Fructose-1,6-bisphosphatase with high temperature stability and use thereof

Abstract

The application provides a fructose-1,6-bisphosphatase with high-temperature stability and an application thereof, and specifically belongs to the technical field of biological enzyme engineering. The prepared fructose-1,6-bisphosphatase simultaneously considers enzyme activity, overcomes the contradiction that stability and activity are difficult to be obtained in traditional modification, synchronously solves the industry problems of heat inactivation and low catalytic efficiency, and provides a transformative enzyme tool for the fields of biological manufacturing, medicine and diagnosis.

Description

Technical Field

[0001] This invention relates to the field of bioenzyme engineering technology, and in particular to a high-temperature stable fructose-1,6-bisphosphatase and its applications. Background Technology

[0002] Fructose-1,6-bisphosphatase (FBPase), a key rate-limiting enzyme in the Calvin cycle, catalyzes the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate, playing a central role in the gluconeogenesis pathway. However, natural FBPase has significant drawbacks: its poor thermal stability leads to easy inactivation in industrial fermentation environments of 40-60°C, forcing the addition of cooling stages in biofuel production processes, significantly increasing energy consumption and costs; simultaneously, its catalytic efficiency is insufficient, exhibiting low affinity for the substrate F-1,6-BP (Km≈7.96 μM) and strong inhibition by AMP (Ki=111.09 μM), severely limiting gluconeogenesis flux. In medical applications, existing FBPase inhibitors (such as sulfonylureas) are prone to off-target effects due to poor selectivity, while enzyme overexpression in gene therapy may lead to metabolic imbalances. Although protein engineering attempts to improve performance through site-directed mutagenesis (such as M251L and Asp32 modification), the improvement in thermal stability is limited; chemical immobilization technology faces challenges such as active site masking and low recovery rate; and metabolic engineering strategies lack universality due to host dependence.

[0003] The industry urgently needs to develop a new FBPase variant that can significantly enhance high-temperature tolerance while maintaining high catalytic activity.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a high-temperature stable fructose-1,6-bisphosphatase and its applications. This invention introduces mutations into the amino acid sequence of wild-type fructose-1,6-bisphosphatase, thereby enhancing its high-temperature tolerance while maintaining catalytic efficiency, thus giving it excellent thermal stability.

[0006] To achieve the above-mentioned objectives of this invention, the following technical solution is adopted: This invention provides a high-temperature stable fructose-1,6-bisphosphatase, which is obtained by mutation based on wild-type fructose-1,6-bisphosphatase. The amino acid sequence of the wild-type fructose-1,6-bisphosphatase is shown in SEQ ID NO.1. The mutation is selected from any one of the following: R193P, K222L, D228H, K229E, A263Y, S264E, E295Y, L305A, N41R, N41Y, G99E, K104R, E143K, E144Y, V154L, A155V, A155M, H176R, Q191Y, C224T, Q225K. Triple mutations of Q225R, T231D, N232D, N250I, E295Q, G314A, S16P, H17G, E55S, Q63M, Q63K, S123T, S183E, E198Q, E207Q, E227N, K229P, E98K, D39Y+V46I+N41L, or double mutations of L305A+D39Y.

[0007] Preferably, the mutation is selected from any one of the following: T231D, N41Y, Q225K, S16P, H17G, K229P.

