A sucrose isomerase mutant and its application

Abstract

This invention relates to the field of enzyme engineering technology, and more particularly to a mutant and its recombinant strain that improves the thermostability of sucrose isomerase. The amino acid profile of the sucrose isomerase mutant is shown in SEQ ID NO. 1. This sucrose isomerase mutant is obtained by mutating the 199th amino acid, Ala, to Leu, from the wild-type sucrose isomerase derived from *Pantoea dispersa*, resulting in a 32.51% improvement in thermostability compared to the wild-type. Catalytic experiments using the sucrose isomerase mutant of this invention show that the purity of isomaltulose in the catalytic product can reach 100%. This invention lays the foundation for the industrial application of sucrose isomerase.

Description

Technical fields:

[0001] This invention relates to the field of enzyme engineering technology, and in particular to a mutant and its recombinant strain that improves the thermal stability of sucrose isomerase. Background technology:

[0002] Isomaltulose, also known as palaginose, is a reducing disaccharide formed by glucose and fructose linked by α-1,6-glycosidic bonds. It is naturally found in sugar beets, molasses, and syrups. It is usually produced by catalysis of sucrose isomerase and is an isomer of sucrose, but the two have different properties. Compared with sucrose, isomaltulose has lower sweetness, lower melting point, lower viscosity, is less hygroscopic, and stronger stability. Isomaltulose also has excellent physiological functions: (1) easy to absorb and highly safe; (2) suitable for people with high blood pressure, high cholesterol, and high blood sugar, as well as obese people; (3) improves concentration; (4) non-cariogenic and prevents tooth decay; (5) does not cause diarrhea; (6) does not cause a rise in blood sugar; (7) anti-fatigue; (8) a flavor inhibitor, which can be added to functional foods as a prebiotic to improve the nutritional value of functional foods.

[0003] Researchers from various countries have discovered a variety of microorganisms capable of synthesizing sucrose isomerases, such as *Serratia plymuthica*, *Pantoeadispersa*, *Protaminobacter rubrum*, *Enterobacters*, *Erwinia rhapontici*, and *Klebsiella* sp., which are major producers of isomaltulose (65%-85%); and *Pseudomonas mesoacidophila* and *Agrocbacterium radiobacter*, which are major producers of trehalose (85%-90%). Currently, the main hosts expressing high sucrose isomerase activity are non-food safe bacteria such as Escherichia coli and Sperm rubrum. Their use in food production poses safety risks. Furthermore, the enzyme itself has poor thermal stability, making it difficult to carry out continuous enzymatic conversion and storage and transportation of sucrose isomerase in industrial applications. As a result, the enzymatic preparation cost of isomaltulose remains high, preventing its large-scale application. Summary of the Invention:

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a mutant with improved thermostability of sucrose isomerase, its recombinant strain, and its application.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a sucrose isomerase mutant, the amino acid sequence of which is shown in SEQ ID NO. 1. The sucrose isomerase mutant is obtained by mutating amino acid Ala to Leu at position 199 of a wild-type sucrose isomerase derived from *Pantoea dispersa*, and the wild-type sucrose isomerase has the amino acid sequence shown in SEQ ID NO. 3.

[0007] The present invention also provides a coding gene for the sucrose isomerase mutant, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0008] The present invention also provides a recombinant expression vector containing the coding gene of the above-mentioned sucrose isomerase mutant, wherein the expression plasmid used in the recombinant expression vector is pWB980.

[0009] The present invention also provides a recombinant bacterium containing the coding gene of the above-mentioned sucrose isomerase mutant, wherein the recombinant bacterium is obtained by transferring the above-mentioned recombinant expression vector into a host bacterium for expression;

[0010] Furthermore, the host bacterium is Bacillus amyloliquefaciens CGMCC No. 11218.

[0011] The present invention also provides the use of the recombinant expression vector or recombinant bacteria containing the coding gene of the above-mentioned sucrose isomerase mutant, particularly its use in the production of the sucrose isomerase mutant shown in SEQ ID NO.1.

[0012] This invention also provides the application of the sucrose isomerase mutant, particularly its application in the catalytic synthesis of isomaltulose;

[0013] Furthermore, the conditions for the catalytic synthesis of isomaltulose are as follows:

[0014] The catalytic temperature is 32–35℃, the catalytic time is 2.5–5h, the pH of the substrate is 6.0–7.1, the substrate is a sucrose solution with a concentration of 60–70%, and 50–60 U of sucrose isomerase is added per gram of sucrose (dry).

