Beta-fructofuranosidase mutant and method for catalytically synthesizing fructosyl rebaudioside A by using beta-fructofuranosidase mutant
By performing site-directed mutation of the amino acid sequence of β-fructosidase, mutants with higher thermal stability and higher enzyme activity are formed, the problem of poor thermal stability of β-fructosidase in the prior art is solved, and the effect of efficient catalytic synthesis of fructosyl rebaudioside A is achieved.
Patent Information
- Application Number
- CN202410984716.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-22
- Publication Date
- 2025-04-25
AI Technical Summary
The thermal stability of β-fructosidase from Arthrobacteria is poor, resulting in serious loss of enzyme activity after catalytic reactions, limiting its efficient application.
By performing site-directed mutations on the amino acid sequence of wild-type β-fructosidase, mutants are formed, and their enzyme activity and thermal stability are improved. Specifically, mutations occur on amino acids at positions 86, 87, 108, 170, 174, 178, 226, 273, 287, 394, 404, 434, 477 and 478.
It improves the thermal stability and enzyme activity of β-fructosidase, and can efficiently catalyze the synthesis of fructose rebaudioside A, which increases the conversion rate of rebaudioside A and the yield of fructose rebaudioside A.
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Figure CN119876093A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of genetic engineering, and in particular to a beta-fructofuranosidase mutant and a method for catalyzing the synthesis of fructosyl rebaudioside A by the mutant. Background Art
[0002] Rebaudioside A (RebA) and steviol glycosides (SGs) are the two main components of stevia extract. Steviosides are formed by combining a glucose group at the "C19-" and "C13-" positions of steviol, and have a bitter and licorice taste. Rebaudioside A has one more glucose group connected to the "C13-" position of steviol than steviol glycosides, so it tastes sweeter and has less unpleasant aftertaste.
[0003] In order to further improve the taste of rebaudioside A, its carbohydrate part can be modified by enzymatic method. β-Fructofuranosidase from Arthrobacter sp.K1 can transfructosylate rebaudioside A to produce fructosyl rebaudioside A, that is, selectively transfer a fructose group to the glucosyl part of the C19 carboxyl group of rebaudioside A through β-2,6-linkage, further removing the bitterness of rebaudioside A. However, the thermal stability of β-fructofuranosidase from Arthrobacter sp.K1 is poor, and the enzyme activity is generally severely lost after 2 hours of catalysis, which limits the efficient application of β-fructofuranosidase. Summary of the invention
[0004] In view of this, the object of the present invention is to provide a β-fructofuranosidase mutant and a method for catalyzing the synthesis of fructosyl rebaudioside A, so as to overcome the problem of poor thermal stability of β-fructofuranosidase in the prior art and efficiently catalyze the synthesis of fructosyl rebaudioside A.
[0005] In a first aspect, the present invention provides a β-fructofuranosidase mutant, which is derived from a mutation in one or more of the 86th, 87th, 108th, 170th, 174th, 178th, 226th, 273rd, 287th, 394th, 404th, 434th, 477th or 478th positions of the amino acid sequence of the wild-type β-fructofuranosidase shown in SEQ ID NO:1.
[0006] Furthermore, the β-fructofuranosidase mutant is obtained by causing any one or more of the following mutations in the amino acid sequence shown in SEQ ID NO: 1:
[0007] The 86th amino acid mutated from D to F;
[0008] The 87th amino acid mutated from A to G;
[0009] The amino acid at position 108 mutated from G to P;
[0010] The 170th amino acid mutates from R to any of M, Y, or L;
[0011] The 174th amino acid mutated from N to D;
[0012] The amino acid at position 178 mutated from K to F;
[0013] The amino acid at position 226 mutated from D to E;
[0014] The amino acid at position 273 mutated from R to F;
[0015] The amino acid at position 287 mutated from Q to P;
[0016] The amino acid at position 394 mutated from A to V;
[0017] The amino acid at position 404 mutated from L to Y;
[0018] The amino acid at position 434 mutated from Q to one of I or V;
[0019] The amino acid at position 477 mutated from S to D;
[0020] The amino acid at position 478 mutated from E to N.
[0021] Furthermore, the amino acid sequence of the β-fructofuranosidase mutant is shown in SEQ ID NO:2 or SEQ ID NO:3.
[0022] In a second aspect, the present invention provides an application of a β-fructofuranosidase mutant, and the application of the β-fructofuranosidase mutant in catalyzing the synthesis of fructosyl rebaudioside A.
