Sucrose isomerase mutant and efficient synthesis of isomaltulose thereof

Abstract

The application discloses a sucrose isomerase mutant and efficient synthesis of isomaltulose, and belongs to the technical field of genetic engineering and enzyme engineering. The mutant M282S / M441F / A470E has a specific activity of 871.3 U / mg at 30 DEG C and pH 6.0, which is 1.33 times higher than that of the wild type, and the stability is improved, and the half-life is about 120 min at 45 DEG C. The recombinant sucrose isomerase mutant is applied to isomaltulose production, the yield and conversion rate are improved, the supernatant of fermentation liquor is used as a crude enzyme solution and is added into a reaction system, when the sucrose concentration is 650 g / L, the yield is 98.7%, and the yield is 641.6 g, so that isomaltulose can be efficiently synthesized and high yield is obtained. The application provides a high-quality sucrose isomerase mutant for the biological catalytic synthesis of isomaltulose, and the yield of prepared isomaltulose reaches the highest level reported at home and abroad.

Description

Technical Field

[0001] This invention relates to a sucrose isomerase mutant and its efficient synthesis of isomaltulose, belonging to the fields of genetic engineering and enzyme engineering technology. Background Technology

[0002] Isomaltulose, also known as 6-O-glucopyranosyl-D-fructose or palaginose, is an isomer of sucrose. Isomaltulose is only half as sweet as sucrose, has no off-flavor, and its sweetness is stable. It is not broken down in the mouth or stomach after consumption, thus preventing tooth decay, effectively reducing the occurrence of oral diseases, and significantly reducing the number of Streptococcus variants in saliva. After entering the small intestine, isomaltulose is slowly hydrolyzed and absorbed by sucrase and isomaltase, without rapid changes in insulin and blood sugar levels. This can effectively prevent diabetes, boost energy and combat fatigue, prevent diarrhea, and accelerate fat metabolism.

[0003] Isomaltulose is naturally found in small amounts in foods such as beets and honey, but its low content and difficulty in extraction make it difficult to meet market demand. Currently, isomaltulose can be prepared using chemical methods and biotransformation. Chemical methods are costly and polluting, while microbial transformation and extraction are difficult and have low production intensity. Enzymatic methods can effectively avoid these problems. Currently, sucrose isomerase is the most effective enzyme for the industrial production of isomaltulose using biological enzymatic methods.

[0004] Sucrose isomerase (SIase, EC 5.4.99.11) is capable of isomerizing sucrose into isomaltulose and trehalulose. The isomerization products are affected by temperature and pH. Sucrose isomerases are mainly found in bacteria, such as *Erwinia rhapontici*, *Serratia plymuthica*, *Klebsiella sp.*, and *Pantoea dispersa*. However, sucrose isomerases from different sources exhibit problems such as poor stability, low conversion rates, and the production of byproducts.

[0005] Bacillus subtilis possesses a relatively clear genetic metabolic pathway, is non-pathogenic, has no codon bias, has a short fermentation cycle, is suitable for high-cell-density culture, and has a strong protein secretion capacity that can directly secrete proteins extracellularly, making it a commonly used food-grade strain. The efficient extracellular expression of recombinant sucrose isomerase in Bacillus subtilis and the efficient synthesis of isomaltulose are of great significance for its industrial production. Summary of the Invention

[0006] Since the synthesis of isomaltulose catalyzed by sucrose isomerase has problems such as poor enzyme stability, low conversion rate, and byproducts, the technical problem to be solved by this invention is how to synthesize isomaltulose efficiently and at high concentration.

[0007] This invention provides a sucrose isomerase mutant, which is obtained by mutating at least one of the 282nd, 441st, and 470th positions of a sucrose isomerase; the sucrose isomerase is a sucrose isomerase with the amino acid sequence shown in SEQ ID NO.1, or a sucrose isomerase having at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the sequence shown in SEQ ID NO.1.

