Immobilized sucrose isomerase and aminated carbon nanotube covalent immobilization method thereof

Abstract

The invention provides immobilized sucrose isomerase and an aminated carbon nanotube covalent immobilization method thereof. The method comprises the following steps: screening out sucrose isomerase with a nucleotide sequence as shown in SEQ ID NO.1; the method comprises the following steps: carrying out fermentation culture on genetically engineered bacteria containing a nucleotide sequence as shown in SEQ ID NO.1 to obtain wet thalli, and carrying out cell disruption and centrifugation to obtain a sucrose isomerase crude enzyme solution; the preparation method comprises the following steps: treating an aminated carbon nanotube in glutaraldehyde to obtain a pretreated aminated carbon nanotube; and taking a sucrose isomerase crude enzyme solution, adding the pretreated aminated carbon nanotubes, and carrying out oscillation immobilization to obtain the immobilized sucrose isomerase. The immobilized sucrose isomerase disclosed by the invention can be used for catalytically preparing isomaltulose, has better activity, good pH tolerance and thermal stability and high conversion rate, can continuously react for 9 batches, and has the conversion rate of more than 99% in each batch.

Description

Technical Field

[0001] The invention relates to immobilized sucrose isomerase and a method for covalently fixing the same to amino-carbon nanotubes. Background Art

[0002] Isomaltulose is a natural sugar found in products such as sugar cane and honey, and is also an internationally recognized safe substitute for sucrose. As a reducing sugar, isomaltulose can be hydrogenated and reduced to produce the functional edible sugar alcohol isomalt. Both have similar physical properties and taste to sucrose, and are anti-caries, do not cause blood sugar and insulin to rise, improve brain function, enhance human memory, inhibit fat accumulation and prevent obesity, so they have broad application prospects in the pharmaceutical, food and other industries.

[0003] The natural isomaltulose content is extremely low, and it is impossible to meet the growing market demand by extraction. Therefore, it is necessary to develop an artificial synthesis method to achieve the large-scale preparation of isomaltulose. At present, the production method of isomaltulose at home and abroad is mainly biological method, that is, sucrose isomerase is used to catalyze sucrose to produce isomaltulose. This technology has mild reaction conditions and high catalytic efficiency, and has become the main way to produce isomaltulose industrially. Zhang et al. heterologously expressed sucrose isomerase from P. dispersa UQ68J in Yarrowia lipolytica and applied it to the synthesis of isomaltulose. When the substrate sucrose concentration is 600g / L, the maximum concentration of isomaltulose can reach 572.1g / L. (Zhang P, Wang ZP, Sheng J, et al. High and efficient isomaltulose production using an engineered Yarrowia lipolyticastrain [J]. Bioresour Technol, 2018, 265: 577-580.). Zhang Rongzhen et al. mutated sucrose isomerase and applied the recombinant sucrose isomerase mutant to isomaltulose production, with improved yield and conversion rate. The fermentation broth supernatant was used as a crude enzyme solution to add to the reaction system. When the sucrose concentration was 650g / L, the yield was 98.7% and the output was 641.6g, which could efficiently synthesize isomaltulose and obtain high yield (CN117821433A). However, in the production of isomaltulose, the enzyme has poor stability, which affects the catalytic efficiency. The use of free enzyme or bacterial cell for production and fermentation will increase the difficulty of product separation. Immobilization can achieve multi-batch use and reduce the cost of enzyme preparations.

