Galactitol dehydrogenase mutant and application thereof in preparation of D-tagatose

Abstract

The invention discloses a galactitol dehydrogenase mutant and application of the galactitol dehydrogenase mutant in preparation of D-tagatose, and relates to the technical field of biology. The amino acid sequence of the galactitol dehydrogenase mutant is as shown in SEQ ID NO. 3. According to the invention, a high-throughput screening method based on NTB-PMS detection is used for carrying out semi-rational transformation on a galactitol dehydrogenase mutant derived from Rhizolium leguminosarum, so that the galactitol dehydrogenase mutant with high catalytic activity and thermal stability is obtained. Among all reported galactitol dehydrogenase at present, the mutant has the highest enzyme activity, the half-life period of the mutant reaches 128 h under the conditions that the pH is 8.0 and the temperature is 35 DEG C, and the mutant has important industrial application prospects.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a galactitol dehydrogenase mutant and application thereof in the preparation of D-tagatose. Background Art

[0002] D-tagatose is a natural low-calorie functional sweetener with 92% of the sweetness of sucrose, but only 38% of the calories of sucrose. Because of its biological activities including lowering blood sugar, anti-oxidation, improving intestinal flora and others, it is widely used in the fields of food and medicine. The synthesis of D-tagatose is mainly through chemical and biological methods. The chemical method has the disadvantages of difficult control of the synthesis process, difficult purification of the product, high by-products and high cost, which hinders the application of chemical methods for the synthesis of D-tagatose. At present, commercial D-tagatose is usually produced by isomerization of D-galactose by L-arabinose isomerase (L-AI, EC 5.3.1.4). However, due to the inherent thermodynamic equilibrium of L-AI and the similar properties between D-galactose and D-tagatose, there are still problems of limited conversion rate and high purification cost.

[0003] Galactitol-2-dehydrogenase (EC 1.1.1.16) has been found in a variety of microorganisms, including Rhizobium leguminosarum, Aspergillus niger, Burkholderia cepacia, Rhodobactersphaeroides, and Pseudomonas sp., and can convert NAD + As a hydrogen acceptor, it oxidizes the C2 hydroxyl group of the substrate galactitol to a keto group to generate D-tagatose. + The regeneration system can completely convert galactitol into D-tagatose. Since the galactose dehydrogenase activity that has been discovered and identified is generally low, with the highest activity being only 15.2U / mg, the key technical difficulties that need to be solved in order to industrialize the above enzyme catalysis process are: 1. Develop galactitol dehydrogenase and its engineered bacteria with industrial application prospects; 2. Establish a system for efficient in-situ regeneration of coenzymes suitable for industrial scale-up. Summary of the invention

[0004] The purpose of the present invention is to provide a galactitol dehydrogenase mutant and its application in the preparation of D-tagatose to solve the problems existing in the above-mentioned prior art. The galactitol dehydrogenase mutant has the advantages of high catalytic activity and thermal stability, can be used for catalytic preparation of D-tagatose, and has important industrial application prospects.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a galactitol dehydrogenase mutant, the amino acid sequence of which is shown in SEQ ID NO.3.

[0007] The present invention also provides a gene encoding the galactitol dehydrogenase mutant.

[0008] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.4.

[0009] The present invention also provides a recombinant vector, comprising the above-mentioned coding gene.

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

[0011] The present invention also provides the use of the above-mentioned encoding gene, recombinant vector or recombinant host cell in preparing the above-mentioned galactitol dehydrogenase mutant.

[0012] The present invention also provides the use of the galactitol dehydrogenase mutant in the preparation of D-tagatose.

[0013] The present invention also provides an enzyme combination for preparing D-tagatose, comprising NADH oxidase and the above-mentioned galactitol dehydrogenase mutant.

[0014] The present invention also provides the use of the above enzyme combination in the preparation of D-tagatose.

[0015] The present invention also provides a method for preparing D-tagatose, comprising using galactitol as a substrate, the above enzyme combination as a catalyst, and NAD + The step of preparing the D-tagatose as a hydrogen acceptor through a catalytic reaction.