[0008] In one embodiment of the present invention, the mutant is obtained by mutating the 282nd and / or 441st and / or 470th amino acids of the sucrose isomerase with an amino acid sequence as shown in SEQ ID NO.1.

[0009] In one embodiment of the present invention, the mutant is obtained by mutating methionine at position 282 of the sucrose isomerase, as shown in SEQ ID NO.1, to serine.

[0010] In one embodiment of the present invention, the mutant is obtained by mutating methionine at position 441 of the sucrose isomerase, as shown in SEQ ID NO.1, to phenylalanine.

[0011] In one embodiment of the present invention, the mutant is obtained by mutating alanine at position 470 of the sucrose isomerase, as shown in SEQ ID NO.1, to glutamic acid.

[0012] In one embodiment of the present invention, the mutant is obtained by mutating methionine at position 282 of the sucrose isomerase shown in SEQ ID NO.1 to serine, methionine at position 441 to phenylalanine, and alanine at position 470 to glutamic acid; named: PdSlase M282S / M441F / A470E The amino acid sequence of the mutant is shown in SEQ ID NO.2.

[0013] In one embodiment of the present invention, the nucleotide sequence encoding the sucrose isomerase parent enzyme is shown in SEQ ID NO. 3, encoding the PdSlase. M282S / M441F / A470E The nucleotide sequence of the mutant is shown in SEQ ID NO.4.

[0014] The present invention also provides a gene encoding the above-mentioned mutant.

[0015] The present invention also provides a recombinant vector containing the above-mentioned mutant.

[0016] In one embodiment of the present invention, the carrier includes, but is not limited to, pMA0911.

[0017] The present invention also provides a recombinant vector carrying the above-mentioned genes.

[0018] In one embodiment of the present invention, the carrier includes, but is not limited to, pMA0911.

[0019] The present invention also provides a vector carrying the gene encoding the base sequence shown in SEQ ID NO.3 or SEQ ID NO.4, the vector including but not limited to pMA0911.

[0020] The present invention also provides recombinant cells expressing the above-mentioned sucrose isomerase mutant, or carrying the above-mentioned gene, or the above-mentioned recombinant vector.

[0021] In one embodiment of the present invention, the recombinant cells are expressed using fungi or bacteria as expression hosts.

[0022] In one embodiment of the present invention, the recombinant cells include, but are not limited to, Bacillus subtilis 168.

[0023] The present invention also provides an enzyme preparation containing the above-mentioned sucrose isomerase mutant, or a mixture of sucrose isomerase mutant enzyme and enzyme protectant.

[0024] In one embodiment of the present invention, the enzyme preparation is a lyophilized powder of the sucrose isomerase, containing the sucrose isomerase and its protectant.

[0025] The present invention also provides a method for improving the activity and / or stability of sucrose isomerase, wherein the method involves mutating methionine at position 282 of the sucrose isomerase, as shown in SEQ ID NO.1, to serine;

[0026] Alternatively, the methionine at position 441 of the sucrose isomerase, as shown in SEQ ID NO.1, may be mutated to phenylalanine;

[0027] Alternatively, the alanine at position 470 of the sucrase isomerase, as shown in SEQ ID NO.1, may be mutated to glutamic acid;

[0028] Alternatively, the amino acid sequence of the sucrose isomerase shown in SEQ ID NO.1 may be mutated by changing methionine at position 282 to serine, methionine at position 441 to phenylalanine, and alanine at position 470 to glutamic acid.

[0029] The present invention also provides a recombinant Bacillus subtilis, wherein the recombinant Bacillus subtilis uses pMA0911 as a vector to express the sucrose isomerase shown in SEQ ID NO.1 or SEQ ID NO.2 in Bacillus subtilis 168.

[0030] The present invention also provides a method for constructing the recombinant bacteria, comprising the following steps: linking the sucrose isomerase gene with the vector pMA0911, transforming the obtained recombinant expression vector into Bacillus subtilis 168, and obtaining the recombinant bacteria.