[0004] Enzyme immobilization technology combines free enzymes with corresponding carriers through physical or chemical methods, thereby enhancing the stability of the enzyme and separating the enzyme from the substrate to achieve the purpose of reuse and reduce costs. The carriers of immobilized enzymes mainly include organic polymer carriers, inorganic carriers and composite carriers. Among them, commonly used inorganic carriers mainly include porous glass, graphite, metal oxides, molecular sieves, etc. Zhang et al. used graphene oxide as a carrier and immobilized free sucrose isomerase (ErSIase) by physical adsorption. With 600 g / L sucrose as a substrate, the reaction was carried out at 40 ° C and pH 6.0 for 180 min. The conversion rate of sucrose to isomaltulose catalyzed by ErSlase-GO reached 95.3% (Zhang, F, Cai, X, Cheng F, et al. Immobilization of Sucrose Isomerase from Erwinia sp. with Graphene Oxide and Its Application in Synthesizing Isomaltulose. Appl Biochem Biotechnol 194, 709–724 (2022)). Hu Yi et al. used magnetic diatomite composites to immobilize lipase to catalyze the synthesis of plant sterol ester compounds. After 8 repeated uses, the enzyme immobilized by magnetic nanocomposite materials can still maintain good activity, retaining 80% to 90% of the initial activity (CN116926057A). This type of carrier material has a large specific surface area, high mechanical strength and strong adsorption capacity. The immobilized enzymes formed by immobilizing the enzymes through different immobilization strategies often have unique advantages such as high catalytic activity, good thermal stability, good organic solvent tolerance and recyclability. Therefore, the use of a strong adsorption carrier to immobilize sucrose isomerase is of great significance for the industrial application of biosynthesis of isomaltulose. Summary of the invention

[0005] The invention aims to provide a highly active immobilized sucrose isomerase and a method for covalently fixing the same using amino-modified carbon nanotubes.

[0006] The technical solution adopted by the present invention is:

[0007] An immobilized sucrose isomerase is prepared according to the following method:

[0008] (1) screening out a sucrose isomerase whose nucleotide sequence is shown in SEQ ID NO.1;

[0009] (2) Fermenting and culturing the genetically engineered bacteria containing the nucleotide sequence shown in SEQ ID NO.1, suspending the obtained wet bacteria in a pH 4.0-9.0 buffer to obtain a bacterial suspension, disrupting the bacterial suspension and centrifuging it, and taking the supernatant to obtain a crude sucrose isomerase enzyme solution;

[0010] (3) treating the amino-treated carbon nanotubes in a glutaraldehyde solution having a volume concentration of 0.1 to 1% for 1 to 4 hours to obtain pretreated amino-treated carbon nanotubes;

[0011] (4) Take the crude sucrose isomerase solution, add the pretreated amino carbon nanotubes, shake and fix at 1000-1200 rpm and 20-45° C. for 0.5-5 h, centrifuge, rinse with buffer, and centrifuge again to remove the buffer to obtain immobilized sucrose isomerase.

[0012] The present invention also provides a method for covalently fixing sucrose isomerase on aminated carbon nanotubes, the method comprising the following steps:

[0013] (1) screening out a sucrose isomerase whose nucleotide sequence is shown in SEQ ID NO.1;

[0014] (2) Fermenting and culturing the genetically engineered bacteria containing the nucleotide sequence shown in SEQ ID NO.1, suspending the obtained wet bacteria in a pH 4.0-9.0 buffer to obtain a bacterial suspension, and crushing and centrifuging the bacterial suspension to obtain a crude sucrose isomerase enzyme solution;

[0015] (3) treating the amino-modified carbon nanotubes in glutaraldehyde having a volume concentration of 0.1 to 1% for 1 to 4 hours to obtain pretreated amino-modified carbon nanotubes;

[0016] (4) Take the crude sucrose isomerase solution, add the pretreated amino carbon nanotubes, shake and fix at 1000-1200 rpm and 20-45° C. for 0.5-5 h, centrifuge, rinse with buffer, and centrifuge again to remove the buffer to obtain immobilized sucrose isomerase.

[0017] Furthermore, the genetically engineered bacteria containing the nucleotide sequence shown in SEQ ID NO.1 are generally constructed by the following method: constructing an expression plasmid containing the nucleotide sequence shown in SEQ ID NO.1; transforming the expression plasmid into the host bacterium Escherichia coli to obtain the genetically engineered bacteria containing the nucleotide sequence shown in SEQ ID NO.1.

[0018] In the step (2), the buffer is a citric acid-disodium hydrogen phosphate buffer having a pH value of 4.0 to 9.0, preferably a pH value of 5.0.