[0016] The present invention discloses the following technical effects:

[0017] 1. The present invention uses a high-throughput screening method based on NTB-PMS detection to semi-rationally transform the galactitol dehydrogenase (GenBank ID: WP_011650422.1) from Rhizobiumleguminosarum, and obtains a galactitol dehydrogenase mutant with high catalytic activity and thermal stability. The enzyme activity of the galactitol dehydrogenase mutant is 50U / mg at a substrate concentration of 100mmol / L, which is 24 times that of the wild type. Among all the galactitol dehydrogenases reported so far, the mutant has the highest enzyme activity. At the same time, the mutant has a half-life of 128h under pH 8.0 and 35°C, and has important industrial application prospects.

[0018] 2. The present invention uses a galactitol dehydrogenase mutant and NADH oxidase as catalysts, NAD +As a hydrogen acceptor, it catalyzes the preparation of D-tagatose from galactitol under weak alkaline conditions. Galactitol can be completely converted into D-tagatose within 12 hours. It has the advantages of high substrate concentration, high conversion efficiency, low cost and no by-products. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The SDS-PAGE test results of RlGDH and T193G / G98C mutants; wherein, M is a marker; 1 is RlGDH; 2 and 3 are T193G / G98C mutants;

[0021] Figure 2 The figure is the result of the test on the optimum reaction temperature of RlGDH (WT) and T193G / G98C mutant;

[0022] Figure 3 The figure is the result of the optimal reaction pH test of RlGDH (WT) and T193G / G98C mutant;

[0023] Figure 4 Figure 2 is the temperature stability test result of RlGDH (WT) and T193G / G98C mutant;

[0024] Figure 5 The figure is the result of pH stability test of RlGDH (WT) and T193G / G98C mutant;

[0025] Figure 6 The result diagram of detecting RlGDH enzyme activity under different galactitol concentrations;

[0026] Figure 7 The results of detecting the enzyme activity of the T193G / G98C mutant under different galactitol concentrations are shown in the figure;

[0027] Figure 8 This is the HPLC detection chart of D-tagatose standard sample;

[0028] Fig. 9 It is the HPLC detection chart of the galactitol standard sample;

[0029] Fig.10 HPLC detection chart of D-tagatose prepared by T193G / G98C mutant combined with NADH oxidase SpNox. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The materials used in the embodiments of the present invention include: Escherichia coli T7 and expression vector pET-30a were purchased from Novagen. Restriction endonucleases, DNA polymerases and other reagents were purchased from TaKaRa. Galactitol, D-tagatose, nitroblue tetrazolium (NBT) and 5-methylphenazinemethosulphate (PMS) were purchased from Sigma-Aldrich. Other consumables and reagents were purchased from Shanghai Bioengineering Technology Co., Ltd.

[0036] Example 1 Screening of galactitol dehydrogenase with high catalytic activity

[0037] 1. Homology modeling and molecular docking of galactitol dehydrogenase

[0038] Galactitol dehydrogenase RlGDH from Rhizobium leguminosarum and its cofactor NAD + The three-dimensional structure of the galactitol was generated by AlphaFold3 Sever. Then, AutoDock Vina was used to dock the galactitol molecule to the substrate pocket active center of the three-dimensional structure, and the docking results were analyzed using the PyMol 2.5.2 visualization tool.

[0039] The amino acid sequence of the RlGDH enzyme is shown in SEQ ID NO.1, and the nucleotide sequence of the RlGDH gene is shown in SEQ ID NO.2.

[0040] SEQ ID NO.1:

[0041] MSYQQKFRLDGERAVVTGGGRAIGLCCTEALAEAGAAVVVIERSEADAEQALALRNRGYDVEVRVGDVTDAARMDAIATELADGGRPATILVNNAGIGQSGIPAQDLTDADWLRMMDVNLNGVFWCSRA FGRSMISMKRGAIVNLGSMSGTICNRPQPQTAYNVSKAAVHHLTRSLAAEWAHHGIRVNAVAPTYIETPMVVAVEANRERIPLWLADTPMARMGTPEEVASAVLFLASGAASLMTGAIVNVDAGFTCW*.

