A modified mannitol dehydrogenase and use thereof
By mutating the amino acid sequence of mannitol dehydrogenase and coupling it with recombinant strains, the coenzyme dependence and stability issues of MDH in biotransformation were resolved, enabling efficient and low-cost D-mannitol production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHEJIANG HUAKANG PHARMA
- Filing Date
- 2026-03-30
- Publication Date
- 2026-07-03
Smart Images
Figure CN121931070B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering technology, and specifically relates to a modified mannitol dehydrogenase and its application. Background Technology
[0002] The biochemical whole-cell conversion of fructose to mannitol requires the participation of mannitol dehydrogenase (MDH). Mannitol dehydrogenase (MDH) plays a crucial role in the bioconversion of D-fructose into mannitol and exhibits coenzyme-dependent barriers. MDH requires the coenzyme factors reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) or reduced nicotinamide adenine dinucleotide (NADH), but these coenzymes are expensive and continuously consumed during conversion, increasing industrial costs. Furthermore, the imperfect coenzyme regeneration system limits large-scale application, requiring the addition of regenerators or coupling with other enzymes. MDH exhibits inhomogeneity in stability and activity. The sources of MDH vary greatly, and different species show significant differences in enzyme activity and stability. Some enzymes have poor thermostability and are easily inactivated at room temperature. Most mannitol dehydrogenases experience a decrease in stability during purification and storage, requiring special protective agents (such as glycerol and BSA) to maintain activity. Although MDH is a highly efficient enzyme source with many excellent properties and great potential for industrial production, the conversion efficiency of most MDH is still insufficient to support large-scale, low-cost production. Furthermore, the production conditions for industrial applications are demanding. Pure enzyme conversion has strict requirements for pH, temperature, and ionic strength, making industrial scale-up difficult and requiring high equipment investment. The high cost of MDH enzyme preparations and the imperfect recycling technology make it difficult to reduce the overall production cost. Summary of the Invention
[0003] The technical problem to be solved by the present invention is to provide a modified mannitol dehydrogenase and its application, which reduces the production cost of the strain, improves enzyme activity, and improves the conversion efficiency of D-mannitol.
[0004] The present invention is achieved by providing a modified mannitol dehydrogenase, wherein the modified mannitol dehydrogenase has an amino acid mutation in the amino acid sequence shown in SEQ ID NO.1, and the amino acid mutation site is at least one of the following positions: 37, 75, 176, 185, 197, 225, 244, and 300.
[0005] Further, the modified mannitol dehydrogenase is made by mutating the amino acid sequence shown in SEQ ID NO.1 to one of the following: (1) cysteine C at position 37 is mutated to alanine A (C37A); (2) glutamine Q at position 75 is mutated to valine V (Q75V); (3) glutamine Q at position 176 is mutated to leucine L (Q176L); (4) glutamine Q at position 185 is mutated to asparagine N (Q185N); (5) glutamate E at position 197 is mutated to arginine R (E197R); (6) phenylalanine F at position 225 is mutated to methionine M (F225M); (7) tyrosine Y at position 244 is mutated to phenylalanine F (Y244F); (8) glutamine Q at position 300 is mutated to leucine L (Q300L).
[0006] This invention is implemented in such a way that it also provides a gene encoding the modified mannitol dehydrogenase as described above.
[0007] This invention is implemented in such a way that a recombinant strain is also provided, comprising the modified mannitol dehydrogenase as described above, or comprising the gene as described above.
[0008] This invention is implemented in such a way that it also provides the use of the modified mannitol dehydrogenase as described above, or the gene as described above, or the recombinant strain as described above in the preparation of D-mannitol.
[0009] This invention is implemented in such a way that it also provides the use of the modified mannitol dehydrogenase as described above, or the gene as described above, or the recombinant strain as described above in the synthesis of L-gulose or 2-amino-2-deoxy-D-mannitol.
[0010] This invention is implemented in such a way that it also provides the use of the modified mannitol dehydrogenase as described above, or the gene as described above, or the recombinant strain as described above in the synthesis of L-gulose or 2-amino-2-deoxy-D-mannitol.