[0008] The amino acid sequence of R193P is shown in SEQ ID NO. 22; The amino acid sequence of K222L is shown in SEQ ID NO. 23; The amino acid sequence of D228H is shown in SEQ ID NO. 24; The amino acid sequence of K229E is shown in SEQ ID NO. 25; The amino acid sequence of A263Y is shown in SEQ ID NO. 26; The amino acid sequence of S264E is shown in SEQ ID NO. 27; The amino acid sequence of E295Y is shown in SEQ ID NO. 28; The amino acid sequence of L305A is shown in SEQ ID NO. 29; The amino acid sequence of N41R is shown in SEQ ID NO. 30; The amino acid sequence of N41Y is shown in SEQ ID NO. 31; The amino acid sequence of G99E is shown in SEQ ID NO. 32; The amino acid sequence of K104R is shown in SEQ ID NO. 33; The amino acid sequence of E143K is shown in SEQ ID NO. 34; The amino acid sequence of E144Y is shown in SEQ ID NO. 35; The amino acid sequence of V154L is shown in SEQ ID NO. 36; The amino acid sequence of A155V is shown in SEQ ID NO. 37; The amino acid sequence of A155M is shown in SEQ ID NO. 38; The amino acid sequence of H176R is shown in SEQ ID NO. 39; The amino acid sequence of Q191Y is shown in SEQ ID NO. 40; The amino acid sequence of C224T is shown in SEQ ID NO. 41; The amino acid sequence of Q225K is shown in SEQ ID NO. 42; The amino acid sequence of Q225R is shown in SEQ ID NO. 43; The amino acid sequence of T231D is shown in SEQ ID NO. 45; The amino acid sequence of N232D is shown in SEQ ID NO. 46; The amino acid sequence of N250I is shown in SEQ ID NO. 47; The amino acid sequence of E295Q is shown in SEQ ID NO. 49; The amino acid sequence of G314A is shown in SEQ ID NO. 50; The amino acid sequence of S16P is shown in SEQ ID NO. 51; The amino acid sequence of H17G is shown in SEQ ID NO. 52; The amino acid sequence of E55S is shown in SEQ ID NO. 53; The amino acid sequence of Q63M is shown in SEQ ID NO. 54; The amino acid sequence of Q63K is shown in SEQ ID NO. 55; The amino acid sequence of S123T is shown in SEQ ID NO. 57; The amino acid sequence of S183E is shown in SEQ ID NO. 58; The amino acid sequence of E198Q is shown in SEQ ID NO. 59; The amino acid sequence of E207Q is shown in SEQ ID NO. 60; The amino acid sequence of E227N is shown in SEQ ID NO. 61; The amino acid sequence of K229P is shown in SEQ ID NO. 62; The amino acid sequence of E98K is shown in SEQ ID NO. 64; The amino acid sequence of D39Y+V46I+N41L is shown in SEQ ID NO. 67; The amino acid sequence of L305A+D39Y is shown in SEQ ID NO. 68.

[0009] The present invention also provides a polynucleotide encoding the above-mentioned high-temperature stable fructose-1,6-bisphosphatase.

[0010] The present invention also provides a recombinant vector comprising the above-mentioned polynucleotides.

[0011] The present invention also provides a host cell comprising the above-mentioned polynucleotides or the above-mentioned recombinant vector.

[0012] Preferably, the host cell is a prokaryotic cell or a eukaryotic cell.

[0013] The present invention also provides a genetically engineered bacterium containing the above-mentioned polynucleotides or a recombinant vector.

[0014] This invention also provides a method for preparing a high-temperature stable fructose-1,6-bisphosphatase thermostable enzyme, comprising the following steps: S1. Select the mutation site and introduce the mutation into the gene for fructose-1,6-bisphosphatase through primer design; S2. Transform the recombinant plasmid into competent cells and screen for positive clones; S3. Induce protein expression and purify the protein to obtain the final product.

[0015] This invention also provides the application of the above-mentioned high-temperature stable fructose-1,6-bisphosphatase in any of the fields of industrial biotechnology, medicine, agriculture and synthetic biology, and immobilized or diagnostic reagents.

[0016] Preferably, the industrial biotechnology specifically includes biofuel production, sugar alcohol synthesis, and cellulose degradation process optimization; Preferably, the pharmaceutical field specifically includes drug target screening, enzyme replacement therapy, and covalent inhibitor development for metabolic diseases such as diabetes and obesity; Preferably, agriculture and synthetic biology specifically include constructing heat-resistant crop or algal energy-producing strains to improve photosynthetic efficiency and stress resistance; Preferably, the preparation of diagnostic reagents specifically includes heat-stable reagents for the detection of fructose-1,6-bisphosphatase activity, supporting genetic disease screening and metabolic research.