[0042] SEQ ID NO.2:

[0043] atgtcttaccagcagaaattccgtctggatggtgaacgtgctgttgttaccggtggtggtcgtgcgatcggtctgtgttgcaccgaagcattagctgaagcgggtgctgccgtagtagtaattgaacgttctgaagctgatgctgaacaggctctggctctgcgtaaccgtggttatgatgttgaagttcgtgttggcgatgttaccgatgcggcgcgtatggatgcgatcgcgaccgaactggctgatggcggtcgtccggcgaccatcctggttaacaacgcgggcatcggccagagcggtatcccggcacaggatctgaccgatgcggattggctgcgtatgatggatgttaacctgaacggtgttttctggtgctctcgtgcgttcggtcgtagcatgatctctatgaaacgtggcgcgatcgttaacctgggcagcatgagcggcaccatctgcaaccgtccgcagccgcagaccgcgtacaacgttagcaaagcggcggttcatcacctgacccgtagcctggcggctgaatgggcgcaccacggtatccgtgttaacgcggttgcgccgacctacatcgaaaccccgatggttgttgcggttgaagcgaaccgtgaacgtatcccgctgtggctggcggataccccgatggcgcgtatgggcaccccggaagaagttgcgagcgcggttctgttcctggcgtctggcgcggcgagcctgatgaccggcgcgatcgttaacgttgatgcgggcttcacctgctggtaa。

[0044] 2. Construction of mutant library

[0045] The RlGDH substrate was selected in the present invention The amino acids in the range (I97, G98, Q99, S100, M148, S149, N154, Q157, Q159, N163, P192, T193, Y194, I195, T197, M199, V200, V203, N206, R209, W213) were selected as mutation sites, and the iterative saturation mutagenesis strategy was used for mutation modification. First, the galactitol substrate was selected by molecular docking model. The first round of site saturation mutagenesis was performed on all amino acids in the range. After the first round of saturation mutagenesis, the mutation sites with high activity mutants were screened, and then the mutants with the highest enzyme activity in the first round were selected as controls for the next round of saturation mutagenesis. All amino acids in the range were subjected to iterative saturation mutagenesis.

[0046] Using pET-30a-RlGDH plasmid as template, site-directed saturation mutagenesis was performed by whole plasmid PCR mediated by NNK primer. After the PCR product was cleaved by Dpn I enzyme to remove the template, it was transformed into Escherichia coli T7 competent cells by heat shock method to obtain mutant library.

[0047] Construction method of pET-30a-RlGDH plasmid: The amino acid sequence of galactitol dehydrogenase from Rhizobium leguminosarum (GenBank ID: WP_011650422.1) was codon optimized to obtain the gene sequence, which was then synthesized by GenScript Biotechnology Co., Ltd. and inserted into the multiple cloning site of plasmid pET-30a(+) using restriction endonucleases Nde I and Xho I to obtain the recombinant plasmid pET-30a-RlGDH.

[0048] 3. High-throughput screening of high enzyme activity mutants

[0049] The high-throughput screening method of NBT-PMS colorimetric reaction was used to screen for galactose-2-dehydrogenase mutants with high catalytic activity. The specific principle is that galactitol will generate D-tagatose under the catalysis of galactitol dehydrogenase, and at the same time, the cofactor NAD + Reduced to NADH, NBT and NADH react under the action of PMS to generate bright blue-purple formazan and NAD + , A-Zan in OD 580 It has a higher absorbance, and the color change of wild-type enzyme and mutant enzyme can be observed by comparing or measuring OD 580 The absorbance value can quickly screen out mutants with high enzyme activity.

[0050] First, 188 mutants (coverage ≥ 99%) and 4 wild-type controls were selected from the amino acid mutation library of each mutation site with a sterilized toothpick and placed in 96-well deep-well plates (2 plates) containing 200 μL TB (50 μg / L kanamycin). The microplate shaker was shaken at 37°C and 1000rpm for 12 hours. Then, 100 μL of culture was transferred from two deep-well plates to another 2 96-well microplates (preservation plates) with a spray gun, and 100 μL of 40% glycerol was added to preserve the strain at -80°C. Then, 900 μL of fresh TB (50 μg / L kanamycin, 0.1 mM IPTG) was added to the two deep-well plate mother plates with a spray gun, and the microplate shaker was shaken at 20°C and 1000rpm for 20 hours. Finally, the culture medium was removed by centrifugation at 4000 rpm, 100 μL of cell lysis buffer was added, and the cells were completely lysed on a microplate shaker at room temperature for 20 min, and the supernatant was collected by centrifugation at 4000 rpm. After the supernatant was diluted with buffer in an appropriate proportion, 50 μL of the dilution was pipetted with an equal volume of colorimetric solution (5 mM galactitol, 1 mM NAD + , 100mMTris-HCl 8.0, 0.3g / L NBT, 0.01g / L PMS) were mixed and reacted in the dark. After 20 minutes of reaction, the color depth was observed and recorded, and the OD was measured with an enzyme marker. 580 Each screening plate selects a sample with a darker color than the control and an OD 580 The first three mutants with higher absorbance than the control were cultured overnight, and the plasmids were extracted and sent to Shanghai Bioengineering Co., Ltd. for sequencing. After the first round of saturation mutagenesis was completed for all selected mutation sites, the mutant with the highest enzyme activity after mutation was used as the control, and then the second round of site-directed saturation mutagenesis and screening was carried out.