[0011] This invention is implemented as follows, and also provides a method for preparing D-mannitol, comprising the following steps: mixing the recombinant strain as described above with... Cb The formate dehydrogenase (AAC49766.1) strain of FDH plasmid was cultured and fermented. The resulting wet cells were ultrasonically disrupted, and crude enzyme solution was extracted. D-fructose was used as the substrate, and phosphate buffered saline (PBS) was used as the reaction medium to form a reaction system to obtain D-mannitol through a transformation reaction.
[0012] Compared with the prior art, the modified mannitol dehydrogenase and its application of the present invention have a significant improvement in the efficacy of mannitol dehydrogenase. AsMDH is subjected to amino acid mutations according to the amino acid sequence shown in SEQ ID NO.1, wherein the mutation sites are at least one of positions 37, 75, 176, 185, 197, 225, 244, and 300. The resulting recombinant strain containing the mutant is then used in conjunction with... Cb By coupling a strain containing the formate dehydrogenase of the FDH plasmid with a dual-enzyme, the efficiency of D-mannitol production from D-fructose was significantly improved. After adding 150 g / L of fructose and reacting for 8 hours, the conversion rate of the dual-enzyme system containing the Y224F mutant reached 90.12%, and the conversion rate of the dual-enzyme system containing the Q300L mutant reached 86.23%. This invention reduces the cost of recombinant strains, increases enzyme activity, and improves the conversion efficiency of D-mannitol. Attached Figure Description
[0013] Figure 1 This invention relates to wild-type mannitol dehydrogenase. As Electrophoretic diagram for verifying the elution of the target protein from MDH and mutants;
[0014] Figure 2 This is a schematic diagram of the NADH standard curve for Embodiment 3 of the present invention;
[0015] Figure 3 This is a schematic diagram showing the growth of different mutant strains and their relative enzyme activities in Example 3 of the present invention;
[0016] Figure 4 This is a schematic diagram illustrating the enzyme activity stability of different strain mutants in Example 3 of the present invention;
[0017] Figure 5 This is a schematic diagram of the enzyme conversion rate of different strain mutants in Example 4 of the present invention. Detailed Implementation
[0018] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.
[0019] Unless otherwise specified, the experimental methods used in the examples are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0020] The culture medium formulations used in the following examples are as follows:
[0021] (1) LB medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, pH adjusted to 7.0, autoclaved at 121℃ for 20 min, and cooled for later use.
[0022] (2) LB solid medium: The solid medium is based on the LB medium formula with the addition of 1.5% agarose. After sterilization, it is poured into plates (LB plates) for later use.
[0023] (3) TB medium: yeast powder 24g / L, peptone 12g / L, glycerol 5g / L, K2HPO4 9.4g / L, KH2PO4 2.2g / L, autoclaved at 121℃ for 20min, and cooled for later use.
[0024] The reagents used in the following examples are as follows:
[0025] (1) Ampicillin stock solution (100mg / mL): Add 10g of ampicillin to 100mL of ultrapure water, filter sterilize with a 0.22μm filter membrane, dispense into aliquots, store at -20℃, and use at a concentration of 0.1%.
[0026] (2) Isopropyl thio-β-D-galactopyranoside (IPTG) stock solution: Dissolve 1.2g of IPTG in 50mL of sterile water, filter sterilize using a 0.22μm filter membrane, dispense into aliquots, and store at -20℃.
[0027] (3) Phosphate Buffered Solution (PBS): Purchase PBS Buffer premixed powder (1X) from Sangon Biotech (Shanghai) Co., Ltd. and prepare it according to the instructions.
[0028] (4) Lysis buffer: Add 1‰ Triton X-100 (about 50µL), 5mM 2-mercaptoethanol, 200~500µL PMSF and 25mM imidazole to 50mL phosphate buffer.
[0029] (5) Protein gradient elution buffer: Prepare imidazole concentrations of 50, 100, 200 and 500 mM using phosphate buffer and adjust the pH to 8.0.
[0030] (6) Protein replacement solution: Phosphate buffer with 5 mM DTT and 30% glycerol.