[0031] The present invention also provides a method for soluble expression of sucrose isomerase, wherein the above-mentioned recombinant cells are inoculated into a culture medium and fermented to obtain the enzyme.

[0032] In one embodiment of the present invention, the above-mentioned recombinant Bacillus subtilis is inoculated into a culture medium and cultured at 30-35°C for 24-48 hours.

[0033] In one embodiment of the present invention, the culture medium contains tryptone, yeast extract, glycerol, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate.

[0034] In one embodiment of the present invention, the culture temperature is 30°C and the culture time is 48 hours.

[0035] In one embodiment of the present invention, the culture medium formulation is 12 g / L peptone, 24 g / L yeast extract, 5 g / L glycerol, 2.3 g / L potassium dihydrogen phosphate, and 12.5 g / L dipotassium hydrogen phosphate.

[0036] The present invention also provides a method for preparing isomaltulose, wherein the method comprises using the above-mentioned mutant, or the above-mentioned recombinant cell, or the above-mentioned enzyme preparation, or the recombinant enzyme prepared by fermentation of the above-mentioned recombinant cell, and using sucrose as a substrate to react and prepare isomaltulose.

[0037] In one embodiment of the present invention, the reaction conditions are as follows: 400-800 g / L sucrose is added to a 50 mM pH 6.0 citrate-disodium hydrogen phosphate buffer, and the amount of enzyme added is one-tenth of the total system of the fermentation broth of recombinant cells. The reaction is carried out at 30°C and 200 rpm for 12 h in a shaker.

[0038] The present invention also provides the application of the recombinant cells, or the sucrose isomerase, or the enzyme preparation in the field of isomaltulose production.

[0039] In one embodiment of the present invention, the method is used to efficiently synthesize isomaltulose and obtain high yield.

[0040] The present invention also provides the application of the above-mentioned mutant, or the above-mentioned gene, or the above-mentioned vector, or the above-mentioned recombinant cell, or the above-mentioned enzyme preparation, or the above-mentioned method in the preparation of isomaltulose or food, pharmaceutical, cosmetic or health products containing isomaltulose.

[0041] Beneficial effects

[0042] (1) This invention utilizes Pantoea dispersa sucrose isomerase PdSlase and its mutant PdSlase. M282S / M441F / A470E The sucrose isomerase mutant was expressed in food-grade Bacillus subtilis strain 168, and a high-efficiency expression was obtained, which simultaneously improved enzyme activity, stability, conversion rate and yield.

[0043] (2) Positive mutant PdSlase M282S PdSlase M441F PdSlase A470E The specific enzyme activities increased to 808.5 U / mg, 772.9 U / mg, and 694.1 U / mg, respectively;

[0044] (3) The obtained mutant PdSlase M282S / M441F / A470E The expression level in recombinant bacteria is no lower than that of wild type, with improved stability and a specific enzyme activity increased to 871.3 U / mg, which is 1.33 times higher than that of wild type. The isomaltulose yield increased to 641.6 g with a conversion rate of 98.7%, which reduced the cost for downstream separation and preparation of recombinant sucrose isomerase.

[0045] (4) The results showed that at 45℃, the mutant PdSlase M282S / M441F / A470E The half-life is approximately 120 min, indicating that the sucrose isomerase mutant has good stability. This recombinant sucrose isomerase was applied in the production of isomaltulose. Attached Figure Description

[0046] Figure 1 The amino acid sequence of PdSlase is compared with that of other sucrose isomerases.

[0047] Figure 2 SDS-PAGE analysis of PdSlase and its mutants.

[0048] Figure 3 The specific enzyme activity of PdSlase and its mutants.

[0049] Figure 4 PdSlase and its mutant PdSlaseM282S / M441F / A470E Optimal pH.

[0050] Figure 5 PdSlase and its mutant PdSlase M282S / M441F / A470E Optimal temperature.