[0023] In a third aspect, the present invention provides a nucleic acid sequence encoding the above-mentioned β-fructofuranosidase mutant.
[0024] The nucleic acid sequence encoding the β-fructofuranosidase mutant in the above technical solution is preferably the sequence shown in SEQ ID NO:5.
[0025] In a fourth aspect, the present invention provides a recombinant expression vector comprising the above-mentioned nucleic acid sequence.
[0026] In a fifth aspect, the present invention provides a recombinant cell, which contains the above-mentioned nucleic acid sequence or the above-mentioned recombinant expression vector.
[0027] In a sixth aspect, the present invention provides a method for synthesizing fructosyl rebaudioside A using a β-fructofuranosidase mutant, comprising the following steps:
[0028] Rebaudioside A, sucrose and an enzyme solution containing the above-mentioned β-fructofuranosidase mutant are added to the catalytic reaction system, reacted for 1 to 10 hours, inactivated the enzyme, and centrifuged to obtain the supernatant, which is fructosyl rebaudioside A.
[0029] Furthermore, the preparation method of the enzyme solution containing the above-mentioned β-fructofuranosidase mutant is as follows:
[0030] (1) constructing the coding gene of the above-mentioned β-fructofuranosidase mutant into an expression vector to obtain a recombinant expression vector;
[0031] (2) transforming the recombinant expression vector into a host bacterium to obtain a recombinant strain containing a β-fructofuranosidase mutant expression system.
[0032] (3) After activating the recombinant strain, inoculate it into LB medium, add isopropyl-β-D-thiogalactoside with a final concentration of 0.02-1 g / L as an inducer, induce culture for 18-36 hours, centrifuge and collect the bacteria, resuspend the bacteria and break the cells, and centrifuge and collect the supernatant, which is the enzyme solution containing the β-fructofuranosidase mutant.
[0033] Furthermore, in the catalytic reaction system, the concentration of rebaudioside A is 1-500 mM / L, the concentration of sucrose is 1-2000 mM / L, and the added amount of the enzyme solution containing the β-fructofuranosidase mutant is 0.1-10 U / mL.
[0034] Beneficial effects:
[0035] Compared with the prior art, the present invention effectively improves the enzyme activity and thermal stability of β-fructofuranosidase through site-directed mutagenesis, and the obtained β-fructofuranosidase mutant can efficiently catalyze the synthesis of fructosyl rebaudioside A with rebaudioside A and sucrose as reaction substrates, thereby improving the conversion rate of rebaudioside A and the yield of fructosyl rebaudioside A, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Figure 1 Schematic diagram of the synthesis of rebaudioside A into fructosyl rebaudioside A catalyzed by β-fructofuranosidase.
[0037] Figure 2 It is the liquid chromatogram of the catalytic reaction in Example 2. DETAILED DESCRIPTION
[0038] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0039] In order to better illustrate the technical solution of the present invention, the present invention also provides the following specific examples. It should be understood that the raw materials used in the following examples are all commercially available raw materials unless otherwise specified.
[0040] Example 1
[0041] Construction of recombinant strain MsFFase
[0042] The gene fragment encoding the wild-type β-fructofuranosidase from Arthrobacter was codon-optimized, and the optimized nucleotide sequence (as shown in SEQ ID NO:4) was inserted between the NcoⅠ and XhoⅠ restriction sites of the expression vector pET28a (produced by Novagen) to obtain the recombinant plasmid pET-MsFFase.
[0043] The above-mentioned recombinant plasmid pET-MsFFase was added to the competent cells of Escherichia coli BL21 (DE3), mixed, placed on ice for 30 minutes, heat-shocked in a 42°C water bath for 45 seconds, ice-bathed for 2 minutes, and activated in a 37°C shaker for 120 minutes. Finally, the bacterial solution was spread on an LB plate containing 50 mg / L kanamycin and cultured in a 37°C incubator for 12 hours to obtain the recombinant strain MsFFase.
[0044] Example 2
[0045] Detection of the products of wild-type β-fructofuranosidase catalytic reaction
[0046] The recombinant strain MsFFase in Example 1 was spread on a LB solid plate (10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, 15 g / L agar) containing 50 mg / L kanamycin and placed in a 37°C incubator for 12 h. The next day, a single colony was selected from the plate and cultured in LB medium at 37°C and 200 rpm. When the absorbance (OD 600 ) reached 0.6-0.8, isopropyl-β-D-thiogalactoside (IPTG) was added to the culture medium with a final concentration of 0.1 mM, and the culture was induced at 20°C for 24 h.