[0031] Example 1: Obtaining the Mutant Recombinant Strains
[0032] 11. Strains and plasmids
[0033] The strain used to construct the expression strain was *Escherichia coli*. Escherichia coliBL21(DE3), carrying the target gene in pETDuet-1 series plasmids, were all purchased from Beijing Qingke Biotechnology Co., Ltd. The target gene is mannitol dehydrogenase. As The amino acid composition of MDH is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.2.
[0034] 12. Mutant design and construction
[0035] For containing As The original strain of MDH (i.e., containing As Plasmid extraction was performed on a wild-type strain of MDH. Plasmid extraction was performed according to the instructions of the plasmid extraction kit from Tiangen Biotech (Beijing) Co., Ltd., to obtain plasmids containing... As The plasmid of the MDH gene (pETDuet-) As MDH-WT). Then site-directed mutagenesis was performed. As MDH gene sequences were analyzed using software to predict mutants.
[0036] As The MDH mutant is formed by mutating the amino acid sequence shown in SEQ ID NO.1 to the following: (1) cysteine C at position 37 is mutated to alanine A (C37A); (2) glutamine Q at position 75 is mutated to valine V (Q75V); (3) glutamine Q at position 176 is mutated to leucine L (Q176L); (4) glutamine Q at position 185 is mutated to asparagine N (Q185N); (5) glutamic acid at position 197 is mutated to arginine (E197R); (6) phenylalanine at position 225 is mutated to methionine (F225M); (7) tyrosine at position 244 is mutated to phenylalanine (Y244F); (8) glutamine at position 300 is mutated to leucine (Q300L).
[0037] The primer designs for site-directed mutagenesis are shown in Table 1.
[0038] Table 1 Primer Design Table
[0039]
[0040] With site-directed mutation As Taking MDH-C37A as an example: First, prepare two reaction systems as shown in Tables 2 and 3. Add 5 μL of high-fidelity DNA polymerase (named 2× Phanta MaxMaster Mix) purchased from Nanjing Novizan Biotechnology Co., Ltd., and 1 μL of pET- AsMDH-WT was used as the template in reaction system 1, and 2 μL of C37A-F was used as the upstream primer. ddH2O was added to bring the total volume of reaction system 1 to 10 μL. The high-fidelity polymerase, template, and ddH2O in reaction system 2 were added in the same way as in reaction system 1, with 2 μL of C37A-R added as the downstream primer. Both reaction systems were first subjected to the PCR reaction procedure shown in Table 4. After the reaction, the PCR products in both reaction systems were mixed and vortexed until no air bubbles remained. The PCR reaction procedure shown in Table 5 was then continued to obtain the final product. As Amplification products of MDH-C37A.
[0041] The reaction systems and procedures for the other seven site-directed mutagenesis methods are the same as those for site-directed mutagenesis. As The MDH-C37A is consistent. Furthermore, the upstream primers of site-directed mutagenesis reaction system 1 are all XF-labeled, and the downstream primers of reaction system 2 are all XR-labeled, where X is a pronoun. That is: Construction As When using MDH-Q75V, the upstream primer is Q75V-F and the downstream primer is Q75V-R;
[0042] Build As When using MDH-Q176L, the upstream primer is Q176L-F and the downstream primer is Q176L-R.
[0043] Build As When using MDH-Q185N, the upstream primer is Q185N-F and the downstream primer is Q185N-R;
[0044] Build As When using MDH-E197R, the upstream primer is E197R-F and the downstream primer is E197R-R;
[0045] Build As When using MDH-F225M, the upstream primer is F225M-F, and the downstream primer is F225M-R;
[0046] Build As When using MDH-Y244F, the upstream primer is Y244F-F and the downstream primer is Y244F-R;
[0047] Build As When using MDH-Q300L, the upstream primer is Q300L-F and the downstream primer is Q300L-R.
[0048] The remaining high-fidelity enzyme, template, ddH2O, and the above-mentioned site-directed mutagenesis in the reaction system As The specific steps in MDH-C37A are completely consistent.
[0049] Table 2 Reaction system 1 (10 μL)
[0050]
[0051] Table 3 Reaction system 2 (10 μL)
[0052]
[0053] Table 4. Two-step first-round PCR reaction procedure
[0054]
[0055] The symbol * indicates that the denaturation, annealing, and extension steps are performed sequentially in the same stage, which constitutes one cycle.