[0015] The solution of the present invention has the following advantages:

[0016] This invention first achieved the efficient expression of sucrose isomerase and its mutant derived from *Pantoea dispersa* in *Bacillus amyloliquefaciens* strain CGMCC No. 11218. The resulting genetically engineered strains were named CGMCC No. 11218-Sim and CGMCC No. 11218-Sim-T, respectively. The CGMCC No. 11218-Sim strain showed an enzyme activity of 301.54 U / mL in a 500 mL shake flask. Secondly, the mutant CGMCC No. 11218-Sim-T showed an enzyme activity of 305.12 U / mL in a 500 mL shake flask. Although the enzyme activity was not significantly different from the control, its thermostability was 32.51% higher than the wild type. Catalytic experiments using the sucrose isomerase mutant of this invention showed that the purity of isomaltulose in the catalytic product reached 100%. This invention lays the foundation for the industrial application of sucrose isomerase. Attached image description:

[0017] Figure 1 Validation diagram of positive transformants

[0018] Lane 1: Contains Sim-T strain; Lane 2: Contains Sim strain.

[0019] Figure 2 Validation diagram of positive transformants

[0020] Lane 1: CGMCC No.11218-Sim-T, Lane 2: CGMCC No.11218-Sim.

[0021] Figure 3 Liquid chromatography chromatogram of 0.5 mg / mL isomaltulose standard.

[0022] Figure 4 Liquid phase diagram of a 0.5 mg / mL crystallized sample. Detailed implementation method:

[0023] The present invention will be further described below through specific embodiments. Unless otherwise specified, the technical means and materials involved in the following embodiments are all known to those skilled in the art, and suitable means and materials that can solve the corresponding technical problems can be selected. In addition, the embodiments should be understood as illustrative, not limiting the scope of the present invention, and the essence and scope of the present invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the present invention also fall within the protection scope of the present invention.

[0024] 1. The following definitions are used in this invention:

[0025] Nomenclature of amino acids and DNA nucleic acid sequences

[0026] 1.1 The IUPAC nomenclature, a recognized system for naming amino acid residues, is used, employing either three-letter or single-letter codes. DNA nucleic acid sequences are named using the IUPAC nomenclature.

[0027] 1.2 Identification of Sucrose Isomerase Mutants

[0028] The term "amino acid replaced at the original amino acid position" is used to represent the mutated amino acid in the mutant. For example, Ala199Leu means that the amino acid at position 199 is replaced by Leu in the wild type, and the position number corresponds to the amino acid sequence number of the wild type in SEQ ID No. 3.

[0029] In this invention, Sim represents wild-type sucrose isomerase, and Sim-T represents a sucrose isomerase mutant, as shown in the table below:

[0030]

[0031] The amino acid sequence SEQ ID NO.1 of the mutant Sim-T is as follows:

[0032] ASPLTKPSTPIAATNIQKSADFPIWWKQAVFYQIYPRSFKDSNGDGIGDIPGIIEKLDYLKMLGVDAIWINPHYESPNTDNGYDISDYRKIMKEYGSMADFDRLVAEMNKRGMRLMIDIVINHTSDRHRWFVQSRSGKDNPYRD YYFWRDGKQGQAPNNYPSFFGGSAWQLDKQTDQYYLHYFAPQQPDLNWDNPKVRLELYDILRFWLDKGVSGLRFDTVATFSKIPGFPDLSKAQLKNFAEAYTEGPNIHKYIHEMNRQVLSKYNVATAGEIFGVPVSAMPDYFDR RREELNIAFTFDLIRLDRYPDQRWRRKPWTLSQFRQVISQTDRAAGEFGWNAFLDNHDNPRQVSHFGDDSPQWRERSAKALATLLLTQRATPFIFQGAELGMTNYPFKNIEEFDDIEVKGFWNDYVASGKVNAAEFLQEVRMT SRDNSRTPMQWNDSVNAGFTQGKPWFHLNPNYKQINAAREVNKPDSVFSYYRQLINLRHQIPALTSGEYRDLDPQNNQVYAYTRILDNEKYLVVVNFKPEQLHYALPDNLTIASSLLENVHQPSLQENASTLTLAPWQAGIYKLN

[0033] 2. Culture medium used in this invention:

[0034] (1) LB medium: Each liter contains 10g peptone, 5g yeast extract, 10g NaCl, and 18g agar added to the solid medium. All other components are the same.