[0019] The wet bacterial cell concentration in the bacterial suspension is 5 to 15 g / L, preferably 7.5 to 15 g / L.

[0020] Furthermore, the fermentation culture step in step (2) is preferably:

[0021] The genetically engineered bacteria containing the nucleotide sequence shown in SEQ ID NO.1 were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance, cultured at 37°C, 180 rpm for 12 h, and then inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance at a volume concentration of 1% inoculum, and cultured at 37°C, 180 rpm until the bacterial OD 600 When the cell culture temperature reaches 6-8, add IPTG with a final concentration of 0.1 mM, induce culture at 25°C for 11 hours, centrifuge the fermentation broth at 4°C and 8000 rpm for 10 minutes, discard the supernatant, collect the precipitate, and obtain the wet cells of sucrose isomerase genetically engineered bacteria.

[0022] As further preferred, in step (3), the volume concentration of the glutaraldehyde solution is 0.2%.

[0023] In the step (3), the mass amount of the amino-modified carbon nanotubes is 2.5 to 25 mg / mL, preferably 5 to 8 mg / mL, based on the volume of the glutaraldehyde solution.

[0024] Preferably, in step (4), the concentration of the sucrose isomerase solution is 3-9 mg / mL, more preferably 4.5 mg / mL.

[0025] As further preferred, in the step (4), the amount of the amino-modified carbon nanotubes added is 1-10 mg / mL, and more preferably the amount of the amino-modified carbon nanotubes added is 2 mg / mL.

[0026] Preferably, in step (4), the reaction temperature is 20-45°C, preferably 25°C.

[0027] In the step (4), the reaction time is 0.5 to 5 h, preferably 1.5 h.

[0028] In the step (4), the pH value of the buffer solution is 4.0 to 9.0, preferably 5.0 to 6.0.

[0029] The present invention also provides a method for preparing isomaltulose by using the immobilized sucrose isomerase as a catalyzer, wherein the method comprises: using the immobilized sucrose isomerase covalently fixed to amino carbon nanotubes as a catalyst, using sucrose as a substrate, reacting in a pH 6.0 citric acid-disodium hydrogen phosphate buffer at a temperature of 20 to 45° C. for 1 to 3 hours to prepare isomaltulose.

[0030] The concentration of the sucrose is 100-500 g / L.

[0031] The dosage of immobilized sucrose isomerase is generally 10 to 20 mg (calculated on dry weight).

[0032] The concentration of the citric acid-disodium hydrogen phosphate buffer is preferably 50 mM.

[0033] The reaction is preferably carried out under stirring at 1000-1200 rpm.

[0034] The beneficial effects of the present invention are mainly reflected in that the immobilized sucrose isomerase of the present invention has good activity, good pH tolerance and thermal stability, high conversion rate, easy separation, reusability, good recyclability, and is used to prepare isomaltulose, and 9 batches are continuously reacted, and the conversion rate of each batch is greater than 99%, thereby increasing its service life, reducing the catalyst cost, and having great market application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the reaction formula for the synthesis of isomaltulose catalyzed by sucrose isomerase.

[0036] Figure 2 This is a comparison of the pH tolerance of the immobilized enzyme and the free enzyme.

[0037] Figure 3 This is a comparison of the thermal stability of the immobilized enzyme and the free enzyme.

[0038] Figure 4 This is a graph showing the effect of substrate concentration on the conversion rate of immobilized enzyme catalyzed reaction.

[0039] Figure 5 Bar graph showing the conversion rate of the batch reaction of isomaltulose synthesis by immobilized sucrose isomerase. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0041] Example 1: Cultivation of Sucrose Isomerase Genetically Engineered Bacterial Cells

[0042] A sucrose isomerase PdSIase (>AY223549.1Sucrose isomerase gene from P. dispersa UQ68J) with high catalytic activity was used as a gene probe to search for homologous sequences in the GenBank database, remove protein sequences with homology greater than 80%, and perform alignment analysis on these sequences to screen enzyme genes with conserved sequence characteristic structures of sucrose isomerase. The optimized nucleotide sequence of sucrose isomerase is shown in SEQ ID NO.1.