[0017] The present invention also provides a method for catalytically hydrolyzing fructose-1,6-bisphosphate to produce fructose-6-phosphate, comprising the step of adding the above-mentioned fructose-1,6-bisphosphatase to the reaction system.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: The prepared fructose-1,6-bisphosphatase increases the upper limit of the working temperature from 40℃ to 48℃-52℃, which can meet the needs of industrial high-temperature fermentation. At the same time, it takes into account enzyme activity, overcomes the contradiction of "stability and activity are difficult to achieve at the same time" in traditional modification, and solves the industry problems of thermal inactivation and low catalytic efficiency simultaneously, providing a transformative enzyme tool for the fields of biomanufacturing, medicine and diagnostics.

[0019] The sequence of wild-type fructose-1,6-bisphosphatase is SEQ ID NO.1: MKTLGEFIVEKQHEFSHATGELTALLSAIKLGAKIIHRDINKAGLVDILGASGAENVQGEVQQKLDLFANEKLKAALKARDIVAGIASEEEDEIVVFEGCEHAKYVVLMDPLDGSSNIDVNVSVGTIFSIYRRVTPVGTPVTEEDFLQPGNKQVAAGYVVYGSSTm LVYTTGCGVHAFTYDPSLGVFCLCQERMRFPEKGKTYSINEGNYIKFPNGVKKYIKFCQEEDKSTNRPYTSRYIGSLVADFHRNLLKGGIYLYPSTASHPDGKLRLLYECNPMAFLAEQAGGKASDGKERILDIIPETLHQRRSFFVGNDHMVEDVERFIREFPDA. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 : SDS-PAGE results of FBP; Figure 2 Results of thermal stability test after incubation at 44℃ for 30 min; Figure 3 Results of thermal stability test after incubation at 44℃-52℃ for 30 min; Figure 4 Results of thermal stability test after superimposed mutations were incubated at 44℃ for 30 min; Figure 5 Kinetic curve of wild-type fructose-1,6-bisphosphatase at 30℃. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0023] The following is a detailed explanation using specific embodiments: The primers used in the embodiments of this invention are as follows: Q12H-F:AAACTCGTGATGCTTTTCGACAATAAATT Q12H-R:GTCGAAAAGCATCACGAGTTTTCTCATGC F15H-F:AGCATGAGAATGCTCGTGCTGCTTTTC F15H-R:CAGCACGAGCATTCTCATGCTACCGGTGA S16H-F:GGTAGCATGATGAAACTCGTGCTGCTTTT S16H-R:CACGAGTTTCCATGCTACCGGTGAG A18Y-F:CTCACCGGTATAATGAGAAAACTCGTGCT A18Y-R:TTTTTCATTATACCGGTGAGCTCACTGC D39Y-F:TTTGTTGATATAGCGATGGATAATCTTGGCG D39Y-R:ATCCATCGCTATATCAACAAAGCAGGACT I40A-F:TGCTTTGTTTGCATCGCGATGGATAATCT I40A-R:CATCGCGATGCAAACAAAGCAGGACTG N41L-F:TCCTGCTTTCAGGATATCGCGATGGATAA N41L-R:CGCGATATCCTGAAAGCAGGACTGGTTGA A43H-F:AACCAGTCCATGTTTGTTGATATCGCGATV A43H-R:ATCAACAACATGGACTGGTTGATATCCT V46I-F:CAGGATATCAATCAGTCCTGCTTTGTTGA V46I-R:GCAGGACTGATTGATATCCTGGGTGCCAG