[0047] Collect the above bacterial solution for refrigerated centrifugation (4°C, 8000rpm centrifugation for 5min), discard the supernatant to obtain bacterial mud, and rinse twice with PBS buffer. Add 5mL PBS buffer per gram of bacterial cells, place in an ice-water mixture, and use an ultrasonic disruptor to ultrasonically disrupt the bacterial cells. The ultrasonic parameters are set to Ф6 (probe diameter is 6mm), 300W, 15min. Centrifuge again with a refrigerated centrifuge, and the centrifugation parameters are set to 4°C, 8000rpm, 20min. After centrifugation, take the supernatant as a crude enzyme solution containing wild-type β-furanosidase, and store it in a 4°C refrigerator for use.
[0048] Catalytic reaction of wild-type β-fructofuranosidase: 100mM / L rebaudioside A, 300mM / L sucrose, 5U / mL crude enzyme solution containing wild-type β-fructofuranosidase, and 50mM / L PBS buffer solution (pH 6.5) were added to the catalytic reaction system (5mL) and reacted at 45°C. After 30 minutes of reaction, 200uL of the reaction solution was inactivated at high temperature, diluted 100 times with pure water, and then detected by HPLC.
[0049] Test results such as Figure 2 As shown, the retention time of the catalytic product fructosyl rebaudioside A is 8.61 min.
[0050] Example 3
[0051] Homology modeling was used to simulate the spatial structure of the wild-type β-fructofuranosidase and predict potential thermal stability-related residues. These amino acid residues were mutated and 17 single-point mutants (A87G, N174D, D226E, Q287P, A394V, S477D, E478N, R170M, Q434I, R170Y, L404Y, D86F, Q434V, K178F, R170L, R273F, G108P) were obtained to improve enzyme activity and stability.
[0052] The recombinant plasmid pET-MsFFase constructed in Example 1 was used as a template, and PCR amplification was performed using the following primers and PrimeSTAR DNA polymerase (produced by TaKaRA). After verification by agarose gel electrophoresis, the template was digested by adding Dpn I restriction enzyme to the PCR reaction product. The digested PCR reaction product was then transformed into Escherichia coli BL21 (DE3) competent cells according to the method in Example 1, and inoculated into LB liquid culture medium containing 50 mg / L kanamycin, and the mutant plasmid was extracted after culturing at 37°C and 200 rpm for 12 h, and sent for testing and identification. The mutant recombinant plasmids pET-MsFFase-R170M, pET-MsFFase-Q434I, pET-MsFFase-R170Y, pET-MsFFase-L404Y, pET-MsFFase-D86F, pET-MsFFase-Q434V, pET-MsFFase-K178F, pET-MsFFase-R170L, pET-MsFFase-R273F, pET-MsFFase-G108P, pET-MsFFase-A87G, pET-MsFFase-N174D, pET-MsFFase-D226E, pET-MsFFase-Q287P, pET-MsFFase-A394V, pET-MsFFase-S477D, and pET-MsFFase-E478N were obtained.
[0053] The primers used in the above steps are as follows:
[0054] The primers for site-directed mutagenesis of mutant R170M are:
[0055] Forward primer: 5′-TAGCGCAATGGTTAGCAAAAACGGCGAAATCA-3′ (SEQ ID NO: 6);
[0056] Reverse primer: 5'-TGCTAACCATTGCGCTACCTGACCATTGGGTC-3' (SEQ ID NO: 7).
[0057] The primers for site-directed mutagenesis of mutant Q434I are:
[0058] Forward primer: 5′-GGTTGGTTATCAGCTTCATTGATACCATTGGCAC-3′ (SEQ ID NO: 8);
[0059] Reverse primer: 5'-GAAGCTGATAACCAACCCACCCGGCATAACAT-3' (SEQ ID NO: 9).
[0060] The primers for site-directed mutagenesis of mutant R170Y are:
[0061] Forward primer: 5′-TAGCGCATACGTTAGCAAAAACGGCGAAATCA-3′ (SEQ ID NO: 10);
[0062] Reverse primer: 5'-TGCTAACGTATGCGCTACCTGACCATTGGGTC-3' (SEQ ID NO: 11).