[0035] (2) Fermentation medium: Each liter contains 64g of corn flour, 40g of soybean meal, 4g of disodium hydrogen phosphate, 0.3g of potassium dihydrogen phosphate, and 70mg of high-temperature amylase.

[0036] (3) Preparation of culture medium for competent Bacillus subtilis cells:

[0037] SP-A salt solution: (NH4)2SO4 4g / L, K2HPO4·3H2O 28g / L, KH2PO4 12g / L, sodium citrate 2g / L;

[0038] SP-B salt solution: MgSO4·7H2O 0.4 g / L;

[0039] 100×CAYE solution: 20 g / L casein hydrolysate, 100 g / L yeast extract;

[0040] SPI (200mL): 98mL SP-A salt solution, 98mL SP-B salt solution, 2mL 50% glucose, 2mL 100×CAYE;

[0041] SPII medium (600 mL): 588 mL SPI, 6 mL 50 mmol / L CaCl2, 6 mL 250 mmol / L MgCl2;

[0042] 100×EGTA solution: 10mmol / L EGTA solution.

[0043] (4) Preparation of Bacillus amyloliquefaciens competent cell solution:

[0044] LBS: Each liter contains 91.085g of sorbitol, 10g of sodium chloride, 10g of peptone, and 5g of yeast powder;

[0045] Washing buffer: Each liter contains 91.085g of sorbitol, 91.085g of mannitol, and 100ml of 10% glycerol;

[0046] Resuspension buffer: Each liter contains 91.085 g of sorbitol, 91.085 g of mannitol, 100 mL of 10% glycerol, and 140 g of 14% PEG6000.

[0047] 3. The sucrose isomerase activity unit (U) used in this invention is defined as follows: the amount of enzyme required to convert sucrose into 1 μmol of isomaltulose per minute at 35°C and pH 6.0 is defined as 1 U.

[0048] Enzyme activity assay: Dilute the fermentation supernatant (crude enzyme solution) to a suitable concentration using pH 6.0 disodium hydrogen phosphate-citric acid buffer. Take 200 μL of the diluted crude enzyme solution and add it to 800 μL of substrate (25% sucrose solution, preheated in a 35℃ water bath for 5-10 min). Incubate at 35℃ for 10 min, then add 2 mL of DNS solution and incubate in a boiling water bath for 5 min. Quickly cool to room temperature and measure the OD value at 540 nm. Calculate the corresponding enzyme activity using the isomaltulose standard curve. The blank is 200 μL of enzyme solution diluted by the same factor and boiled at 100℃ for 10 min.

[0049] Accurately weigh 0.1000 g of isomaltulose standard and dissolve it in pH 6.0 disodium hydrogen phosphate-citrate buffer, bringing the volume to 50 ml to prepare a 2 mg / ml isomaltulose standard stock solution. Dilute the isomaltulose standard stock solution with pH 6.0 disodium hydrogen phosphate-citrate buffer to obtain a series of standard solutions of different concentrations (0.1, 0.15, 0.2, 0.25, 0.3, 0.35 mg / ml). Take 1 ml of each concentration standard solution and add 2 ml of DNS solution (two or three replicates). Simultaneously, use 1 ml of pH 6.0 disodium hydrogen phosphate-citrate buffer as a blank and process as above. Boil for 5 min, then immediately cool to room temperature in cold water. Measure the absorbance (A) at 540 nm. The expected absorbance should be within the linear detection range (absorbance A between 0.25 and 0.8). The standard equation for isomaltulose (Y = aX - b) is Y = 3.617X - 0.2842; R 2 =0.9889.

[0050] The formula for calculating the activity of sucrose isomerase is: Enzyme activity S=(A+b)×1000×5×n / (a×342.3×t) Where, A is the absorbance of the solution at 540nm; 1000 is the conversion factor (milligrams to micrograms); n is the dilution factor of the enzyme solution; t is the reaction time (min).

[0051] Note: When adding 200 μL of enzyme solution to 800 μL of substrate for enzyme activity assay, it is also a 5-fold dilution.

[0052] The present invention will be further explained and illustrated below through specific embodiments.

[0053] Example 1: Obtaining the mutant gene for sucrose isomerase

[0054] 1.1 Synthesis and amplification of Sim and Sim-T genes, derived from Pantoea dispersa, for sucrose isomerase.