[0043] According to the codon preference of the expression host E.coli BL21 (DE3), the codons were optimized, and the optimized target gene fragments were inserted into the middle of the NcoⅠ and XhoⅠ restriction enzyme sites in the vector pET28a (+) to obtain the recombinant plasmid; 3 μL of the plasmid to be transformed was added to the thawed competent cells under sterile conditions, slowly mixed and placed on ice for 30 minutes, and the competent cells without the recombinant plasmid were set as the control group 1. After heat shock in a 42℃ water bath for 90 seconds, immediately put in an ice bath at 0℃ for 5 minutes. Add 800 μL of LB medium without antibiotics under sterile conditions, mix well and culture at 37℃, 180rpm for 40 minutes. Centrifuge at 8000rpm for 1 minute, discard 700 μL of supernatant, and resuspend the precipitate with the remaining culture medium. At 37℃, spread the resuspended bacterial solution on an LB plate with kanamycin resistance and culture overnight, and set the LB plate without antibiotics as the control group 2. Colony PCR was performed to verify the results, and positive transformants were selected for sequencing. The genetically engineered bacteria containing sucrose isomerase were successfully obtained through sequencing.

[0044] The genetically engineered bacteria were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance, cultured at 37°C, 180 rpm for 12 h, and then inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance at a volume concentration of 1%, and cultured at 37°C, 180 rpm until the bacterial OD 600 When the cell culture temperature reaches 6-8, add IPTG with a final concentration of 0.1mM, induce culture at 25℃ for 11h, centrifuge the fermentation broth at 4℃ and 8000rpm for 10min, discard the supernatant, collect the precipitate, and obtain the wet bacterial cells of sucrose isomerase genetic engineering bacteria. The formula of LB liquid culture medium is: peptone 20g / L, yeast powder 15g / L, NaCl 10g / L.

[0045] Example 2: Preparation of crude sucrose isomerase solution

[0046] The bacterial cells were suspended in a pH 6.0 citric acid-sodium hydrogen phosphate buffer (pH 6.0) at a concentration of 5 g / L and ultrasonically disrupted. After ultrasonic disruption, the cells were centrifuged at a speed of 12000 rpm / min for 10 min and the supernatant was taken as the crude sucrose isomerase enzyme solution.

[0047] Example 3: Detection method of sucrose and isomaltulose

[0048] Detection of substrate conversion rate and product yield: The liquid chromatograph was Agilent 1260 InfinityⅡLC, the mobile phase was a mixture of acetonitrile and water in a volume ratio of 3:1, and the flow rate was 1.0 mL / min; the chromatographic column was Elite Supersil NH2 plus 5um 4.6mm*250mm; the detector was a differential refractometer; the injection volume was set to 10 μl; the column temperature was 30°C, and the detector temperature was 31.5°C.

[0049] Example 4: Detection of Sucrose Isomerase Activity

[0050] Enzyme activity detection catalytic system: 10 mg (dry weight) of immobilized sucrose isomerase was added to 1 mL of 50 mM, pH 6.0 citric acid-Na2HPO4, the substrate sucrose concentration in the reaction system was 200 g / L, the reaction was carried out at 40°C for 5 min, and the reaction was terminated by boiling water bath treatment for 10 min. Centrifuge at 12000 rpm for 5 min, collect the supernatant and dilute it, and filter it with a filter membrane (0.22 μm) before HPLC detection.

[0051] Enzyme activity definition: At 40°C, the amount of enzyme required to catalyze the production of 1 μmol of product isomaltulose within 1 minute is 1 unit of enzyme activity, expressed as U. The definition of unit enzyme activity is as follows

[0052]

[0053] Relative enzyme activity: The activity of an equal amount of free enzyme is taken as 100%.