S52T-F:CTCAGCACCGGTGGCACCCAGGATATCAA S52T-R:CTGGGTGCCACCGGTGCTGAGAACGTG Q62H-F:GAGTTTCTGATGAACCTCGCCCTGCAC Q62H-R:GGCGAGGTTCATCAGAAACTCGACTTGTT V96Y-F:GCCTTCAAAATAGACAATCTCATCTTCTT V96Y-R:GAGATTGTCTATTTTGAAGGCTGTGAACACG G99K-F:GTGTTCACATTTTTCCAAAGACGACAATCT G99K-R:GTCTTTGAAAAATGTGAACACGCAAAATACGT C100T-F:TGCGTGTTCGGTGCCTTCAAAGACGACAA C100T-R:TTTGAAGGCACCGAACACGCAAAATAC V137L-F:CGGCGTGCCCAGAGGCGTAACGCGGCGGT V137L-R:GTTACGCCTCTGGGCACGCCGGTAACGGA E143V-F:GAAATCTTCAACCGTTACCGGCGTGCCAA E143V-R:CCGGTAACGGTTGAAGATTTCCTCCAG E144L-F:GAGAATCCAGTTCCGTTACCGGCGTG E144L-R:GTAACGGAACTGGATTTCCTCCAGCCTG Q191T-F:CATCCGTTCGGTGCACAGGCAGAAAAC Q191T-R:TGCCTGTGCACCGAACGGATGCGCTTC Q191H-F:CATCCGTTCATGGCACAGGCAGAAAAC Q191H-R:TGCCTGTGCCATGAACGGATGCGCTTC E192H-F:GCGCATCCGATGCTGGCACAGGCAGAAAAA E192H-R:CTGTGCCAGCATCGGATGCGCTTCCCGGA R193P-F:GAAGCGCATCGGTTCCTGGCACAGGCAGA R193P-R:TGCCAGGAACCGATGCCTTCCCGGAGAA K222L-F:CTGGCAGAACAGATGTACTTCTTCACCC K222L-R:AAGTACATTCTGTTCTGCCAGGA D228H-F:GGTGGATTTATGTTCTTCCTGGCAGAATT D228H-R:GGAGAAACAAATCCACCAACCGCC K229E-F:GTTGGTGGATTCATCTCTTCCTGGCAGA K229E-R:GAAGAAGATGAATCCACCAACCGCCCTTA A263Y-F:CGGGTGGCTATAGGTGCTTGGGTAGAGAT A263Y-R:CCAAGCACCTATAGCCACCCGGACGGCAA S264E-F:GTCCGGGTGTTCGGCGGTGCTTGGTAGA S264E-R:AGCACCGCCGAAACCCGGACGGCAAACT E295Y-F:CAGAATACGATATTGCCATCGCTCGCTT E295Y-R: GATGGCAAATATCGTATTCTGGATATCATCCCG L305A-F:GCGCTGGTGTGCGGTTTCCGGGATGATAT L305A-R:CCGGAAACCGCACACCAGCGCCGTTCATT A155M-F:CAAACAGGTTATGGCAGGTTACGTGGTATACGGCTCCTCTAC A155M-R: AACCTGTTTGTTACCAGGCTGGAGG A155V-F:CAAACAGGTTGTTGCAGGTTACGTGGTATACGGCTCCTCTAC A155V-R: AACCTGTTTGTTACCAGGCTGGAGG C224T-F:GTACATTAAATTCACCCAGGAAGAAGATAAATCCACCAACCGCC C224T-R:GAATTTAATGTACTTCTTCACCCCGTTCGGAAAC E143K-F:CGCCGGTAACGAAAGAAGATTTCCTCCAGCC E143K-R:CTTCCGTTACCGGCGTGCCAACAGG E144Y-F:CCGGTAACGGAATACGATTTCCTCCAGCCTGGTAACAAAC E144Y-R:CTTCTTCCGTTACCGGCGTGCCAAC E295Q-F:GAGCGATGGCAAACAACGTATTCTGGATATCATCCCGG E295Q-R:TTTGCCATCGCTCGCTTTACCGC G99E / E98K-F:GTCGTCTTTAAAGAATGTGAACACGCAAAATACGTG G99E / E98K-R:CACAGCCTTCAAAGACGACAATCTCATCTTCTTCTTC G314A-F:GTTCATTCTTTGTCGCGAACGACCATATGGTTGAAGATGTCG G314A-R:GACAAAGAATGAACGGCGCTGGTGC H176A-F:GATGCGGTGTTCGGGCCTTTACTTACGATCCTTCGC H176A-R:AACACCGCATCCGGTGGTGTAAAC K104R-F:GTATTTTGCGTGTTCACAGCCTTCAAAGAC K104R-R:GAACACGCACGCTACGTGGTGCTGATGGACCCCCTG N41R-F:CCATCGCGATATCCGCAAAGCAGGACTGGTTGATATCC N41R-R:GATATCGCGATGGATAATCTTGGCGCCCAG N41Y-F:CCATCGCGATATCTACAAAGCAGGACTGGTTGATATCC N41Y-R:GATATCGCGATGGATAATCTTGGCGCCCAG N232D-F:GATAAATCCACCGATCGCCCTTATACCTCACGTTATATC N232D-R:GGTGGATTTATCTTCTTCCTGGCAG