[0055] The Sim gene sequence of sucrose isomerase from *Pantoea dispersa* was obtained from GenBank: AY223549.1. Its signal peptide was predicted and deleted to obtain the mature protein gene sequence of sucrose isomerase (as shown in SEQ ID NO: 4). Simultaneously, based on the required mutation sites, the gene sequence of the sucrose isomerase mutant Sim-T was designed (as shown in SEQ ID NO: 2), and its sequence was synthesized by a biotechnology company. The synthesized sequence was amplified by PCR using the following primer sequences:

[0056] Primer P1: F 5'-GCAACGAATATACAAAAGTCCGCTGATT-3'

[0057] Primer P2: R 5'-TCAGTTCAGCTTATAGATCCCGGCTT-3'

[0058] Using P1 / P2 as upstream and downstream primers, and the commissioned synthetic sucrose isomerase Sim and Sim-T genes as templates, amplification was performed.

[0059] The amplification reaction system is as follows:

[0060]

[0061]

[0062] The PCR program was set as follows: pre-denaturation: 95℃ for 5 min; denaturation: 98℃ for 10 min; annealing: 59℃ for 20 min; extension: 72℃ for 10 s, 32 cycles.

[0063] 1.2 Linearization of expression vectors

[0064] The pWB980 plasmid was extracted according to the kit's instruction manual. After double digestion with BamHI and SmaI, the product was subjected to agarose gel electrophoresis, and then recovered using a DNA gel recovery kit to obtain the linearized vector sequence.

[0065] 1.3 The target fragments (Sim and Sim-T) digested with BamHI and SmaI were ligated with the vector fragments to form recombinant plasmids pWB980-Sim-T and pWB980-Sim, respectively. The recombinant plasmids were then transformed into Bacillus subtilis WB600.

[0066] The ligation product was transferred to Bacillus subtilis WB600 as follows:

[0067] 1) Pick a single colony of newly activated Bacillus subtilis WB600 and incubate it overnight at 37°C and 220 rpm.

[0068] 2) Transfer 100 μL of culture medium to 5 mL of SPI medium and incubate at 37 °C and 220 r / min until the end of the logarithmic growth phase with OD600 = 1.2 (approximately 3–4 h);

[0069] 3) Take 200 μL of the culture medium that has grown to the end of the logarithmic phase and add it to 2 mL of SPII medium. Incubate at 37 °C and 100 r / min for 1.5 h.

[0070] 4) Add 20 μL of 10 mmol / L EGTA to the bacterial cells in the above SPII medium, and incubate at 37℃ and 100 r / min for 10 min;

[0071] 5) Add the ligation product to the SPII and incubate at 37℃ and 100r / min for 30min;

[0072] Adjust the rotation speed to 220 r / min and continue culturing for 1.5 h. Spread the bacterial culture onto LB selection plates containing 100 μg / mL kanamycin and incubate at 37℃ for 12 h. Screen for positive transformants and verify their selection by gene amplification using P3 / P4 primers. The results are as follows: Figure 1 As shown (the verification band is 2081bp).

[0073] Primer P3: F 5'-AACTGACAAACATCACCCTCTTGC-3'

[0074] Primer P4: R 5'-AAAGCTTGAGTTGCGCCTCCT-3'.

[0075] Example 2: Construction of recombinant strains of sucrose isomerase

[0076] The recombinant plasmids pWB980-Sim-T and pWB980-Sim from Example 1 were extracted and then transferred into Bacillus amyloliquefaciens CGMCC No.11218 by electroporation.

[0077] The electro-spinning method is as follows:

[0078] 1) Clean the electric rotary cup with 75% alcohol, irradiate it under ultraviolet light for more than 20 minutes, and pre-cool it on ice;

[0079] 2) Mix 100 μL of competent cells and 10 ng of plasmid DNA and add to the electroporation vessel, then place on ice for 2 min;

[0080] 3) 2500V electric shock, the shock time is generally 4-6 minutes;

[0081] 4) Immediately after electric shock, add 1 ml of resuscitation medium and incubate at 37°C for 3 hours. Plate the mixture and incubate at 37°C for 12 hours. Screen for positive transformants for verification (using P3 and P4 as upstream and downstream primers for gene amplification; verification diagram shown). Figure 2 Finally, recombinant strains expressing sucrose isomerase were obtained and named CGMCC No.11218-Sim and CGMCC No.11218-Sim-T, respectively.