[0054] Example 5: Study on the isomaltulose selectivity of recombinant sucrose isomerase

[0055] Four 10g / L recombinant strains containing sucrose isomerase (nucleotide sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, and the corresponding amino acid sequences are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8) were screened and reacted for 0.5h at pH 6.0 and 40°C to catalyze 100g / L sucrose to synthesize isomaltulose. As shown in Table 1, the enzyme activity of SEQ ID NO.1 is much higher than that of the other three sucrose isomerases, which is 29.24U / g wet bacteria. The substrate sucrose can be completely converted in 0.5h, and the isomaltulose selectivity is also the highest, which is 81.65%. Therefore, sucrose isomerase SEQ ID NO.1 was selected for subsequent research.

[0056] Table 1: Isomaltulose selectivity of different recombinant sucrose isomerases

[0057]

[0058] Example 6: Screening of carrier materials

[0059] Take 10 mg of activated carbon, silicon dioxide, β-molecular sieve, amino carbon nanotubes, carbon nanotubes, and diatomaceous earth respectively, add 2 mL of 0.2% glutaraldehyde and treat for 3 hours to obtain a pretreated carrier. Take the 3 mg / mL crude enzyme solution obtained in Example 2, add 1 mg / mL of different pretreated carriers, and oscillate on an oscillator for 5 hours at 25°C and 180 rpm; immobilization is completed; centrifuge to remove the supernatant, wash twice with 50 mM, pH 6.0 citric acid-Na2HPO4 buffer solution, and centrifuge to remove excess water to obtain immobilized sucrose isomerase. The relative enzyme activity is determined by the method of Example 4. It can be seen from Table 2 that the immobilized enzyme with amino carbon nanotubes as the carrier has the largest relative enzyme activity, so the carrier is selected as amino carbon nanotubes.

[0060] Table 2: Enzyme activity of sucrose isomerase immobilized on different carriers

[0061]

[0062] Example 7: Optimization of glutaraldehyde concentration

[0063] Take 10 mg of amino carbon nanotubes, add 2 mL of 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1% glutaraldehyde for 3 hours, and obtain pretreated amino carbon nanotubes. Take the 3 mg / mL crude enzyme solution obtained in Example 2, add 1 mg / mL amino carbon nanotubes pretreated with different glutaraldehyde concentrations, and oscillate on an oscillator for 5 hours at 25°C and 180 rpm; immobilization is completed; centrifuge to remove the supernatant, wash twice with 50 mM, pH 6.0 citric acid-Na2HPO4 buffer solution, centrifuge to remove excess water, and obtain immobilized sucrose isomerase. The relative enzyme activity is determined by the method of Example 3. It can be seen from Table 3 that the relative enzyme activity reaches the maximum when the glutaraldehyde concentration is 0.2%, so the optimal glutaraldehyde concentration is 0.2%.

[0064] Table 3: Enzyme activity of sucrose isomerase immobilized on carbon nanotubes treated with different glutaraldehyde concentrations

[0065]

[0066] Example 8: Optimization of buffer pH

[0067] 5 g / L bacterial suspensions were prepared from buffers of pH 4.0, 5.0, 6.0, citric acid-Na2HPO4; pH 7.0, 8.0 Tris-HCl; pH 9.0, Gly-NaOH, and ultrasonically disrupted according to the method of Example 2. After ultrasonic disruption, the suspensions were centrifuged at 12000 rpm / min for 10 min, and 10 mL of supernatant was taken, which was the crude sucrose isomerase solution (3 mg / mL). Then, 1 mg / mL of amino-modified carbon nanotubes pretreated with 0.2% glutaraldehyde were added, and the suspensions were shaken on an oscillator at 25°C and 180 rpm for 5 h. Immobilization was completed. The supernatant was removed by centrifugation, and the suspensions were washed twice with 50 mM, pH 6.0 citric acid-Na2HPO4 buffer solution. After the excess water was removed by centrifugation, the immobilized sucrose isomerase was obtained. The relative enzyme activity was determined by the method of Example 4. It can be seen from Table 4 that the activity of sucrose isomerase is highest when it is immobilized at pH 5.0, so the optimal immobilization pH is pH 5.0.