[0063] The primers for site-directed mutagenesis of mutant L404Y are:
[0064] Forward primer: 5′-GAATTTTTACGGAGGCCAGCCGTTTGCCCCGG-3′ (SEQ ID NO: 12);
[0065] Reverse primer: 5'-GGCCTCCGTAAAAATTCAAGTTCGTCGGGTTG-3' (SEQ ID NO: 13).
[0066] The primers for site-directed mutagenesis of mutant D86F are:
[0067] Forward primer: 5′-CGACGAATTTGCGAATCAGTATAGTGTTAATGGTTGG-3′ (SEQ ID NO: 14);
[0068] Reverse primer: 5'-GATTCGCAAATTCGTCGGTCAGAGGCCAGGTA-3' (SEQ ID NO: 15).
[0069] The primers for site-directed mutagenesis of mutant Q434V are:
[0070] Forward primer: 5'-GTTGGTTGTGAGCTTCATTGATACCATTGGCAC-3' (SEQ ID NO: 16);
[0071] Reverse primer: 5'-TGAAGCTCACAACCAACCCACCCGGCATAACA-3' (SEQ ID NO: 17).
[0072] The primers for site-directed mutagenesis of mutant K178F are:
[0073] Forward primer: 5′-CGGCGAAATCTTTCTGTTCTTCACCGACGTTGCG-3′ (SEQ ID NO: 18);
[0074] Reverse primer: 5'-GAAGAACAGAAAGATTTCGCCGTTTTTGCTAACACGT-3' (SEQ ID NO: 19).
[0075] The primers for site-directed mutagenesis of mutant R170L are:
[0076] Forward primer: 5′-TAGCGCACTGGTTAGCAAAAACGGCGAAATCA-3′ (SEQ ID NO: 20);
[0077] Reverse primer: 5'-TTTGCTAACCAGTGCGCTACCTGACCATTGGGTC-3' (SEQ ID NO: 21).
[0078] The primers for site-directed mutagenesis of mutant R273F are:
[0079] Forward primer: 5′-ATGCAGTTTGAAACCGCTACCTGTAATGAGGC-3′ (SEQ ID NO: 22);
[0080] Reverse primer: 5'-GCGGTTTCAAACTGCATAGCGCTGTTGCCCTC-3' (SEQ ID NO: 23).
[0081] The primers for site-directed mutagenesis of mutant G108P are:
[0082] Forward primer: 5′-AACCTGCCGTTCGATGACCGTCATGTCTTTGC-3′ (SEQ ID NO: 24);
[0083] Reverse primer: 5'-TCATCGAACGGCAGGTTCCGGTCGGCCACCAG-3' (SEQ ID NO: 25).
[0084] The primers for site-directed mutagenesis of mutant A87G are:
[0085] Forward primer: 5′-CGAAGATGGCAATCAGTATAGTGTTAATGG-3′ (SEQ ID NO: 26);
[0086] Reverse primer: 5'-ACTGATTGCCATCTTCGTCGGTCAGAGGCC-3' (SEQ ID NO: 27).
[0087] The primers for site-directed mutagenesis of mutant N174D are:
[0088] Forward primer: 5′-TAGCAAAGACGGCGAAATCAAGCTGTTCTT-3′ (SEQ ID NO: 28);
[0089] Reverse primer: 5'-TTTCGCCGTCTTTGCTAACACGTGCGCTAC-3' (SEQ ID NO: 29).
[0090] The primers for site-directed mutagenesis of mutant D226E are:
[0091] Forward primer: 5′-GGTGACGGAATTGCTGCAGGCTGATGGTAC-3′ (SEQ ID NO: 30);
[0092] Reverse primer: 5'-GCAGCAATTCCGTCACCTTGTTGAAACCGG-3' (SEQ ID NO: 31).
[0093] The primers for site-directed mutagenesis of mutant Q287P are:
[0094] Forward primer: 5′-TTATCGCCCGGGTGACCCGTATGCCGAAAC-3′ (SEQ ID NO: 32);
[0095] Reverse primer: 5'-GGTCACCCGGGCGATAACCCAGGTCTGCCT-3' (SEQ ID NO: 33).