[0056] Table 5. Two-step second-round PCR reaction procedure
[0057]
[0058] The symbol * indicates that the denaturation, annealing, and extension steps are performed sequentially in the same stage, which constitutes one cycle.
[0059] Take 7.5 μL of amplification product and add 1.5 μL of [unspecified ingredient]. Dpn Mix 1 μL of the rapid digestion enzyme and 1 μL of CutSmart rapid digestion enzyme buffer, and incubate at 37°C for 30 min. The template in the digestion system is the processed PCR product, which can be used for subsequent transformation.
[0060] 13. Preparation of competent Escherichia coli cells
[0061] The competent E. coli cells used in the experiment were all prepared using the CaCl2 method, which specifically included the following steps:
[0062] (1) Take out the two types of Escherichia coli stored at -80℃ Escherichia coli ( E.coli TOP10 and E.coli BL21(DE3) glycerol bacteria were streaked on non-resistant LB solid medium and incubated overnight at 37°C.
[0063] (2) Pick a single colony from the LB plate and inoculate it into a container of 10 mL of liquid LB medium. Incubate at 37°C and 200 rpm for 12-14 h.
[0064] (3) Inoculate 1% into 50 mL of antibiotic-free LB medium and incubate at 37°C and 200 rpm for 2-3 hours. Wait for OD... 600 When the value reaches 0.4~0.5, let it stand in an ice bath for 10 minutes.
[0065] (4) Centrifuge at 4℃ and 5000×g for 5 min and collect the bacterial precipitate.
[0066] (5) Resuspend the bacterial cells in a 0.1M CaCl2-MgCl2 solution (the final concentrations of the two components, CaCl2 and MgCl2, in the solution are both 0.1M and are mixed together) and let stand in an ice bath for 15 minutes.
[0067] (6) Centrifuge at 4℃ and 2000×g for 10 min and collect the bacterial precipitate.
[0068] Repeat steps (5) and (6) twice.
[0069] (7) Add 2 mL of 0.1 M CaCl2 (using 15% glycerol as solvent) to resuspend.
[0070] (8) Aliquot 50 μL and store in a -80℃ freezer to obtain Escherichia coli. E.coli TOP10 competent cells and E.coli BL21(DE3) competent cells.
[0071] 14. Transformation of recombinant products and screening of positive clones
[0072] Take 10 μL of the obtained processed PCR products (i.e., the aforementioned eight mutant strains) and add them to E. coli. E.coli Top 10 competent cells were incubated at 100 μL on ice for 30 min. Then, they were heat-shocked at 42°C for 90 s. After an ice incubation of 5 min, 1 mL of LB medium was added. The cells were then incubated at 37°C on a shaker for 1 h, plated onto LB agar plates containing 100 μg / mL ampicillin resistance, and incubated overnight for 8–10 h. Single clones were picked for sequencing. Sequencing results were used to identify correctly mutated mutants. The strains were then preserved and plasmids were extracted according to the instructions of the Tiangen Biotech plasmid mini-prep kit.
[0073] The mutant plasmid extracted in the previous step was transformed into E. coli. E.coli BL21(DE3) competent cells were used, and subsequent procedures were the same as those for the transformation of the recombinant product in the previous step. After culturing on plates for 8-10 hours, mutant strains were selected for preservation at -80°C for later use.
[0074] The aforementioned eight mutant strains underwent the steps described above.
[0075] Example 2: Obtaining crude enzyme solution and purifying protein from mutant recombinant bacteria
[0076] 21. Target protein expression induction
[0077] The bacterial strains containing eight mutants obtained in Example 1 were subjected to shake-flask fermentation to investigate the target protein.As MDH expression induction includes the following steps:
[0078] (1) Seed activation: Select the seeds containing the eight mutant strains mentioned above. As A single colony of MDH engineered bacteria was inoculated into 10 mL of LB liquid medium containing 100 μg / mL ampicillin resistance and cultured at 37°C and 200 rpm for 12-14 h. This process eliminates bacteria without the target plasmid by adding 100 μg / mL ampicillin to the medium, retaining only engineered bacteria that have successfully transferred the mannitol dehydrogenase gene and the ampicillin resistance gene, thus avoiding contamination by other bacteria or the use of nutrients by ineffective bacteria.