N250H-F:GCGGATTTCCACCGTCACCTGCTGAAAGGCGGTATTTATC N250H-R:ACGGTGGAAATCCGCGACCAGTG N250I-F:CGGATTTCCACCGTATTCTGCTGAAAGGCGGTATTTATC N250I-R:ACGGTGGAAATCCGCGACCAGTG Q191Y-F:CTGCCTGTGCTACGAACGGATGCGCTTCCCG Q191Y-R:GCACAGGCAGAAAACGCCGAG Q225K-F:CATTAAATTCTGCAAAGAAGAAGATAAATCCACCAACCGCCC Q225K-R:GCAGAATTTAATGTACTTCTTCACCCCGTTCGG Q225R-F:CATTAAATTCTGCCGTGAAGAAGATAAATCCACCAACCGCCC Q225R-R:GCAGAATTTAATGTACTTCTTCACCCCGTTCGG T231D-F:GAAGATAAATCCGATAACCGCCCTTATACCTCACGTTATATCGG T231D-R:GGATTTATCTTCTTCCTGGCAGAATTTAATGTAC T231E-F:GAAGATAAATCCGAAAACCGCCCTTATACCTCACGTTATATCGG T231E-R:GGATTTATCTTCTTCCTGGCAGAATTTAATGTAC V154L-F:GTAACAAACAGCTCGCGGCAGGTTACGTGGTATACGGC V154L-R:CTGTTTGTTACCAGGCTGGAGGAAATC E55S-F:CAGCGGTGCTAGCAACGTGCAGGGCGAGGTTC E55S-R:AGCACCGCTGGCACCCAGG E143K-F:GGCACGCCGGTAACGAAAGAAGATTTCCTCCAGCCTGG E143K-R:CGTTACCGGCGTGCCAACAGGC E198Q-F:GATGCGCTTCCCGCAGAAAGGCAAAACCTACTCC E198Q-R:CGGGAAGCGCATCCGTTCCTGG E207Q-F:CCTACTCCATCAACCAGGGAAACTACATTAAGTTTCCGAACGG E207Q-R:GTTGATGGAGTAGGTTTTGCCTTTCTCCG E227N-F:CTGCCAGGAAAACGATAAATCCACCAACCGCCCTTATAC E227N-R:TTCCTGGCAGAATTTAATGTACTTCTTCACCCCG H17G-F:CACGAGTTTTCTGGTGCTACCGGTGAGCTCACTGC H17G-R:AGAAAACTCGTGCTGCTTTTCGAC K229P-F:CCAGGAAGAAGATCCTTCCACCAACCGCCCTTATACCTCAC K229P-R:ATCTTCTTCCTGGCAGAATTTAATGTACTTCTTCAC N121E-F:CCAACATCGATGTTGAAGTCTCTGTCGGTACCATTTTCTCC N121E-R:AACATCGATGTTGGACGAGCCATCC Q63K-F:CAGGGCGAGGTTCAGAAAAAACTCGACTTGTTCGCTAATG Q63K-R:CTGAACCTCGCCCTGCACGTTCTC Q63M-F:CAGGGCGAGGTTCAGATGAAACTCGACTTGTTCGCTAATG Q63M-R:CTGAACCTCGCCCTGCACGTTCTC S16P-F:CAGCACGAGTTTCCCCATGCTACCGGTGAGCTCACTGC S16P-R:AAACTCGTGCTGCTTTTCGACAATAAATTC S123T-F:GATGTTAACGTCACCGTCGGTACCATTTTCTCCATCTACCGCC S123T-R:GACGTTAACATCGATGTTGGACGAGC S183E-F:CTTTACTTACGATCCTGAACTCGGCGTTTTCTGCCTGTGCC S183E-R:AGGATCGTAAGTAAAGGCGTGAACACCGCATC S310P-F:GCACCAGCGCCGTCCGTTCTTTGTCGGCAACGACC S310P-R:ACGGCGCTGGTGCAGGGTTTCCGGGATG This invention employs five rational design approaches. Molecular dynamics simulations of the FBP were performed at three temperatures: 300K, 340K, and 380K. By comparing the differences in structural fluctuations (RMSF) at various sites of the protein after simulation, hotspot regions with relatively significant thermal structural changes were identified. Next, the trajectory file was processed using the cpptraj program in Amber Tools, and IDEA analysis was performed on the calculated protein structural motion to obtain near-field and far-field residue sites associated with the vibrational modes of these structural hotspots. Finally, mutation tools were used to mutate these hotspots.