[0068] Table 4: Effect of pH on the immobilization of sucrose isomerase during sucrose isomerase immobilization

[0069]

[0070]

[0071] Example 9: Optimization of carrier addition amount

[0072] Take the crude enzyme solution (3 mg / mL) obtained in Example 2, and then add 1, 2, 3, 4, 6, 8, 10 mg / mL of pretreated amino carbon nanotubes (dry weight), respectively, and shake the oscillator for 5 hours at 25°C and 180 rpm; immobilization is completed; centrifuge to remove the supernatant, wash twice with 50mM, pH 6.0 citric acid-Na2HPO4 buffer solution, and centrifuge to remove excess water to obtain immobilized sucrose isomerase. The relative enzyme activity was determined by the method of Example 4. The results are shown in Table 5. When the carrier addition amount is 2 mg / mL, the enzyme activity reaches the maximum, so the optimal carrier addition amount is 2 mg / mL.

[0073] Table 5: Effect of different carrier addition amounts on the activity of immobilized sucrose isomerase

[0074]

[0075] Example 10: Optimization of enzyme concentration

[0076] The wet bacteria were resuspended at 5g / L, 7.5g / L, 10g / L, 12.5g / L, and 15g / L to obtain bacterial suspensions, and then crude enzyme solution was obtained by the method of Example 2, and the treated amino carbon nanotubes (2mg / mL) were added, and the oscillator was oscillated for 5h at 25°C and 180rpm; immobilization was completed; centrifugation was performed and washed twice with 50mM, pH 6.0 citric acid-Na2HPO4 buffer solution. The relative enzyme activity was determined by the method of Example 4. It can be seen from Table 6 that the relative enzyme activity reached the maximum when it was 7.5g / L, so the optimal initial bacterial concentration was 7.5g / L.

[0077] Table 6: Effect of different enzyme concentrations on the activity of immobilized sucrose isomerase

[0078]

[0079] Example 11: Optimization of the temperature of sucrose isomerase immobilized on amino carbon nanotubes

[0080] Take the crude enzyme solution (4.5 mg / mL) obtained in Example 2, and then add the pretreated amino carbon nanotubes (dry weight) (2 mg / mL) respectively, and oscillate on an oscillator for 5 hours at 20°C, 25°C, 30°C, 35°C, 40°C, and 45°C 180rpm; end the immobilization; centrifuge to remove the supernatant, wash twice with 50mM, pH 6.0 citric acid-Na2HPO4 buffer solution, centrifuge to remove excess water, and obtain immobilized sucrose isomerase. The relative enzyme activity was determined by the method of Example 4. The results are shown in Table 7. The enzyme activity reaches the maximum at a temperature of 25°C, so the optimal immobilization temperature is 25°C.

[0081] Table 7: Effects of different temperatures on the activity of immobilized sucrose isomerase

[0082]

[0083] Example 12: Optimization of the time for immobilizing sucrose isomerase on amino-modified carbon nanotubes

[0084] Take the crude enzyme solution (4.5 mg / mL) obtained in Example 2, and then add (2 mg / mL) of pre-treated amino carbon nanotubes (dry weight). Under the conditions of 25°C and 180rpm, the oscillator was oscillated for 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 4h, and 5h; immobilization was completed; the supernatant was removed by centrifugation, and the solution was washed twice with 50mM, pH 6.0 citric acid-Na2HPO4 buffer solution, and the excess water was removed by centrifugation to obtain immobilized sucrose isomerase. The enzyme activity was determined by the method of Example 4, and it was obtained from Table 8 that the enzyme activity was the highest when the immobilization time was 1.5h. Therefore, the optimal immobilization time is 1.5h.