[0051] A galactitol dehydrogenase mutant with high catalytic activity was obtained by screening. The mutant was found to be T193G / G98C through plasmid extraction and sequencing. Its amino acid sequence is shown in SEQ ID NO.3, and its nucleotide sequence of the encoding gene is shown in SEQ ID NO.4.

[0052] SEQ ID NO.3:

[0053] MSYQQKFRLDGERAVVTGGGRAIGLCCTEALAEAGAAVVVIERSEADAEQALALRNRGYDVEVRVGDVTDAARMDAIATELADGGRPATILVNNAGICQSGIPAQDLTDADWLRMMDVNLNGVFWCSRAFGRSMISMKRGAIVNLGSMSGTICNRPQPQTAYNVSKAAVHHLTRSLAAEWAHHGIRVNAVAPGYIETPMVVAVEANRERIPLWLADTPMARMGTPEEVASAVLFLASGAASLMTGAIVNVDAGFTCW*。

[0054] SEQ ID NO.4:

[0055] .

[0056] Example 2 Induction, expression and purification of R1GDH and T193G / G98C mutants

[0057] The recombinant E. coli expressing R1GDH or T193G / G98C mutant constructed in Example 1 was cultured, induced, expressed and purified. The specific methods are as follows:

[0058] The recombinant E. coli was inoculated into 100 mL of TB medium containing kanamycin (50 μg / mL) and cultured at 37°C with shaking until OD600 When the pH is 0.4, IPTG is added to make the final concentration 0.1mmol / L, and the induction is carried out at 20℃ for 18h. After the induced expression, the bacterial solution is centrifuged at 8000rpm for 10min, the supernatant is discarded, the bacteria are collected, and the bacteria are resuspended with lysis buffer (pH 8.0, 10mmol / L imidazole, 300mmol / LNaCl, 50mmol / L Tris-HCl), and 1mL lysozyme (10mg / mL) is added and left to stand on ice for 15min. Then the cells are ultrasonically lysed (power 250w, working time 5s, interval time 10s, total time 30min, ice bath), and the cell lysate is centrifuged at 4℃12000rpm for 25min. The supernatant is the crude enzyme solution. The crude enzyme solution and the nickel-containing filler in the treated chromatography column are all transferred to a clean finger-shaped bottle, sealed with a sealing film, buried in ice, and shaken on a shaker for 1h to allow the enzyme to fully combine with the nickel in the filler and the enzyme protein with the histidine tag. Pour the filler and enzyme mixture onto the column for purification. Elute the impurity protein 4 times with wash buffer (pH 8.0, 20mmol / L imidazole, 300mmol / L NaCl, 50mmol / LTris-HCl). Add 1mL Elution buffer (pH 8.0, 250mmol / L imidazole, 300mmol / L NaCl, 50mmol / LTris-HCl) to elute the protein. Collect the eluted protein solution and verify it by denaturing polyacrylamide gel electrophoresis (SDS-PAGE).

[0059] The results are as follows Figure 1 As shown, there is a single band at 28 KDa, that is, purified RlGDH and T193G / G98C mutant enzymes were obtained.

[0060] Example 3 Study on the enzymatic properties of R1GDH and T193G / G98C mutants

[0061] Definition of galactitol dehydrogenase activity unit: Under the optimal temperature and optimal pH conditions, the amount of enzyme required to generate 1 μmol of NADH per minute is one enzyme activity unit (1U).