[0024] Mutations were designed to fill structural gaps, enhance internal hydrophobic interactions, increase internal hydrogen bonding, harden proline residues in loop structures, deglycinate helix structures, and increase surface salt bridge interactions. Sequence identity analysis was also performed. By comparing sequences with thermostable homologs, residues that differed from existing sequences at specific sites in thermostable sequences and had a high probability of appearing in thermostable sequences were selected for mutation. The mutated sequences are listed in SEQ ID NO. 2-SEQ ID NO. 68.

[0025] Example 1 (1) Construction of FBPase mutants. Different mutant sequences of FBPase were designed as primers. The PCR product and linearized vector were mixed in a certain proportion using a DNA Assembly Cloning Kit and transformed at 50°C for 15 min under the catalysis of recombinase. The assembled plasmid was transformed into DH5a competent cells and plated on plates containing AMP resistance and cultured overnight at 37°C. After culture, single clones were picked for PCR verification. Positive clones were cultured overnight in test tubes, and the plasmid was extracted, transformed into BL21 competent cells, plated on plates containing AMP resistance, and cultured overnight at 37°C to obtain protein expression strains.

[0026] (2) Cell culture and protein purification. Inoculate 5 mL of LB medium with AMP resistance during fermentation and incubate overnight at 37°C. Transfer 5 mL of the seed culture to 1 L of LB medium with AMP resistance and incubate at 37°C and 220 rpm for 2-4 hours. When the bacterial concentration is around 0.6-0.8, begin cooling. After cooling to 16°C, add 0.5 mM IPTG to begin low-temperature induction culture. After overnight culture, centrifuge to remove the supernatant. Collect the precipitate in a 50 mL centrifuge tube for protein purification. Resuspend the culture in 30 mL of 25 mM Tris-HCl and 500 mM NaCl buffer, and vortex to mix. Add 3-5 mL of TieChui lysis buffer to the bacterial suspension, mix well, and incubate at room temperature for 30 min-1 h until the bacterial suspension becomes clear. Centrifuge at 4℃ to remove precipitate. Pour the supernatant into a pre-treated nickel column for binding. Wash with 50 mL of 25 mM Tris-HCl, 500 mM NaCl, and 25 mM imidazole buffer. Elute the protein with 15 mL of 25 mM Tris-HCl (500 mM NaCl, 250 mM imidazole) buffer. Dialyze the 15 mL eluent overnight in 25 mM Tris-HCl and 500 mM NaCl buffer. Concentrate the 15 mL eluent through an ultrafiltration tube at 3500 tpm and 4℃ to 0.5–1 mL. Determine protein concentration using the Bradford method, ensuring all mutant protein concentrations are uniform before detection. Experimental results are as follows: Figure 1 As shown, Figure 1 The image shows the SDS-PAGE results of FBP. All FBPase mutants were purified, and their molecular weights were as expected. The products were single bands (only a portion is shown in the image).