[0051] Figure 6 PdSlase and its mutant PdSlase M282S / M441F / A470E pH stability.

[0052] Figure 7 PdSlase and its mutant PdSlase M282S / M441F / A470E Temperature stability.

[0053] Figure 8 PdSlase M282S / M441F / A470E Yield and production of isomaltulose synthesized under high sucrose concentration. Detailed Implementation

[0054] The culture media involved in the following examples are as follows:

[0055] LB liquid medium: sodium chloride 10 g / L, peptone 10 g / L, yeast extract 5 g / L.

[0056] TB liquid medium: peptone 12g / L, yeast extract 24g / L, glycerol 5g / L, potassium dihydrogen phosphate 2.3g / L, dipotassium hydrogen phosphate 12.5g / L.

[0057] The detection methods involved in the following embodiments are as follows:

[0058] Method for determining sucrose isomerase activity:

[0059] Add 0.5 μM sucrose isomerase to 1 mL of 50 mM pH 6.0 citrate-disodium hydrogen phosphate buffer containing 200 g / L sucrose; react at 30 °C for 10 min, then stop the enzymatic reaction by boiling in a water bath at 100 °C for 10 min, centrifuge and collect the supernatant, and use HPLC to detect the content of isomaltulose in the reaction solution.

[0060] Isomaltulose content determination: Isomaltulose was determined using an Agilent 1260 liquid chromatograph and a Thermo amino column. The mobile phase was acetonitrile:water (80:20), the injection volume was 10 μL, the flow rate was 0.8 mL / min, the column temperature was 30 °C, and the detector was a refractive index detector (RID).

[0061] Sucrose isomerase activity is defined as the amount of enzyme required to catalyze the formation of 1 μmol of isomaltulose from sucrose per minute, expressed as one enzyme activity unit (U). Specific enzyme activity is defined as the enzyme activity per unit protein (U / mg).

[0062] Example 1: Construction of recombinant Bacillus subtilis 168 / pMA0911-PdSlase

[0063] The specific steps are as follows:

[0064] (1) The amino acid sequence encoding sucrose isomerase PdSlase from Pantoea dispersa was modified by removing the first 1-21 amino acids of the signal peptide (SEQ ID NO.1) and sent to Sangon Biotech (Shanghai) Co., Ltd. for codon optimization. A 6×His tag was inserted at the C end of the gene and cloned into the vector pMA0911 at the BamHI / EcoRI cloning site to obtain the recombinant plasmid pMA0911-PdSlase, which was stored at -20℃.

[0065] SEQ ID NO.1:

[0066] ASPLTKPSTPIAATNIQKSADFPIWWKQAVFYQIYPRSFKDSNGDGIGDIPGIIEKLDYLKMLGVDAIWINP

[0067] HYESPNTDNGYDISDYRKIMKEYGSMADFDRLVAEMNKRGMRLMIDIVINHTSDRHRWFVQSRSGKDNP

[0068] YRDYYFWRDGKQGQAPNNYPSFFGGSAWQLDKQTDQYYLHYFAPQQPDLNWDNPKVRAELYDILRFW

[0069] LDKGVSGLRFDTVATFSKIPGFPDLSKAQLKNFAEAYTEGPNIHKYIHEMNRQVLSKYNVATAGEIFGVPV

[0070] SAMPDYFDRRREELNIAFTFDLIRLDRYPDQRWRRKPWTLSQFRQVISQTDRAAGEFGWNAFFLDNHDN

[0071] PRQVSHFGDDSPQWRERSAKALATLLLTQRATPFIFQGAELGMTNYPFKNIEEFDDIEVKGFWNDYVASG

[0072] KVNAAEFLQEVRMTSRDNSRTPMQWNDSVNAGFTQGKPWFHLNPNYKQINAAREVNKPDSVFSYRQ

[0073] LINLRHQIPALTSGEYRDLDPQNNQVYAYTRILDNEKYLVVVNFKPEQLHYALPDNLTIASSLLENVHQPSL

[0074] QENASTLTLAPWQAGIYKLN

[0075] (2) Add 10 μL of plasmid to 500 μL of B. subtilis 168 competent cells, mix gently, and heat in a 45℃ metal bath for 90 s. Immediately after the heat shock, place on ice for 1 min. Incubate at 37℃, 200 rpm, and shake for 2.5 h. Centrifuge at 6,000 rpm for 5 min to collect the cells. Resuspend the cells in 50 μL of the supernatant and spread them on LB agar plates containing 50 μg / mL kanamycin sulfate. Invert the plates and incubate overnight at 37℃.

[0076] Recombinant Bacillus subtilis 168 / pMA0911-PdSlase was prepared.

[0077] Example 2: Mutant Design and Construction of Sucrose Isomerase

[0078] according to Figure 1 Based on the amino acid sequence alignment, site-directed mutagenesis was performed at specific amino acid sites. Primers as shown in Table 1 were designed, and sucrose isomerase mutants were obtained by site-directed mutagenesis using the pMA0911-PdSlase expression vector prepared in Example 1 as a template via whole plasmid PCR. The obtained positive mutants were then subjected to combined mutagenesis.

[0079] Table 1: Primers used in sucrose isomerase mutant design

[0080]

[0081]

[0082] The PCR amplification reaction system consisted of: 25 μL of 2×PrimeSTAR, 1 μL of pMA0911-PdSlase plasmid, 10 μL of ddH2O, and 2 μL of each primer. The PCR program was: 98℃ for 5 min; 98℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s, for 30 cycles; 72℃ for 10 min, 16℃ for 10 min.

[0083] After PCR, the amplified fragments were purified using a kit and added to 100 μL of E. coli BL21(DE3) competent cells. The cells were incubated on ice for 30 min, then heat-shocked in a 42℃ metal bath for 90 s. Immediately after the heat shock, the cells were placed on ice for 5 min. 700 μL of LB liquid medium was added to a centrifuge tube, and the cells were incubated at 37℃, 200 rpm, with shaking, for 1 h. The cells were collected by centrifugation at 5,000 × g for 5 min, and 50 μL of the supernatant was used to resuspend the cells. The cells were then spread onto LB agar plates containing 100 μg / mL ampicillin. The plates were incubated upside down at 37℃ overnight (approximately 10 h). Single colonies were picked and placed in 5 mL of LB liquid medium containing 100 μg / mL ampicillin. The cells were incubated at 37℃, 200 rpm, with shaking, for approximately 12 h. The plasmids were extracted and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to verify their correctness.

[0084] plasmid pMA0911-PdSlase was prepared separately. N109K pMA0911-PdSlase G112N pMA0911-PdSlase R127Q pMA0911-PdSlase L171K pMA0911-PdSlase P185K pMA0911-PdSlase N267D pMA0911-PdSlase M282S pMA0911-PdSlase Y307D pMA0911-PdSlase S359R pMA0911-PdSlase L374T pMA0911-PdSlase F384Y pMA0911-PdSlase A387S pMA0911-PdSlase E428N pMA0911-PdSlase M441F pMA0911-PdSlase Q466E pMA0911-PdSlase A470E pMA0911-PdSlase Q554D pMA0911-PdSlase M282S / M441F / A470E .

[0085] Example 3: Expression and purification of recombinant sucrose isomerase and its mutants

[0086] The specific steps are as follows:

[0087] (1) Preparation of crude enzyme solution

[0088] Take 10 μL of the plasmid prepared in Example 2 and add it to 500 μL of B. subtilis 168 competent cells. Gently mix and place in a 45°C metal bath for 90 s for heat shock, then immediately place on ice for 1 min. Incubate at 37°C, 200 rpm with shaking for 2.5 h. Centrifuge at 6,000 × g for 5 min to collect the cells. Resuspend the cells in 50 μL of the supernatant and spread on LB agar plates containing 50 μg / mL kanamycin sulfate. Invert the plates and incubate overnight at 37°C.