[0062] Determination method: Add 990 μL of galactitol prepared with Tris-HCl buffer as substrate to a 1.5 mL EP tube, then add 10 μL of appropriately diluted enzyme solution, add 10 μL of boiled inactivated enzyme solution to the blank control tube, react for 3 minutes under optimal conditions, then immediately take out and pour into a colorimetric dish to detect OD using a spectrophotometer 340 The amount of NADH generated by the reaction was calculated according to the standard curve, and then the enzyme activity of galactitol dehydrogenase was calculated.

[0063] (1) Optimal temperature detection of RlGDH and T193G / G98C mutants

[0064] 100 mM galactitol in pH 8.0 Tris-HCl buffer was used as substrate to measure the enzyme activities of RlGDH and T193G / G98C mutant at 25°C-55°C. The temperature at which the enzyme activity was highest was taken as 100%, and then the relative enzyme activities at other temperatures were calculated. The results are shown in Figure 2 As shown, the optimum temperature of the wild enzyme RlGDH is 35°C, and the optimum temperature of the T193G / G98C mutant is 40°C, but the enzyme activity of the T193G / G98C mutant still maintains more than 60% of the maximum enzyme activity at 50°C.

[0065] (2) Detection of the optimal pH value of RlGDH and T193G / G98C mutants

[0066] The enzyme activities of RlGDH and T193G / G98C mutants were measured at 30°C using buffers with different pH ranges, i.e., 50 mM Tris-HCl buffer (pH 7.0-8.9) and 50 mM Tris-glycine-NaOH buffer (pH 9-11.5), with 100 mM galactitol as substrate. The pH value with the highest enzyme activity was taken as 100%, and then the relative enzyme activities under other pH conditions were calculated. The results are shown in Table 1. Figure 3 As shown, the optimum pH of the wild enzyme RlGDH is 9.5, and the optimum pH of the T193G / G98C mutant is 10.5. However, the enzyme activity of the T193G / G98C mutant still maintains more than 60% of the maximum enzyme activity in the pH range of 9.0-11.5.

[0067] (3) Temperature stability test of RlGDH and T193G / G98C mutants

[0068] The purified RlGDH and T193G / G98C mutants were dissolved in 50 mM Tris-HCl (pH 8.0) and then incubated in a 35°C water bath. Samples were taken at different time intervals to determine the residual enzyme activity. The temperature stability curves of RlGDH and the mutants were drawn with the activity of the untreated enzyme as 100%. The half-life (t 1 / 2 ) is defined as the time when the activity drops to 50% of the initial activity and is calculated by nonlinear regression. Figure 4 As shown, the wild enzyme RlGDH has poor temperature stability with a half-life of only 7.5 hours, while the T193G / G98C mutant has a half-life of 128 hours at 35°C, showing extremely strong thermal stability.

[0069] (4) pH stability test of RlGDH and T193G / G98C mutants

[0070] The purified RlGDH and T193G / G98C mutants were dissolved in a buffer solution of pH 7.0-11.5 and incubated at 4°C for 12 h. The residual enzyme activity was then determined under the optimal conditions, and the pH stability curves of RlGDH and the mutants were drawn with the activity of the untreated enzyme as 100%. Figure 5 As shown, the wild enzyme RlGDH remained stable between pH 7.0 and 9.5. The T193G / G98C mutant showed stronger pH stability, and the enzyme activity still maintained more than 70% of the highest enzyme activity under a wide pH range of 7.0-11.0.

[0071] (5) R1GDH and T193G / G98C mutant V max and K m Determination of value

[0072] The V of RlGDH and T193G / G98C mutant were determined under the optimal reaction temperature and pH of wild-type enzyme RlGDH. max and K m The reaction time was 3 minutes. The enzyme activity was determined by reacting the same concentration of enzyme solution with 2.5mmol / L, 5mmol / L, 10mmol / L, 25mmol / L, 50mmol / L, 75mmol / L, 100mmol / L, 150mmol / L, 200mmol / L, and 300mmol / L galactitol substrates. Nonlinear fitting was performed using GraphPad Prism 9, and K was calculated using the Michaelis-Menten equation. m Value and V max value.

