D-tagatose 4-epimerase mutant for efficient synthesis of d-tagatose and application thereof
By mutating the amino acid sequence of D-tagatose 4-epimerase, a highly efficient D-tagatose 4-epimerase mutant was obtained, solving the problems of enzyme library scarcity and low catalytic efficiency, and realizing the efficient preparation of D-tagatose, which has industrial application potential.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG UNIV OF TECH
- Filing Date
- 2026-03-19
- Publication Date
- 2026-06-09
AI Technical Summary
The existing D-tagatose 4-epimerase enzyme library is scarce, has low catalytic efficiency, and is difficult to modify, making it difficult to meet the needs of industrial production.
By performing single or combined mutations in the amino acid sequence of D-tagatose 4-epimerase, a highly efficient D-tagatose 4-epimerase mutant was obtained. A recombinant vector and recombinant genetically engineered bacteria were then constructed to catalyze the preparation of D-tagatose from D-fructose.
The conversion efficiency of D-tagatose was improved, and the conversion rate of the mutant at 70℃ was more than 3 times that of the wild type. This expands the application potential of the enzyme library, overcomes the environmental pollution problems of chemical synthesis methods, and has important industrial application prospects.
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Abstract
Description
(I) Technical Field
[0001] This invention belongs to the field of bioengineering, specifically relating to a D-tagatose 4-epimerase for the efficient synthesis of D-tagatose. Eb T4E mutants and their applications. (II) Background Technology
[0002] D-Tagatose is a rare, naturally occurring monosaccharide belonging to the hexulose family, with the chemical formula C6H. 12 O6 is a diastereomer of fructose, with the hydroxyl group at the C-4 position of fructose facing a different direction. Tagash has approximately 92% the sweetness of sucrose but only one-third the calories. It also has virtually no unpleasant aftertaste and is known to lower blood sugar, reduce obesity, and improve gut microbiota. It was approved as a new food ingredient by the U.S. Food and Drug Administration in 2001 and by my country in 2014, respectively, thus possessing broad market prospects in the pharmaceutical and health industries.
[0003] The chemical synthesis of tagatose involves using soluble alkali or alkaline earth metal salts as catalysts to catalyze the isomerization reaction of D-galactose with metal hydroxides under alkaline conditions. This method suffers from drawbacks such as numerous byproducts, low product purity, and the potential for heavy metal and acidic wastewater pollution. Biological methods for tagatose preparation offer advantages such as fewer byproducts, milder reaction conditions, and environmental friendliness, making them a promising mainstream method for future industrial production. Currently, biological tagatose preparation primarily relies on the synergistic catalysis of lactose by β-galactosidase and L-arabinose isomerase. This reaction requires two steps, and the reported L-arabinose isomerases generally exhibit poor thermal stability. Therefore, exploring other efficient and low-cost routes for tagatose synthesis is of significant research and application value.
[0004] Tagatose 4-epimerase (T4E) can achieve the interconversion between D-fructose and D-tagatose, and has the advantages of simple reaction, low cost and simple separation and purification. However, there are few reported tagatose 4-epimerases, and their activities are generally insufficient for industrial production. (III) Summary of the Invention
[0005] The purpose of this invention is to provide a novel, highly efficient D-tagatose 4-epimerase mutant for the synthesis of D-tagatose and its application, thereby solving the technical problems of the scarcity of D-tagatose 4-epimerase enzyme libraries, low catalytic efficiency, and high difficulty in modification.
[0006] The technical solution adopted in this invention is:
[0007] This invention provides a novel, highly efficient D-tagatose 4-epimerase mutant for the synthesis of D-tagatose. The mutant is obtained by single or combined mutations at positions 131, 17, 18, 31, 32, 70, 73, 95, 119, 120, 122, 130, 172, 181, 201, 220, 234, 256, 258, 265, 267, 197, 279, 280, 294, 320, 334, 358, 359, 372, 410, 414, 453, and 463 of the amino acid sequence shown in SEQ ID NO.2.