[0082] Example 3: Shake-flask verification of CGMCC No. 11218-Sim and CGMCC No. 11218-Sim-T

[0083] The recombinant bacteria obtained in Example 2 were scraped from a glycerol tube and inoculated onto antibiotic-free LB solid medium, where they were densely streaked. After approximately 12 hours of growth, single colonies were transferred to LB liquid medium (Kana: final concentration 0.05 mg / mL) and cultured at 37°C and 250 rpm for 12 hours. Then, 2% inoculum was added to three 500 mL fermentation media (Kana: final concentration 0.05 mg / mL) from the LB primary seed culture and cultured at 37°C and 250 rpm for 48 hours. Samples were taken periodically, and sucrose isomerase activity was measured. The sucrose isomerase activity of recombinant strain CGMCC No.11218-Sim reached a maximum of 301.54 U / mL after 48 hours of fermentation, while that of recombinant strain CGMCC No.11218-Sim-T reached a maximum of 305.12 U / mL after 48 hours of fermentation.

[0084] Example 4: Purification and thermal stability analysis of Sim and Sim-T

[0085] A certain amount of fermentation broth sample from Example 3 was centrifuged to obtain the fermentation supernatant. The supernatant was then precipitated with 60% ammonium sulfate, centrifuged again to obtain the precipitate, and reconstituted with a certain amount of buffer solution followed by dialyzing for 24 hours. The dialyzed enzyme solution was purified using an SP-sepharose FF strong cation exchange chromatography column, and the thermal stability of the purified sample was determined. The purified sample was incubated at 50°C for 30 minutes, and the residual enzyme activity was measured. With the untreated enzyme activity as 100%, it was found that the thermal stability of Sim-T was 32.51% higher than that of Sim.

[0086]

[0087]

[0088] Example 5: Preparation of isomaltulose using Sim-T in a 250 mL saccharification system

[0089] (1) Preparation of sucrose standard solution: Dissolve sucrose in pH 6.0 disodium hydrogen phosphate-citric acid buffer to prepare a sucrose solution with a mass fraction of 67%.

[0090] (2) The conditions for producing isomaltulose using sucrose isomerase were as follows: 55 U of enzyme was added per gram of dry sucrose. The sucrose isomerase solution was added to a 67% sucrose solution. The reaction was controlled at pH 6.0, temperature 35℃, and stirring speed 200 rpm for 3 hours, until the sucrose conversion rate reached 100%. After the conversion rate reached 100%, the temperature was gradually lowered to 25℃ and maintained for 10 hours for crystallization. The sample was centrifuged at 12000 r / min for 15 min, the precipitate was collected, and lyophilized for later use. The purity of the sample was determined by HPLC and calculated according to the isomaltulose content formula below. Figure 3 and Figure 4 The images show the liquid chromatography (LC) chromatograms of a 0.5 mg / mL isomaltulose standard and a 0.5 mg / mL crystalline sample from this example, respectively. The purity of isomaltulose in the prepared product can reach 100%.

[0091]

[0092] HPLC conditions:

[0093] Mobile phase: 82% acetonitrile, 0.8 mL / min, column temperature 35℃, injection volume 10 μL, ELSD conditions: 90℃, 2.2 mL / min.

[0094] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions and variations in form and detail to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A sucrose isomerase mutant, characterized in that, The amino acids of the sucrose isomerase mutant are shown in SEQ ID NO.

1.

2. The encoding gene of the sucrose isomerase mutant according to claim 1.

3. The encoding gene as described in claim 2, characterized in that, It has the nucleotide sequence shown in SEQ ID NO.

2.

4. A recombinant expression vector or recombinant strain containing the encoding gene of claim 2.

5. The recombinant expression vector as described in claim 4, characterized in that, The expression plasmid used was pWB980.

6. The recombinant strain according to claim 4, characterized in that, The host bacterium used was Bacillus amyloliquefaciens CGMCC No. 11218.

7. The use of the recombinant expression vector or recombinant strain according to claim 4 in the production of the sucrose isomerase mutant according to claim 1.

8. The application of the sucrose isomerase mutant of claim 1 in the catalytic synthesis of isomaltulose.

9. The application as described in claim 8, characterized in that, The conditions for the catalytic synthesis of isomaltulose are as follows: The catalytic temperature is 32–35℃, the catalytic time is 2.5–5h, the pH of the substrate is 6.0–7.1, the substrate is a sucrose solution with a concentration of 60–70%, and 50–60 U of sucrose isomerase is added per gram of sucrose.

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