[0089] Recombinant Bacillus subtilis containing different mutants were prepared separately.

[0090] Single colonies of recombinant Bacillus subtilis from solid culture plates were picked and placed into 5 mL LB liquid medium tubes containing 50 μg / mL kanamycin sulfate. The culture was incubated at 37°C and 200 rpm with shaking for approximately 8 hours. The seed culture grown in the tubes was then inoculated into shake flasks containing 50 μg / mL kanamycin sulfate at a 1% inoculation rate. The culture was incubated at 37°C and 200 rpm with shaking for 2 hours. The shake flasks were then transferred to 30°C and 200 rpm for 48 hours to induce fermentation. The fermentation broth was centrifuged at 10,000 × g for 10 min to separate the supernatant and bacterial cells. The supernatant containing more than 10 g / L of wet bacterial cells was considered the crude enzyme solution.

[0091] (2) Preparation of pure enzyme solution

[0092] The fermentation broth supernatant was filtered through a 0.22 μm aqueous filter, and the filtrate was purified using a nickel column. The HisTrap HP column (GE Healthcare, Chicago, USA) was equilibrated for 5 column volumes with protein purification buffer A (20 mM Tris, 150 mM NaCl, pH 7.0). After loading the sample, contaminating proteins were washed with 3% protein purification buffer B (20 mM Tris, 150 mM NaCl, 1 M imidazole, pH 7.0), followed by elution of the target protein with 30% protein purification buffer B and collection.

[0093] The target protease solution collected after purification from the nickel column was desalted using a Sephadex G-25 desalting column. First, three column volumes were equilibrated by washing with ultrapure water and buffer C (50 mM pH 6.0 citrate-disodium hydrogen phosphate buffer), then the sample was loaded, and the protein was eluted with buffer C to obtain pure sucrose isomerase and pure sucrose isomerase mutant enzyme solutions. The concentrations of the pure enzymes obtained after desalting were all >0.4 mg / mL, the total amount of pure enzyme was >50 mL, and the total expression level of sucrose isomerase was >20 mg / L. After concentrating the pure enzyme solution to a concentration of 1 mg / mL, SDS-PAGE electrophoresis was performed, and the band results were observed to determine whether a single purified target protein was obtained.

[0094] The results showed that the protein band was consistent with the theoretical value of 68 kDa. Figure 2 The recombinant isomerase and its mutant were both successfully expressed, and pure enzymes, PdSlase, were obtained respectively. WT PdSlase N109K PdSlase G112N PdSlase R127Q PdSlase L171K PdSlase P185K PdSlase N267D PdSlase M282S PdSlase Y307D PdSlase S359R PdSlase L374T PdSlase F384Y PdSlase A387S PdSlase E428N PdSlase M441F PdSlase Q466E PdSlase A470E PdSlase Q554D PdSlase M282S / M441F / A470E .

[0095] Example 4: Enzyme activity assay of sucrose isomerase and its mutants

[0096] The specific enzyme activity of the pure enzymes prepared in Example 3 was measured, and the specific enzyme activity of PdSlase and its mutants was analyzed. The results are shown in Table 2 and below. Figure 3 As shown:

[0097] Table 2: Specific enzyme activities of wild-type sucrose isomerase and its different mutant pure enzymes

[0098]

[0099]

[0100] The results showed that the specific enzyme activity of wild-type PdSlase was 657.3 U / mg, while that of the positive mutant PdSlase was higher. M282S PdSlase M441F PdSlase A470E The specific enzyme activities were increased to 808.5, 772.9, and 694.1 U / mg, respectively. Combined mutagenesis of the positive mutant yielded the triple mutant PdSlase. M282S / M441F / A470E The specific enzyme activity was 871.3 U / mg. Figure 3 It is 1.33 times higher than that of the wild type.