[0079] (2) Expanded culture: At an inoculum rate of 3%, the activated bacterial culture was transferred to 50 mL of TB liquid medium containing 100 μg / mL ampicillin resistance and cultured at 37℃ and 200 rpm for 2-3 h. OD was monitored during the culture period. 600 value.
[0080] (3) Induced expression: When OD 600 When the value reaches 0.6~0.8, add IPTG to a final concentration of 0.2mM and incubate at 20℃ and 200rpm for 20h.
[0081] (4) Centrifugation to collect bacteria: Centrifuge the induced bacterial culture at 4℃ and 8000rpm for 10min, collect the bacterial precipitate, resuspend and wash twice with PBS, discard the supernatant, and store at -20℃.
[0082] Before harvesting the bacteria, the biomass of the bacteria was first determined: the bacterial solution was diluted to a suitable concentration, and the diluted bacterial solution was placed in a 1cm quartz cuvette and the absorbance of the bacterial solution at a wavelength of 600nm was measured.
[0083] 22. Crude enzyme extraction includes the following steps:
[0084] (1) Resuspend the bacterial cells collected in the previous step with lysis buffer.
[0085] (2) The bacterial resuspension was subjected to low-temperature ultrasonic disruption.
[0086] (3) Centrifuge the cell lysate at 4°C and 8000 rpm for 20 min to remove the bottom cell debris and collect the supernatant.
[0087] (4) The supernatant was filtered through a 0.22 μm cellulose acetate membrane to obtain the crude enzyme solution.
[0088] (5) Run protein electrophoresis on the precipitate and crude enzyme solution to check the expression status.
[0089] 23. Nickel column protein purification
[0090] Using Ni 2+ -Chelating Sepharose Fast Flow affinity chromatography for rapid purification of target proteins, the specific method includes the following steps:
[0091] (1) Rinse and balance: Rinse Ni with ultrapure water 2+ The chromatography column was run twice, and the Ni was washed with nickel column equilibration solution. 2+ One chromatography column;
[0092] (2) Nickel column chromatography: The filtered crude enzyme solution was re-coated onto the column three times. Impurities were eluted with 10 mL of 25 mM, 50 mM, and 100 mM imidazole solutions, respectively. Then, the target protein was eluted with 5 mL of 200 mM imidazole solution. Finally, residual protein was thoroughly eluted with 500 mM imidazole solution. Approximately 10 mL of sample was collected into each EP tube, and the protein concentration was determined.
[0093] (3) Column washing: Finally, wash the nickel column twice with ultrapure water and store it at 4°C with 20% ethanol.
[0094] (4) Ultrafiltration concentration: Use a 10~30kDa ultrafiltration tube to ultrafilter the eluent containing the target protein. Concentrate at 4℃ and 3500rpm by centrifugation. Dilute with protein replacement buffer more than 50 times. Finally, keep 1~2mL. Add glycerol and dispense into aliquots. Quick freeze in liquid nitrogen and store at -80℃.
[0095] 24. Protein Analysis
[0096] In the protein purification process, after elution with different gradient elution buffers, 20 μL of the collected eluent was added to 5 μL of 5× Protein Loading Dye (purchased from Shanghai Sangon Biotech Co., Ltd.) and mixed well. The mixture was boiled in water for 5 min, allowed to stand at room temperature, centrifuged, and 10 μL of the supernatant was loaded onto the sample. A standard protein marker was used for comparison. The runnated gel was stained with Coomassie Brilliant Blue for 10 min, and then destained overnight on a shaker using destaining buffer. Images were taken for analysis. Successfully eluted eluent was selected for ultrafiltration concentration. The protein concentration of the concentrated purified protein was determined using a micro-ultraviolet spectrophotometer.
[0097] SDS-PAGE analysis using polyacrylamide gel electrophoresis showed that all mutant strains expressed the target protein normally, with a distinct protein band around 35 kDa. Furthermore, most of the protein was eluted at a concentration of 500 mM imidazole. (See [link to SDS-PAGE]). Figure 1 As shown in Table 6, the results indicate that all eight successfully constructed mutant recombinant strains were able to express the target protein normally and could be successfully purified.