[0008] Further, the D-tagatose 4-epimerase mutant is one of the following: S131D, S131D / D17A, S131D / G18A, S131D / E31G, S131D / G32D, S131D / G70P, S131D / A73H, S131D / P95D, S131D / A119G, S131D / K120D, S131D / G122A, S131D / Q130N, S131D / K172P, S131D / S181A, S131D / H197D, S131D / F201W, S131D / L220H, S131D / G234A, S131D / R256I, S131D / C258I, S131D / I265K, S131D / P267A, S131 D / S275A, S131D / T279C, S131D / T279G, S131D / T279M, S131D / T279N, S131D / T279S, S131D / T279V, S131D / R280A, S1 31D / S294E, S131D / A320I, S131D / V359I, S131D / T358I, S131D / A372S, S131D / H410C, S131D / H410E, S131D / H410F, S131D / H410T, S131D / H410W, S131D / K414D, S131D / E453D, S131D / L220H / A320I, S131D / L220H / Q334A, S131D / L220 H / M463L, S131D / S294E / A320I, S131D / Q334A / S294E, S131D / Q334A / A320I, S131D / Q334A / M463L, S131D / M463L / S2 94E, S131D / M463L / A320I, S131D / H410W / S294E, S131D / H410W / T279C, S131D / H410W / T279S, S131D / H410W / T279V.
[0009] Due to the specificity of amino acid sequences, any variant of a polypeptide with an amino acid sequence, such as its conserved variants, bioactive fragments, or derivatives, is within the scope of protection of this invention, provided that the polypeptide fragment or variant shares more than 75% homology with the aforementioned amino acid sequence. The alterations may include the deletion, insertion, or substitution of amino acids in the amino acid sequence; for conserved alterations of variants, the substituted amino acid has a similar structure or chemical properties to the original amino acid, such as replacing isoleucine with leucine; variants may also have non-conserved alterations, such as replacing glycine with tryptophan.
[0010] This invention also relates to the encoding gene of a D-tagatose 4-epimerase mutant, a recombinant vector containing said encoding gene, and a recombinant genetically engineered bacterium. The recombinant vector uses pET28a(+) as the base plasmid, and the recombinant genetically engineered bacterium uses... E.coli BL21(DE3) is the host bacterium.
[0011] This invention also relates to the application of the D-tagatose 4-epimerase mutant in the microbial catalytic isomerization of D-fructose to prepare D-tagatose. A recombinant vector containing the gene of the D-tagatose 4-epimerase mutant is constructed, and the recombinant vector is transformed into *Escherichia coli*. Escherichia coli In this process, the recombinant genetically engineered bacteria are induced and cultured. The culture medium is then used to separate wet bacterial cells containing D-tagatose 4-epimerase or pure enzyme solution extracted from broken wet bacterial cells. This enzyme can be used as a catalyst for the microbial catalytic reaction of D-fructose isomerization to prepare D-tagatose.
[0012] Preferably, the application is as follows: using wet bacterial cells or pure enzyme solution extracted from the crushed wet bacterial cells obtained by fermentation culture of recombinant genetically engineered bacteria containing the encoding gene of D-tagatose 4-epistoisase mutant as catalyst, D-fructose as substrate, nickel ions as co-catalyst, and a buffer solution of pH 7-9 (preferably 50 mM sodium phosphate buffer solution (pH 8.0)) as reaction medium to form a reaction system. After the reaction is completed at 50-80℃ and 500-1200 r / min, a mixture of D-fructose and D-tagatose is obtained.
[0013] Preferably, in the reaction system, the initial concentration of the substrate is 10-100 g / L (preferably 50 g / L), the final concentration of the wet bacterial cells is 10-150 g / L (preferably 40 g / L), the final concentration of the pure enzyme solution is 1-20 mg / mL (preferably 10 mg / mL) based on the protein content, and the final concentration of nickel ions is 0.1-10 mM (preferably 1 mM).