[0027] (3) Construction and detection of FBPase reaction system. The PCR instrument temperature was set to the highest incubation temperature and incubated for 30 minutes. All mutants prepared in this application were incubated using the above method at different temperatures (30℃, 44℃, 48℃, 52℃). Protein samples after incubation were then used for detection.

[0028] The enzyme activity detection system utilizes a multi-enzyme coupling reaction to combine the reaction catalyzed by FBP (fructose-1,6-bisphosphatase) with the amplification of the NAD+ reduction signal. Finally, FBP activity is indirectly determined by detecting the amount of NADH generated via OD340. The specific reaction steps are as follows: A. FBP catalytic reaction F1,6-2P F6P+Pi; FBP hydrolyzes fructose 1,6-bisphosphate (F1,6-2P) to produce fructose 6-phosphate (F6P) and inorganic phosphate (Pi), which is the rate-limiting step of gluconeogenesis.

[0029] B. Phosphoglucose isomerase (PGI) reaction F6P G6P; PGI isomerizes F6P to glucose-6-phosphate (G6P), providing a substrate for subsequent reactions.

[0030] C. Glucose-6-phosphate dehydrogenase (G6PDH) signal amplification G6P+NAD+ 6-Phosphoglucate + NADH + H+ + ; G6PDH oxidizes G6P to generate NADH, and the amount generated is directly proportional to FBP activity. NADH has a characteristic absorption peak at 340 nm, and FBP activity can be quantified by dynamically monitoring changes in OD340 using a microplate reader.

[0031] The production of NADH was detected spectrophotometrically at 340 nm, and a kinetic curve was generated. The reaction system contained 100 mM HEPES (pH 7.5), 100 mM NaCl, and 5 mM Mg. 2+ 0.1 mM EDTA, 2 mM NAD + 10 μM Zn 2+ , 0.004mg / mL pgi, 1 mM F1,6-2P, 0.006mg / mL G6PDH and 0.44mg / mL FBPase.

[0032] The kinetic curve of wild-type fructose-1,6-bisphosphatase at 30℃ is as follows: Figure 5 As shown. Analysis and calculations showed that the specific activity of wild-type fructose-1,6-bisphosphatase at 30℃ was 3.14 u / mg. One unit of enzyme activity (U) is defined as the amount of enzyme required to break down a substrate to produce 1 μmol of product per minute under given conditions.

[0033] A rational design screened out 64 single-point mutations and 4 combined mutations, and all mutation sites were detected, resulting in good protein purification quality. Thermal stability tests were performed on the 64 single-point mutations and 4 combined mutations (D39Y+V46I, L305A+E143L, D39Y+V46I+N41L, and L305A+D39Y). The specific calculation method was as follows: the high temperature value of the mutant / the 30-degree value of the mutant, yielding value A; then, the high temperature value at wt / the low temperature value at wt, yielding value B; and finally, A / B, obtaining the thermal stability ratio. Using the wild-type fructose-1,6-bisphosphatase activity at 30℃ as a reference (i.e., the wild-type fructose-1,6-bisphosphatase activity is 100% at 30℃), the relative enzyme activities of 64 single-point mutations and 4 combined mutations were measured at 30℃ and 44℃. Specifically, the relative enzyme activities of the six mutation sites T231D, N41Y, Q225K, S16P, H17G, and K229P were measured at 30℃, 44℃, 48℃, and 52℃. The experimental results are shown in Tables 1-3. The results show that not all mutations yield good results; unfavorable mutations can occur during the mutation process, ultimately leading to lower relative enzyme activities (at 30℃ or 44℃) compared to the wild type. Therefore, the method of this invention has unpredictability.