[0098] Table 6. Concentration of purified protein from different mutants
[0099]
[0100] Example 3: Determination of the enzyme activity and thermal stability of pure enzymes from mutant recombinant bacteria.
[0101] The purified protein was then subjected to enzyme activity assays. First, a NADH standard curve was constructed, including the following steps: 200 μL of NADH standard solutions (0, 0.25, 0.5, 1, and 2 mM) were prepared, and the absorbance at 340 nm was recorded to construct the NADH standard curve. Figure 2 As shown.
[0102] MDH enzyme activity assay reaction system: Prepare 1 mM DTT solution and 0.1 mM Zn2 solution respectively. 2+ Ionic liquid, 20 g / L fructose solution, 1 mM NADH solution, and 0.4 mg / mL pure enzyme. The total reaction volume for enzyme activity assay was 200 μL, including 10 μL DTT and Zn. 2+ 10 μL of naphthalene, 20 μL of fructose, 10 μL of NADH, 50 μL of purified enzyme, and 100 μL of PBS buffer. The reaction time was controlled at 1 min. The absorbance at 340 nm was then immediately measured. The blank group consisted of the reaction solution with added buffer, measured under the same conditions. Figure 3 As shown, the relative enzyme activities of different mutant strains revealed that the Y224F and Q300L mutants had higher relative enzyme activities, which better met the requirements for screening mutant strains with higher enzyme activities.
[0103] Simultaneously with enzyme activity measurement, enzyme stability testing was continuously performed. The pure enzyme was incubated at 50°C, and samples were taken at 0, 0.5, 1, 1.5, 2, 2.5, and 3 hours for MDH thermal stability determination. The results are as follows: Figure 4 As shown, the Y224F and Q300L mutants also exhibit better stability at 50℃.
[0104] Example 4: Determination of MDH catalytic activity of mutant recombinant bacteria
[0105] 41. Acquisition of mutant enzymes MDH and FDH
[0106] Using E. coli E.coli BL21(DE3) was constructed to express formate dehydrogenase. Cb FDH strain (AAC49766.1), formate dehydrogenase Cb FDH uses Escherichia coli. E.coli BL21(DE3) acts as an expression host, expressing Candida boidinii FDH gene of origin Cbfdh contains Cb fdh's pETDuet- Cb The fdh plasmids were all synthesized by Beijing Qingke Biotechnology Co., Ltd., and the codons were optimized according to the codon preferences of the E. coli expression host.
[0107] The two strains were fermented, and the shake-flask fermentation and cell collection processes were carried out in accordance with the corresponding steps in Example 2.
[0108] 42. Catalytic reaction
[0109] Will As MDH and Cb The enzyme conversion rate of FDH was determined by a two-enzyme coupling assay. The conversion reaction was carried out with 150 g / L fructose, 68 g / L sodium formate, and 5 mM NADH. The reaction was conducted at pH 6.5 and temperature 50℃. As MDH and Cb FDH was added to the reaction solution. 600 The reaction time was 8 hours. After dilution and membrane filtration, the sample was analyzed by High Performance Liquid Chromatography (HPLC). A RID-20A differential refractive index detector and a Shodex SUGAR SC1011 column were used to analyze the product D-mannitol and the substrate D-fructose. The mobile phase was ultrapure water. Parameters were set as follows: flow rate 0.8 mL / min, column oven temperature 80℃, detector temperature 40℃, and injection volume 10 μL. The conversion rate of D-mannitol was calculated using the external standard method. The MDH catalytic activity results are as follows: Figure 5 As shown, the transformation efficiency of the Y224F mutant was better, reaching 90.12% at 8 hours, which was 19.73% higher than that of the WT mutant. The transformation efficiency of the Q300L mutant reached 86.23%, which was 14.56% higher than that of the WT mutant.