[0096] The primers for site-directed mutagenesis of mutant A394V are:
[0097] Forward primer: 5′-CGGTTTGGTTCTGGGCAACCCGACGAACTT-3′ (SEQ ID NO: 34);
[0098] Reverse primer: 5'-TGCCCAGAACCAAACCGCTGCCGCCGTTCA-3' (SEQ ID NO: 35).
[0099] The primers for site-directed mutagenesis of mutant S477D are:
[0100] Forward primer: 5′-GTACGGCGATGAAGGCTTGGGTGGTTGGGC-3′ (SEQ ID NO: 36);
[0101] Reverse primer: 5'-AGCCTTCATCGCCGTACGAGTAATCCACAG-3' (SEQ ID NO: 37).
[0102] The primers for site-directed mutagenesis of mutant E478N are:
[0103] Forward primer: 5′-CGGCTCTAATGGCTTGGGTGGTTGGGCAGA-3′ (SEQ ID NO: 38);
[0104] Reverse primer: 5'-CCAAGCCATTAGAGCCGTACGAGTAATCCA-3' (SEQ ID NO: 39).
[0105] Example 4
[0106] Preparation of enzyme solution containing β-fructofuranosidase mutant
[0107] The recombinant strain containing the β-fructofuranosidase mutant plasmid in Example 3 was spread on LB solid plates (10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, 15 g / L agar) containing 50 μg / L kanamycin and placed in a 37°C incubator for 12 h. The next day, single colonies were selected from the plates and cultured in LB medium at 37°C and 200 rpm. When the absorbance (OD 600 ) reached 0.6-0.8, isopropyl-β-D-thiogalactoside (IPTG) was added to the culture medium with a final concentration of 0.1 mM, and the cells were induced at 20°C for 24 h.
[0108] Collect the above bacterial liquids and centrifuge them at 4°C, 8000rpm, 5min, discard the supernatant to obtain bacterial mud, and rinse twice with PBS buffer. Add 5mL PBS buffer per gram of bacterial cells, place in an ice-water mixture, and use an ultrasonic disruptor to ultrasonically disrupt the bacterial cells, with the parameters set to Ф6 (probe diameter is 6mm), 300W, 15min. Centrifuge again with a refrigerated centrifuge, with the centrifugation parameters set to 4°C, 8000rpm, 20min, and take the supernatant as the crude enzyme solution containing the β-fructofuranosidase mutant, and store it in a 4°C refrigerator for later use.
[0109] Example 5
[0110] Detection of enzyme activity and thermal stability
[0111] The single point mutants obtained by the above screening were compared with the wild-type β-fructofuranosidase for enzyme activity and thermal stability to evaluate the effect of the single point mutation.
[0112] Enzyme activity assay: Add 10mM / L rebaudioside A, 30mM / L sucrose and 10μg / mL crude enzyme solution to 1mL β-fructofuranosidase activity assay system, and then make up the remaining volume with PBS buffer (pH 6.5), and react for 10min at 45℃ and 200rpm. Finally, inactivate the reaction solution in a 95℃ water bath for 15min. Dilute the inactivated reaction solution ten times with pure water for HPLC detection.
[0113] The activity of β-fructofuranosidase is defined as: the amount of enzyme required to convert sucrose to produce 1 μmol of fructosyl rebaudioside A within 1 minute is 1 enzyme activity unit (U).
[0114] Thermal stability assay: 10 μg / mL crude enzyme solution was placed in PBS (pH 6.5) buffer. After incubation at 45°C for 1 hour, the enzyme activity was detected under the above experimental conditions. The activity of the wild-type β-fructofuranosidase without incubation at 45°C was taken as 100%, and the relative enzyme activity of the wild-type β-fructofuranosidase and the mutant at different times was calculated. The results are shown in Table 1.
[0115] Table 1 Relative enzyme activities of wild type and single point mutants
[0116]
[0117]
[0118] As shown in Table 1, the initial relative enzyme activities of some single-point mutants were better than those of the wild type. The relative enzyme activities of mutants A87G, D226E, Q287P, S477D, D86F, G108P, K178F and L404Y exceeded those of wild-type β-fructofuranosidase, among which mutant L404Y had the highest enzyme activity, which was 1.46 times that of wild-type β-fructofuranosidase. The enzyme activities of mutants R170Y, Q434V, A394V and R170L were significantly reduced, while the enzyme activities of mutants R170M, N174D, Q434I, E478N and R273F remained basically unchanged.