[0034] Table 1: Thermal stability results of 64 single-point mutations at 30℃ and 44℃ Table 2: Thermal stability results of the four combined mutations at 30℃ and 44℃ Table 3: Thermal stability results of T231D, N41Y, Q225K, S16P, H17G and K229P at 30℃, 44℃, 48℃ and 52℃ After incubation at 44℃ for 30 min, the thermal stability test showed that K229P retained 117.71% of its activity. The experimental results are as follows: Figure 2 As shown in the figure; after incubation at 44℃-52℃ for 30 min, the thermal stability test showed that K229P retained 53.37% of its activity after incubation at 48℃. The experimental results are as follows. Figure 3 As shown, after incubation at 44℃ for 30 min, the thermal stability test showed that D39Y+V46I+N41L retained 103.61% of its activity.

[0035] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fructose-1,6-bisphosphatase with high-temperature stability, characterized in that, It was obtained by mutation based on wild-type fructose-1,6-bisphosphatase, and the amino acid sequence of the wild-type fructose-1,6-bisphosphatase is shown in SEQ ID NO.1; The mutation is selected from any one of the following: R193P, K222L, D228H, K229E, A263Y, S264E, E295Y, L305A, N41R, N41Y, G99E, K104R, E143K, E144Y, V154L, A155V, A155M, H176R, Q191Y, C224T, Q225K. Triple mutations of Q225R, T231D, N232D, N250I, E295Q, G314A, S16P, H17G, E55S, Q63M, Q63K, S123T, S183E, E198Q, E207Q, E227N, K229P, E98K, D39Y+V46I+N41L, or double mutations of L305A+D39Y.

2. The high-temperature stable fructose-1,6-bisphosphatase according to claim 1, characterized in that, The mutated amino acid sequences are shown in SEQ ID NO. 22-SEQ ID NO. 43, SEQ ID NO. 45-SEQ ID NO. 47, SEQ ID NO. 49-SEQ ID NO. 55, SEQ ID NO. 57-SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 67, and SEQ ID NO.

68.

3. A polynucleotide, characterized in that, The enzyme is a high-temperature stable fructose-1,6-bisphosphatase according to any one of claims 1 or 2.

4. A recombinant vector, characterized in that, It contains the polynucleotide described in claim 3.

5. A host cell, characterized in that, It comprises the polynucleotide of claim 3 or the recombinant vector of claim 4.

6. The host cell according to claim 5, characterized in that, The host cell is a prokaryotic cell or a eukaryotic cell.

7. A genetically engineered bacterium, characterized in that, It comprises the polynucleotide of claim 3, or the recombinant vector of claim 4.

8. A method for preparing a high-temperature stable fructose-1,6-bisphosphatase as described in any one of claims 1-2, characterized in that, Includes the following steps: S1. Select the mutation site and introduce the mutation into the gene for fructose-1,6-bisphosphatase through primer design; S2. Transform the recombinant plasmid into competent cells and screen for positive clones; S3. Induce protein expression and purify the protein to obtain the final product.

9. The application of a high-temperature stable fructose-1,6-bisphosphatase as described in any one of claims 1-2 in industrial biotechnology, pharmaceuticals, agriculture and synthetic biology, and in the immobilization or preparation of diagnostic reagents.

10. A method for catalytically hydrolyzing fructose-1,6-bisphosphate to produce fructose-6-phosphate, characterized in that, The step of adding the fructose-1,6-bisphosphatase as described in any one of claims 1-2 to the reaction system.

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