[0110] It should be noted that, in addition to its application in the preparation of D-mannitol, the modified mannitol dehydrogenase, gene, and recombinant strain of this invention also have the following applications:
[0111] 1. Applications in industrial production: High-efficiency synthesis and co-production technology of D-mannitol, such as the co-production of sodium gluconate from mannitol.
[0112] 2. Applied to the medical and health fields
[0113] Direct pharmaceutical applications: Injection: as a dehydrating agent, used to reduce intracranial pressure, intraocular pressure and diuresis; Tablet excipient: used to prepare chewable tablets and stimulants, utilizing their stability and low hygroscopicity; Novel drug delivery systems: as drug carriers to improve drug stability and bioavailability;
[0114] Synthesis of bioactive substances: Synthesis of rare sugars:
[0115] Used for the synthesis of L-gulose: a precursor to the anticancer drug bleomycin and a nucleoside antiviral drug, it is synthesized by coupling a modified mannitol-1-dehydrogenase with NADH oxidase.
[0116] Used for the synthesis of 2-amino-2-deoxy-D-mannitol: a precursor of DNJ, a hypoglycemic substance from mulberry trees, synthesized via a modified aminomannitol dehydrogenase.
[0117] 3. Applied to the food and beverage industry
[0118] Special sweeteners: Low-calorie sweeteners suitable for diabetics and people on a diet; they are not utilized by oral microorganisms and help prevent tooth decay.
[0119] Chewing gum base: Improves taste, extends shelf life, and global demand is growing rapidly;
[0120] Food additives: Used in baked goods and dairy products to improve moisture retention and stability.
[0121] 4. Applications in the fields of agriculture and biotechnology
[0122] Crop improvement: Enhanced stress resistance:
[0123] Transgenic plants (potato, tomato, millet, etc.) that overexpress the mannitol dehydrogenase gene show significantly enhanced tolerance to various abiotic stresses such as drought, salt stress, and high temperature.
[0124] The expression of the mannitol dehydrogenase gene in nematodes enhances resistance to pests and diseases;
[0125] Flower preservation: Introducing the celery mannitol dehydrogenase gene into roses extends the shelf life to one month and resists petal blight;
[0126] Microbial engineering: Fermentation optimization:
[0127] Modify Corynebacterium glutamicum / Corynebacterium obliterans to enhance mannitol utilization and increase L-ornithine / L-tyrosine production;
[0128] By modifying Yersinia lipolyticis, 2'-fucosylated lactose (a breast milk additive) can be synthesized efficiently using mannitol as a carbon source.
[0129] 5. Applied to fine chemical and environmental protection fields
[0130] Chiral synthesis: used to synthesize optically active alcohols and ketones as pharmaceutical intermediates;
[0131] Environmental monitoring: As a biosensor component, it detects the inhibitory effects of pollutants such as heavy metals and polycyclic aromatic hydrocarbons on microbial metabolism;
[0132] Biodegradation: Used in the synthesis of biodegradable plastics and the treatment of organic waste, promoting the development of green chemistry.
[0133] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. An engineered mannitol dehydrogenase, characterized in that, The modified mannitol dehydrogenase has an amino acid mutation in the amino acid sequence shown in SEQ ID NO.1, at position 244 or 300. The modified mannitol dehydrogenase is made by mutating the amino acid sequence shown in SEQ ID NO.1 to one of the following: (1) Tyrosine at position 244 is mutated to phenylalanine; (2) Glutamine at position 300 is mutated to leucine.
2. A gene encoding the modified mannitol dehydrogenase as described in claim 1.
3. A recombinant bacterial strain, characterized in that, It contains the modified mannitol dehydrogenase as described in claim 1, or it contains the gene as described in claim 2.
4. The use of the modified mannitol dehydrogenase as described in claim 1, or the gene as described in claim 2, or the recombinant strain as described in claim 3 in the preparation of D-mannitol.
5. A process for the preparation of D-mannitol, characterized in that, Includes the following steps: The recombinant strain as described in claim 3 and containing Cb The formate dehydrogenase strains containing FDH plasmids were cultured and fermented to obtain wet cells which were then ultrasonically disrupted to extract crude enzyme solution. D-fructose was used as a substrate and phosphate buffer as a reaction medium to form a reaction system, and D-mannitol was obtained through a conversion reaction.