[0119] The residual enzyme activity retention rates of mutants D86F, G108P, K178F, L404Y, and A394V exceeded those of wild-type β-fructofuranosidase, among which mutant L404Y had the highest residual enzyme activity retention rate and had higher thermal stability. The residual enzyme activity retention rates of mutants R170M, Q434I, A87G, D226E, and Q287P were significantly lower than those of the wild-type, and their thermal stability was poor.
[0120] The comprehensive results of enzyme activity and thermal stability tests showed that the mutation sites of mutants L404Y, D86F, G108P and K178F were positive mutation points that improved the performance of β-fructofuranosidase.
[0121] Example 6
[0122] The forward single-point mutations were combined to evaluate the mutation effect of the double-point combination mutation. The combined mutation method is as follows:
[0123] Double point mutant K178F / L404Y:
[0124] Using the recombinant plasmid pET-MsFFase-K178F as a template, the mutation method in Example 3 was used to perform combined mutation with L404Y.
[0125] The primers for K178F / L404Y site-directed mutagenesis are:
[0126] Forward primer: 5′-GAATTTTTACGGAGGCCAGCCGTTTGCCCCGG-3′ (SEQ ID NO: 12);
[0127] Reverse primer: 5'-GGCCTCCGTAAAAATTCAAGTTCGTCGGGTTG-3' (SEQ ID NO: 13).
[0128] Double point mutant D86F / G108P:
[0129] Using the recombinant plasmid pET-MsFFase-D86F as a template, the mutation method in Example 3 was used to perform combined mutation with G108P.
[0130] The primers for D86F / G108P site-directed mutagenesis are:
[0131] Forward primer: 5′-AACCTGCCGTTCGATGACCGTCATGTCTTTGC-3′ (SEQ ID NO: 24);
[0132] Reverse primer: 5'-TCATCGAACGGCAGGTTCCGGTCGGCCACCAG-3 (SEQ ID NO: 25).
[0133] Double point mutant D86F / K178F:
[0134] Using the recombinant plasmid pET-MsFFase-D86F as a template, the mutation method in Example 3 was used to perform combined mutation with K178F.
[0135] The primers for D86F / K178F site-directed mutagenesis are:
[0136] Forward primer: 5′-CGGCGAAATCTTTCTGTTCTTCACCGACGTTGCG-3′ (SEQ ID NO: 18);
[0137] Reverse primer: 5'-GAAGAACAGAAAGATTTCGCCGTTTTTGCTAACACGT-3' (SEQ ID NO: 19).
[0138] Double point mutant D86F / L404Y:
[0139] Using the recombinant plasmid pET-MsFFase-D86F as a template, the mutation method in Example 3 was used to perform combined mutation with L404Y.
[0140] The primers for D86F / L404Y site-directed mutagenesis are:
[0141] Forward primer: 5′-GAATTTTTACGGAGGCCAGCCGTTTGCCCCGG-3′ (SEQ ID NO: 12);
[0142] Reverse primer: 5'-GGCCTCCGTAAAAATTCAAGTTCGTCGGGTTG-3' (SEQ ID NO: 13).
[0143] Double point mutant G108P / K178F:
[0144] Using the recombinant plasmid pET-MsFFase-G108P as a template, the mutation method in Example 3 was used to perform combined mutation with K178F.
[0145] The primers for G108P / K178F site-directed mutagenesis are:
[0146] Forward primer: 5′-CGGCGAAATCTTTCTGTTCTTCACCGACGTTGCG-3′ (SEQ ID NO: 18);
[0147] Reverse primer: 5'-GAAGAACAGAAAGATTTCGCCGTTTTTGCTAACACGT-3' (SEQ ID NO: 19).
[0148] Double point mutant G108P / L404Y:
[0149] Using the recombinant plasmid pET-MsFFase-G108P as a template, the mutation method in Example 3 was used to perform combined mutation with L404Y.
[0150] The primers for G108P / L404Y site-directed mutagenesis are:
[0151] Forward primer: 5′-GAATTTTTACGGAGGCCAGCCGTTTGCCCCGG-3′ (SEQ ID NO: 12);
[0152] Reverse primer: 5'-GGCCTCCGTAAAAATTCAAGTTCGTCGGGTTG-3' (SEQ ID NO: 13).