[0101] Example 5: Optimal pH for sucrose isomerase and mutant enzyme activity

[0102] The specific steps are as follows:

[0103] The specific enzyme activities of recombinant sucrose isomerase and its mutants were determined according to the enzyme activity assay method of Example 4 under 50 mM citrate-disodium hydrogen phosphate buffer with different pH conditions. The results are shown in Table 3 and... Figure 4 As shown:

[0104] Table 3: PdSlase and its mutant PdSlase M282S / M441F / A470E Enzyme activity at different pH levels

[0105]

[0106]

[0107] The results showed that wild-type PdSlase and mutant PdSlase M282S / M441F / A470E Both enzymes exhibited the highest specific activity at pH 6.0. Figure 4 Sucrose isomerase is an acidic protease, which is more suitable for the synthesis of isomaltulose under acidic conditions.

[0108] Example 6: Optimal Temperature for Sucrose Isomerase and Mutant Enzyme Activity

[0109] The specific steps are as follows:

[0110] The specific enzyme activities of recombinant sucrose isomerase and its mutants were determined under different temperature conditions according to the enzyme activity assay method in Example 4. The results are shown in Table 4. Figure 5 As shown:

[0111] Table 4: PdSlase and its mutant PdSlase M282S / M441F / A470E Enzyme activity at different temperatures

[0112]

[0113] The results showed that wild-type PdSlase and mutant PdSlase M282S / M441F / A470E All enzymes exhibited the highest activity at 30℃.

[0114] Example 7: Sucrose isomerase and mutant enzyme activity pH stability

[0115] The specific steps are as follows:

[0116] After incubating the recombinant sucrose isomerase at 4°C for 1 day in 50mM citrate-disodium hydrogen phosphate buffer at different pH values, the specific enzyme activity of the recombinant sucrose isomerase and its mutants was determined at different pH values ​​according to the enzyme activity assay method in Example 4.

[0117] The specific enzyme activity measured under reaction conditions of pH 6.0 and 30℃ was taken as 100%. The results are shown in Table 5. Figure 6 As shown:

[0118] Table 5: PdSlase and its mutant PdSlase M282S / M441F / A470E Residual relative enzyme activity after incubation at different pH levels

[0119]

[0120]

[0121] The results showed that wild-type PdSlase and mutant PdSlase M282S / M441F / A470E Both were most stable at pH 6.0, and the mutants showed improved stability compared to the wild type.

[0122] Example 8: Temperature stability of sucrose isomerase and mutant enzyme activity

[0123] The specific steps are as follows:

[0124] After incubating the recombinant sucrose isomerase at different temperatures for 2 hours, the specific enzyme activity of the recombinant sucrose isomerase and its mutants was measured at different temperatures according to the enzyme activity assay method in Example 4. The specific enzyme activity measured under reaction conditions of pH 6.0 and 30℃ was taken as 100%. The results are shown in Table 6 and... Figure 7 As shown:

[0125] Table 6: PdSlase and its mutant PdSlase M282S / M441F / A470E Residual relative enzyme activity after incubation at different temperatures

[0126]

[0127] The results showed that wild-type PdSlase and mutant PdSlase M282S / M441F / A470E All were most stable at 30℃, and the mutant showed improved stability compared to the wild type. At 45℃, the mutant PdSlase... M282S / M441F / A470E Its half-life is approximately 120 minutes.

[0128] Example 9: Application of sucrose isomerase and mutant crude enzyme solution in high-yield isomaltulose production

[0129] The specific steps are as follows:

[0130] A 100 mL reaction system was prepared in a 500 mL shake flask using 50 mM pH 6.0 citrate-disodium hydrogen phosphate buffer. The enzyme added was one-tenth of the total system volume, i.e., according to the PdSlase mutant in Example 3. M282S / M441F / A470EThe supernatant of 10 mL of fermentation broth obtained by the crude enzyme solution preparation method was mixed with sucrose at final concentrations of 400, 450, 500, 550, 600, 650, 700, 750, and 800 g / L, and reacted in a shaker at 30℃ and 200 rpm for 12 h to prepare the reaction solution. The content of isomaltulose in the reaction solution was detected by HPLC.