[0085] Table 8: Effect of immobilization time on the activity of immobilized sucrose isomerase

[0086]

[0087] Example 13: pH tolerance of immobilized sucrose isomerase

[0088] Free enzyme and immobilized sucrose isomerase were prepared according to the optimal conditions optimized in Example 2 and Examples 5 to 12, respectively. Specifically, immobilized sucrose isomerase was prepared according to the following steps:

[0089] 10 mg of amino-modified carbon nanotubes were added to 2 mL of 0.2% glutaraldehyde and treated for 3 h to obtain pretreated amino-modified carbon nanotubes;

[0090] The crude enzyme solution (4.5 mg / mL, pH 5.0) obtained in Example 2 was added with 2 mg / mL pretreated amino carbon nanotubes, and the mixture was shaken and fixed for 1.5 h at 25°C and 180 rpm to complete the immobilization. The supernatant was removed by centrifugation, and the mixture was washed twice with 50 mM, pH 6.0 citric acid-Na2HPO4 buffer solution. After centrifugation to remove excess water, immobilized sucrose isomerase was obtained.

[0091] 1 mL of pH 4.0, 5.0, 6.0, 7.0, and 8.0 (50 mM citric acid-Na2HPO4) buffer systems were prepared, and 2 mg of immobilized sucrose isomerase (dry weight) was added to each of them. The samples were incubated at 4°C for 4 h, and the residual enzyme activity was determined using the method of Example 3, with the initial enzyme activity of the untreated free enzyme and the immobilized enzyme being 100%. Figure 2 It can be seen that with the increase of pH, the activities of the immobilized enzyme and the free enzyme first increased and then decreased. At pH 4 and pH 6-8, the enzyme activity of the immobilized enzyme was significantly higher than that of the free enzyme.

[0092] Example 14: Temperature stability of immobilized sucrose isomerase

[0093] Free enzyme and immobilized sucrose isomerase were prepared according to the optimal process of Example 2 and Example 13. The immobilized enzyme and free enzyme were placed in a constant temperature water bath at 20°C, 25°C, 30°C, 35°C, 40°C, and 45°C for 4 hours, and the residual enzyme activity was determined by the method of Example 3, with the initial enzyme activity of the untreated free enzyme and immobilized enzyme being 100%. Figure 3 It can be seen that with the increase of temperature, the thermal stability of both immobilized enzyme and free enzyme showed a downward trend. The temperature stability of immobilized enzyme was significantly higher than that of free enzyme. At 45°C, the residual enzyme activity of immobilized enzyme was about 35.66%, while the residual enzyme activity of free enzyme was only 11.97%.

[0094] Example 15: Effect of substrate concentration on the catalytic reaction of immobilized sucrose isomerase

[0095] 10mL system: Immobilized sucrose isomerase (dry weight 20mg) was prepared according to the optimal process of Example 2 and Example 13, and used for catalytic reaction. The buffer system was 50mM, pH 6.0 citric acid-disodium hydrogen phosphate buffer. Sucrose of different concentrations (100g / L, 200g / L, 300g / L, 400g / L, 500g / L, 600g / L) was added respectively. The reaction was stirred at 180rpm and 40℃ for 3h. Samples were taken at regular intervals. The reaction was terminated by boiling water bath treatment for 10min. HPLC detection was performed after dilution and filtration. Figure 4 It can be seen that when the sucrose concentration increased to 500 g / L, the conversion rate reached 99.43%.

[0096] Example 16: Amplified reaction of sucrose catalyzed by immobilized sucrose isomerase

[0097] 1L system: Immobilized sucrose isomerase (dry weight 20 mg) was prepared according to the optimal process of Example 2 and Example 13, respectively, and used for catalytic reaction. The buffer system was 50 mM, pH 6.0 citric acid-disodium hydrogen phosphate buffer, the sucrose substrate concentration was 500 g / L, and the reaction was stirred at 180 rpm and 40°C for 3 h. Samples were taken at regular intervals, and the reaction was terminated by boiling water bath treatment for 10 min. After dilution and filtration, HPLC detection was performed. The sucrose conversion rate reached 99.12%.