[0153] Enzyme activity assay: Add 10mM / L rebaudioside A, 30mM / L sucrose and 10μg / mL crude enzyme solution to 1mL β-fructofuranosidase activity assay system, and then make up the remaining volume with PBS buffer (pH 6.5), and react for 10min at 45℃ and 200rpm. Finally, inactivate the reaction solution in a 95℃ water bath for 15min. Dilute the inactivated reaction solution ten times with pure water for HPLC detection.
[0154] The activity of β-fructofuranosidase is defined as: the amount of enzyme required to convert sucrose to produce 1 μmol of fructosyl rebaudioside A within 1 minute is 1 enzyme activity unit (U).
[0155] Thermal stability assay: 10 μg / mL crude enzyme solution was placed in PBS (pH 6.5) buffer. After incubation at 45°C for 1 hour, the enzyme activity was measured under the above experimental conditions. The activity of the wild-type β-fructofuranosidase without incubation at 45°C was taken as 100%, and the relative enzyme activity of the wild-type β-fructofuranosidase and the mutant at different times was calculated. The results are shown in Table 2.
[0156] Table 2 Relative enzyme activities of wild type and multi-point combination mutants
[0157] 0h relative enzyme activity (%) 1h relative enzyme activity (%) Remaining enzyme activity retention rate (%) wild type 100 63.27 63.27 D86F / G108P 69.48 49.53 71.29 D86F / K178F 43.79 34.30 78.33 D86F / L404Y 138.18 101.36 73.35 G108P / K178F 72.87 31.05 42.61 G108P / L404Y 58.02 37.56 64.74 K178F / L404Y 184.92 165.76 89.64
[0158] As shown in Table 2, the initial relative enzyme activity of some multi-point combination mutants is better than that of the wild type. The relative enzyme activities of K178F / L404Y and D86F / L404Y exceed those of the wild type β-fructofuranosidase. The enzyme activities of D86F / L404Y and K178F / L404Y are 1.38 times and 1.85 times of those of the wild type, respectively. The enzyme activities of mutants G108P / L404Y, D86F / G108P, G108P / K178F and D86F / K178F are significantly lower than those of the wild type.
[0159] The residual retained enzyme activity of the combined mutant G108P / K178F was lower than that of the wild type, indicating that its thermal stability was reduced; the thermal stability of the combined mutants D86F / G108P, D86F / K178F, K178F / L404Y and D86F / L404Y were significantly higher than that of the wild type, among which K178F / L404Y had the highest residual enzyme activity of 89.64%, which was 1.42 times that of the wild type.
[0160] Example 7
[0161] β-Fructofuranosidase mutant K178F / L404Y catalyzes the synthesis of fructosyl rebaudioside A
[0162] The gene fragment encoding the β-fructofuranosidase mutant K178F / L404Y was codon-optimized (as shown in SEQ ID NO: 5) and inserted into the expression vector pET28a (produced by Novagen) between the NcoⅠ and XhoⅠ restriction sites to obtain the recombinant plasmid pET-MsFFase-L404Y-K178F.
[0163] The recombinant plasmid pET-MsFFase-L404Y-K178F was added to the competent cells of E. coli BL21 (DE3), mixed, placed in a 42°C water bath for 45 seconds after ice bathing for 30 minutes, placed in a 37°C shaker for activation for 120 minutes after ice bathing for 2 minutes. The bacterial solution was spread on an LB plate containing 50 mg / L kanamycin, and cultured in a 37°C incubator for 12 hours to obtain the recombinant strain MsFFase-L404Y-K178F.
[0164] The recombinant strain MsFFase-L404Y-K178F was spread on a LB solid plate (10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, 15 g / L agar) containing 50 mg / L kanamycin and placed in a 37°C incubator for 12 h. The next day, a single colony was selected from the plate and cultured in LB medium at 37°C and 200 rpm. When the absorbance (OD 600 ) reached 0.6-0.8, isopropyl-β-D-thiogalactoside (IPTG) was added to the culture medium with a final concentration of 0.1 mM, and the culture was induced at 20°C for 24 h.
[0165] The fermented bacterial liquid was collected and centrifuged at 4°C, 8000rpm, 5min, and the supernatant was discarded to obtain bacterial mud, which was washed twice with PBS buffer. 5mL PBS buffer was added per gram of bacterial cells, placed in an ice-water mixture, and the bacterial cells were ultrasonically disrupted with an ultrasonic disruptor, with the parameters set to Ф6 (probe diameter 6mm), 300W, 15min. Centrifuge again with a refrigerated centrifuge, with the centrifugation parameters set to 4°C, 8000rpm, 20min, and the supernatant was taken as the crude enzyme solution containing the β-fructofuranosidase mutant K178F / L404Y, and stored in a 4°C refrigerator for later use.