[0131] The results are shown in Table 7 and... Figure 8 As shown:

[0132] Table 7: Isomaltulose yield and conversion rate of wild-type enzymes and their mutants at different sucrose concentrations

[0133]

[0134] The results showed that, using the wild-type enzyme, the conversion rate was above 90% when the substrate sucrose concentration was below 600 g / L, and 91.7% with a yield of 550.2 g when the sucrose concentration was 600 g / L. When the sucrose concentration was above 600 g / L, the yield increased slightly, but the conversion rate decreased. At a sucrose concentration of 650 g / L, the conversion rate was 85.4% with a yield of 581.1 g / L. Using the mutant PdSlase... M282S / M441F / A470E Catalysis was performed, and when the sucrose concentration was below 650 g / L, the yield was above 98%. When the sucrose concentration was 650 g / L, the conversion rate was 98.7% and the yield was 641.6 g. When the sucrose concentration was above 650 g / L, the yield increased slightly, but the conversion rate decreased.

[0135] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A sucrose isomerase mutant, characterized in that, The mutant is obtained by mutating methionine at position 282 of the sucrose isomerase, as shown in SEQ ID NO.1, to serine.

2. The sucrose isomerase mutant according to claim 1, characterized in that, The mutant is obtained by mutating methionine at position 282 of the sucrose isomerase shown in SEQ ID NO.1 to serine, methionine at position 441 to phenylalanine, and alanine at position 470 to glutamic acid.

3. The gene encoding the mutant of claim 1 or 2.

4. A vector carrying the gene of claim 3.

5. A recombinant cell expressing the sucrose isomerase mutant of claim 1 or 2, or carrying the gene of claim 3, or containing the vector of claim 4; wherein the recombinant cell uses Bacillus subtilis as the expression host.

6. An enzyme preparation, characterized in that, The enzyme preparation contains the sucrose isomerase mutant as described in claim 1.

7. The enzyme preparation according to claim 6, characterized in that, The enzyme preparation contains a mixture of the sucrose isomerase mutant and an enzyme protectant.

8. A method for improving the activity of sucrose isomerase, characterized in that, The method involves mutating methionine at position 282 of the sucrose isomerase, as shown in SEQ ID NO. 1, to serine. Alternatively, the amino acid sequence of the sucrose isomerase shown in SEQ ID NO.1 may be mutated by changing methionine at position 282 to serine, methionine at position 441 to phenylalanine, and alanine at position 470 to glutamic acid.

9. A method for soluble expression of sucrose isomerase, characterized in that, The method involves inoculating the recombinant cells described in claim 5 into a culture medium and then fermenting them to obtain the cells.

10. A method for preparing isomaltulose, characterized in that, The method is as follows: using the mutant described in claim 1 or 2, or the recombinant cell described in claim 5, or the enzyme preparation described in claim 6 or 7, or the recombinant enzyme prepared by fermentation of the recombinant cell described in claim 5, and using sucrose as a substrate, isomaltulose is prepared by reaction.

11. The method according to claim 10, characterized in that, The reaction conditions were: 50 mM citric acid at pH 6.

0. The sucrose content added to the disodium hydrogen phosphate buffer was 400~800 g / L, and the reaction was carried out at 200 rpm for 12 h in a shaker at 30 ℃.

12. The use of the mutant of claim 1 or 2, or the gene of claim 3, or the vector of claim 4, or the recombinant cell of claim 5, or the enzyme preparation of claim 6 or 7, or the method of any one of claims 9 to 11 in the preparation of isomaltulose or food, pharmaceutical, cosmetic or health product containing isomaltulose.

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