclusion bodies, BS3 cross-linked inclusion bodies, and BS (PEG) 5 cross-linked inclusion bodies can reach 75% to 86% or more, which is higher than the relative hydrolysis rate of oligosaccharides of glutaraldehyde cross-linked inclusion bodies of 69%. Taking the initial enzyme activity as 100%, the relative enzyme activity of the four cross-linked inclusion bodies after 15 batches of reaction is still retained at 50% to 65%, while the relative enzyme activity of glutaraldehyde cross-linked inclusion bodies is reduced to 40%, which is much lower than the new cross-linked inclusion bodies prepared by the present invention. The above results confirm that the cross-linked inclusion bodies prepared by the present invention have good repeated reaction activity.

Claims

1. A method for preparing cross-linked inclusion bodies, characterized in that: The steps include: (1) Cultivating recombinant Escherichia coli containing the α-galactosidase gene, collecting the inclusion body precipitate and suspending it in potassium phosphate buffer to obtain an inclusion body suspension; (2) adding a cross-linking agent solution to the inclusion body suspension to construct a cross-linking system; Then the cross-linking system was placed in an ice water bath and stirred for cross-linking for 0.5 to 4 hours, the supernatant was removed by centrifugation, and the precipitate was collected and washed to obtain the cross-linked inclusion body; The cross-linking agent is dimethyl pimelimidate dihydrochloride, dimethyl suberimidate dihydrochloride, di(sulfosuccinimidyl) suberate or polyethylene glycol di(sulfosuccinimidyl) suberate; The final concentration of the inclusion body in the cross-linking system is 5-15 mg / mL; the final concentration of the cross-linking agent in the cross-linking system is 1-10 mM.

2. The method for preparing cross-linked inclusion bodies according to claim 1, characterized in that: In step (1), the inclusion bodies are inclusion bodies having α-galactosidase catalytic activity.

3. The method for preparing cross-linked inclusion bodies according to claim 1, characterized in that: In step (2), when the cross-linking agent is dimethyl pimelimidate dihydrochloride and dimethyl suberimidate dihydrochloride, the stirring cross-linking time is 1 hour; when the cross-linking agent is di(sulfosuccinimidyl) suberate, the stirring cross-linking time is 2 hours; when the cross-linking agent is polyethylene glycol di(sulfosuccinimidyl) suberate, the stirring cross-linking time is 1.5 hours.

4. The method for preparing cross-linked inclusion bodies according to claim 1, characterized in that: In step (2), the final concentration of the inclusion bodies is 10 mg / mL.

5. The method for preparing cross-linked inclusion bodies according to claim 1, characterized in that: In step (2), the concentration of the cross-linking agent is 1 mM.

6. Use of the cross-linked inclusion bodies prepared by the method of claim 1 in hydrolyzing soybean oligosaccharides in soybean milk.

7. The use according to claim 6, characterized in that The steps include: (a) Grinding soybeans into powder with a grinder, adding hexane to defatted and then drying, suspending the defatted soybean powder in distilled water, mixing thoroughly, boiling at 100° C. for 10 min, centrifuging at 10000 g for 10 min, and collecting the supernatant to obtain a soymilk solution containing soybean oligosaccharides; (b) adding the cross-linked inclusion bodies to a soymilk stock solution containing soy oligosaccharides to prepare a soy oligosaccharide hydrolysis reaction system; then reacting the hydrolysis reaction system at pH 5-6 and 55-65° C. for 0.5-3 hours, centrifuging at 10,000 g for 10 minutes, and collecting the supernatant to obtain soymilk after the soy oligosaccharide hydrolysis.

8. The use according to claim 7, characterized in that In step (a), the mass volume ratio of the soybean to hexane is 1:1, in g / mL; the mass volume ratio of the defatted soybean powder to distilled water is 1:10, in g / mL.

9. The use according to claim 7, characterized in that In step (b), the soybean oligosaccharide hydrolysis reaction system includes 1 mL of soybean milk stock solution and 4-8 U / mL of cross-linked inclusion bodies.

10. The use according to claim 9, characterized in that The cross-linked inclusion bodies were 6 U / mL and reacted at 65° C. for 1 hour.

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