[0166] Catalytic reaction: Add 100 mM / L rebaudioside A, 300 mM / L sucrose, 5 U / mL crude enzyme solution containing β-fructofuranosidase mutant K178F / L404Y, and 50 mM / L PBS buffer solution (pH 6.5) to the catalytic reaction system (5 mL), react at 45°C for 5 h, inactivate at high temperature, and centrifuge to obtain the supernatant, which is fructosyl rebaudioside A.
[0167] After 5 h of reaction, the conversion rate of rebaudioside A was 74.21%, and the yield of fructosyl rebaudioside A was 83.78 g / L.
[0168] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A β-fructofuranosidase mutant, characterized in that: The β-fructofuranosidase mutant is derived from a mutation in one or more of the 86th, 87th, 108th, 170th, 174th, 178th, 226th, 273rd, 287th, 394th, 404th, 434th, 477th or 478th positions of the amino acid sequence of the wild-type β-fructofuranosidase shown in SEQ ID NO:
1.
2. The β-fructofuranosidase mutant according to claim 1, characterized in that The β-fructofuranosidase mutant is obtained by causing any one or more of the following mutations to the amino acid sequence shown in SEQ ID NO: 1: The 86th amino acid mutated from D to F; The 87th amino acid mutated from A to G; The amino acid at position 108 mutated from G to P; The 170th amino acid mutates from R to any of M, Y, or L; The 174th amino acid mutated from N to D; The amino acid at position 178 mutated from K to F; The amino acid at position 226 mutated from D to E; The amino acid at position 273 mutated from R to F; The amino acid at position 287 mutated from Q to P; The amino acid at position 394 mutated from A to V; The amino acid at position 404 mutated from L to Y; The amino acid at position 434 mutated from Q to one of I or V; The amino acid at position 477 mutated from S to D; The amino acid at position 478 mutated from E to N.
3. The β-fructofuranosidase mutant according to claim 2, characterized in that The amino acid sequence of the β-fructofuranosidase mutant is shown in SEQ ID NO:2 or SEQ ID NO:
3.
4. An application of a β-fructofuranosidase mutant, characterized in that: Use of the β-fructofuranosidase mutant according to any one of claims 1 to 3 in catalyzing the synthesis of fructosyl rebaudioside A.
5. A nucleic acid sequence, characterized in that The nucleic acid sequence encodes the β-fructofuranosidase mutant according to any one of claims 1 to 3.
6. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleic acid sequence according to claim 5.
7. A recombinant cell, characterized in that The recombinant cell contains the nucleic acid sequence of claim 5 or the recombinant expression vector of claim 6.
8. A method for synthesizing fructosyl rebaudioside A using a β-fructofuranosidase mutant, characterized in that: The following steps are involved: Rebaudioside A, sucrose and an enzyme solution containing the β-fructofuranosidase mutant according to any one of claims 1 to 3 are added to the catalytic reaction system, reacted for 1 to 10 hours, inactivated the enzyme, and centrifuged to obtain the supernatant, which is fructosyl rebaudioside A.
9. The method according to claim 8, characterized in that The preparation method of the enzyme solution containing the β-fructofuranosidase mutant according to any one of claims 1 to 3 is as follows: (1) constructing a gene encoding the β-fructofuranosidase mutant according to any one of claims 1 to 3 into an expression vector to obtain a recombinant expression vector; (2) transforming the recombinant expression vector into a host bacterium to obtain a recombinant strain containing a β-fructofuranosidase mutant expression system. (3) After activating the recombinant strain, inoculate it into LB medium, add isopropyl-β-D-thiogalactoside with a final concentration of 0.02-1 mM / L as an inducer, and induce culture for 18-36 hours. Centrifuge and collect the bacteria, resuspend the bacteria and break the cells, and collect the supernatant by centrifugation, which is the enzyme solution containing the β-fructofuranosidase mutant.
10. The method according to claim 8, characterized in that In the catalytic reaction system, the concentration of rebaudioside A is 1-500 mM / L, the concentration of sucrose is 1-2000 mM / L, and the added amount of the enzyme solution containing the β-fructofuranosidase mutant is 0.1-10 U / mL.