[0073] The results are as follows Figure 6 As shown, the K of wild-type enzyme RlGDH m The value was 23.14±0.82mM, V max The value was 5.43±0.4U / mg. Figure 7 As shown, the K of the T193G / G98C mutant m The value is 181.5±13.39mM, V max The value was 134.2±5.21U / mg. The enzyme activity of the T193G / G98C mutant reached 50U / mg at a substrate concentration of 100mmol / L, which was 24 times that of the wild type. At the same time, the T193G / G98C mutant had the highest enzyme activity among all reported galactitol dehydrogenases.

[0074] Example 4 Preparation of D-tagatose by Combination of T193G / G98C Mutant and NADH Oxidase

[0075] D-tagatose was prepared by combining the T193G / G98C mutant with NADH oxidase-SpNOX enzyme (amino acid sequence as shown in SEQ ID NO.5) as follows:

[0076] Crude enzyme reactions were performed at a 100 mL scale in 500 mL baffled Erlenmeyer flasks.

[0077] Reaction system: 1g T193G / G98C mutant crude enzyme, 1g SpNOX crude enzyme, 0.067g NAD + , 360 g / L galactitol substrate, pH 8.0 Tris-HCl buffer. After shaking the flask at 220 rpm for 12 h under an oxygen environment, the amount of D-tagatose produced was detected by HPLC.

[0078] HPLC conditions were as follows: instrument: Waters 1525 chromatograph; chromatographic column: MARS MCa HPX-87C; mobile phase: ultrapure water; flow rate: 0.6 mL / min; detector: Waters 2414 refractometer.

[0079] Figure 8 and Fig. 9 HPLC profiles of D-tagatose and galactitol standards, respectively; Fig.10 This is the HPLC chart of the preparation of D-tagatose by coupling the T193G / G98C mutant with NADH oxidase. The results of HPLC detection show that 360g / L of galactitol is completely converted into D-tagatose. This is the highest yield among all reports on the production of D-tagatose using galactitol dehydrogenase as a catalyst.

[0080] In summary, the galactitol mutant T193G / G98C of the present invention has high catalytic activity for galactitol and good thermal stability, and can be coupled with NADH oxidase to completely convert high-concentration substrate galactitol into D-tagatose.

[0081] SEQ ID NO.5:

[0082] MGSKIVVVGANHAGTACIKTMLTNYGDANEIVVFDQNSNISFLGCGMALWIGEQIAGPEGLFYSDKEELESLGAKVYMESPVQSIDYDAKTVTALVDGKNHVETYDKLIFATGS QPILPPIKGAEIKEGSLEFEATLENLQFVKLYQNSADVIAKLENKDIKRVAVVGAGYIGVELAEAFQRKGKEVVLIDVVDTCLAGYYDRDLTDLMAKNMEEHGIQLAFGETVKEV AGNGKVEKIITDKNEYDVDMVILAVGFRPNTTLGNGKIDLFRNGAFLVNKRQETSIPGVYAIGDCATIYDNATRDTNYIALASNAVRTGIVAAHNACGTDLEGIGVQGSNGISIY GLHMVSTGLTLEKAKRLGFDAAVTEYTDNQKPEFIEHGNFPVTIKIVYDKDSRRILGAQMAAREDMSMGIHMFSLAIQEGVTIEKLALTDIFFLPHFNKPYNYITMAALGAKD*.

[0083] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A galactitol dehydrogenase mutant, characterized in that: The amino acid sequence is shown in SEQ ID NO.

3.

2. A gene encoding the galactitol dehydrogenase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO.

4.

4. A recombinant vector, characterized in that: Comprising the coding gene described in claim 2 or 3.

5. A recombinant host cell, characterized in that Comprising the recombinant vector according to claim 4.

6. Use of the encoding gene according to claim 2 or 3, the recombinant vector according to claim 4 or the recombinant host cell according to claim 5 in preparing the galactitol dehydrogenase mutant according to claim 1.

7. Use of the galactitol dehydrogenase mutant according to claim 1 in the preparation of D-tagatose.

8. An enzyme combination for preparing D-tagatose, characterized in that: The method comprises NADH oxidase and the galactitol dehydrogenase mutant according to claim 1.

9. Use of the enzyme combination according to claim 8 in the preparation of D-tagatose.

10. A method for preparing D-tagatose, characterized in that: The method comprises using galactitol as a substrate, using the enzyme combination according to claim 8 as a catalyst, and using NAD + The step of preparing the D-tagatose as a hydrogen acceptor through a catalytic reaction.

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