[0098] Example 17: Batch reaction of immobilized sucrose isomerase

[0099] 10mL system: Immobilized sucrose isomerase (dry weight 20mg) was prepared according to the optimal process of Example 2 and Example 13 respectively, and used for catalytic reaction. The concentration of sucrose was 200g / L, and the buffer system was 50mM, pH6.0 citric acid-disodium hydrogen phosphate buffer. The reaction was stirred at 180rpm and 40℃ for 3h. A certain amount of reaction solution was taken and treated in a boiling water bath for 10min to terminate the reaction. After dilution and filtration, HPLC detection was performed. The conversion rate of sucrose was 99%. The immobilized enzyme was recovered by centrifugation and washed twice by centrifugation with 50mM, pH6.0 citric acid-disodium hydrogen phosphate buffer, and a batch reaction was performed. The above batch reaction was repeated until the conversion rate was lower than 80%. By Figure 5 It can be seen that the conversion rate of the 9 batches did not decrease and could reach about 99%. When the reaction was carried out to 10 batches, the conversion rate could reach about 80%.

Claims

1. A method for covalently fixing sucrose isomerase on amino-modified carbon nanotubes, characterized in that The method comprises the following steps: (1) screening out a sucrose isomerase whose nucleotide sequence is shown in SEQ ID NO.1; (2) Fermenting and culturing the genetically engineered bacteria containing the nucleotide sequence shown in SEQ ID NO.1 to obtain wet bacteria, suspending the wet bacteria in a pH 4.0-9.0 buffer to obtain a bacterial suspension, and crushing and centrifuging the bacterial suspension to obtain a crude sucrose isomerase enzyme solution; (3) treating the amino-modified carbon nanotubes in glutaraldehyde having a volume concentration of 0.1 to 1% for 1 to 4 hours to obtain pretreated amino-modified carbon nanotubes; (4) Take the crude sucrose isomerase solution, add the pretreated amino carbon nanotubes, shake and fix at 1000-1200 rpm and 20-45° C. for 0.5-5 h, centrifuge, rinse with buffer, and centrifuge again to remove the buffer to obtain immobilized sucrose isomerase.

2. The method according to claim 1, characterized in that The genetically engineered bacteria containing the nucleotide sequence shown in SEQ ID NO.1 is constructed by the following method: constructing an expression plasmid containing the nucleotide sequence shown in SEQ ID NO.1; transforming the expression plasmid into the host bacterium Escherichia coli to obtain the genetically engineered bacteria containing the nucleotide sequence shown in SEQ ID NO.

1.

3. The method according to claim 1, characterized in that In the step (2), the wet bacterial cell concentration in the bacterial suspension is 5 to 15 g / L.

4. The method according to claim 1, characterized in that In the step (2), the buffer solution is a citric acid-disodium hydrogen phosphate buffer solution with a pH value of 5.0; in the step (3), the volume concentration of the glutaraldehyde solution is 0.2%.

5. The method according to claim 1, characterized in that In the step (3), the mass amount of the amino-modified carbon nanotubes is 2.5-25 mg / mL based on the volume of the glutaraldehyde solution.

6. The method according to claim 1, characterized in that In the step (4), the concentration of the sucrose isomerase solution is 3-9 mg / mL, and the amount of the amino-modified carbon nanotubes added is 1-10 mg / mL.

7. The method according to claim 1, characterized in that In the step (4), the reaction temperature is 25° C. and the reaction time is 1.5 h.

8. Immobilized sucrose isomerase prepared by the method according to any one of claims 1 to 7.

9. A method for preparing isomaltulose catalyzed by using the immobilized sucrose isomerase as claimed in claim 8, characterized in that The method comprises the following steps: using immobilized sucrose isomerase covalently fixed by amino carbon nanotubes as a catalyst, using sucrose as a substrate, reacting in a pH 6.0 citric acid-disodium hydrogen phosphate buffer at a temperature of 20-45° C. for 1-3 hours to prepare isomaltulose.

10. The method according to claim 9, characterized in that The concentration of the sucrose is 100-500 g / L; the amount of the immobilized sucrose isomerase is 10 mg calculated by dry weight.

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