[0014] Preferably, the wet bacterial cells are prepared as follows: recombinant genetically engineered bacteria containing the D-tagatose 4-epimerase mutant gene are inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 200 r / min until OD. 600 =0.8–1.0, to obtain seed culture; the seed culture is transferred at an inoculum concentration of 1–5% to LB liquid medium containing a final concentration of 50 μg / mL kanamycin, and cultured at 37℃ and 180 r / min until OD. 600 =0.6~0.8, add IPTG to a final concentration of 0.1 mM, and induce culture at 28℃ and 180 r / min for 12~14 h to obtain the induced culture suspension. Centrifuge the induced culture suspension at 8000 rpm for 10 min to collect the wet cells.
[0015] Preferably, the pure enzyme solution is prepared according to the following method:
[0016] The wet bacterial cells were resuspended in a pH 8.0, 50 mM sodium phosphate buffer (preferably 50 g / L), sonicated on ice for 1 h at 200 W for 1 s, paused for 2 s, and centrifuged at 8000 r / min for 10 min. The supernatant was filtered through a 0.45 μm filter membrane, and the filtrate was the crude enzyme solution.
[0017] A Ni-NTA chromatography column was used. After washing with deionized water and equilibration buffer for 5-15 column volumes, crude enzyme solution was loaded at a flow rate of 1 mL / min. After loading, the column was washed with equilibration buffer for 5-10 column volumes to remove contaminating proteins. Then, the column was washed with equilibration buffer containing 100 mM imidazole for 5-15 column volumes. Finally, the target protein was eluted with equilibration buffer containing 500 mM imidazole for 5-15 column volumes. The eluent was collected and the protein concentration was adjusted with 50 mM sodium phosphate buffer (pH 8.0) to obtain pure enzyme solution. The equilibration buffer was 50 mM sodium phosphate buffer (pH 8.0) containing 300 mM sodium chloride.
[0018] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:
[0019] (1) In this invention, the wild-type D-tagatose 4-epimerase was first screened to obtain wild-type enzymes that could be used to further improve activity through mutation. Then, the wild-type enzymes were mutated and modified to obtain multiple D-tagatose 4-epimerase mutants that were highly efficient in synthesizing D-tagatose. These mutants improved the conversion efficiency of D-fructose to D-tagatose. The conversion rate of the mutants at 70°C was more than 3 times that of the wild type. The conversion rates of whole-cell catalysis and pure enzyme solution catalysis reached 26.22% and 29.46%, respectively. Compared with the wild type, the mutants could catalyze the conversion of higher concentrations of substrates.
[0020] (2) This invention improves the problem of poor catalytic activity of wild enzymes at high temperatures by modifying them, expands the scarce enzyme library, demonstrates the potential of D-fructose in the synthesis of D-tagatose, overcomes the problem of easy generation of three wastes by chemical synthesis method, and has important industrial application prospects. (iv) Description of the attached drawings
[0021] Figure 1 This is a schematic diagram of the enzyme-catalyzed synthesis of D-tagatose from D-fructose.
[0022] Figure 2 The image shows the high-performance liquid chromatography (HPLC) results for D-fructose and D-tagatose standards.
[0023] Figure 3 This is a high-performance liquid chromatography (HPLC) chromatogram of the reaction solution sample. (V) Detailed Implementation Methods
[0024] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:
[0025] Example 1: Wild-type D-tagatose 4-epimerase genetically engineered bacteria E.coli Construction of BL21(DE3) / T4E
[0026] Gene bank from Eubacteriales bacterium The gene sequence of MAG: tagaturonate epimerasefamily protein (NCBI accession number: MDD7397597.1) was codon-optimized (nucleotide sequence as shown in SEQ ID NO.1, amino acid sequence encoding the protein as shown in SEQ ID NO.2), and a 6×His-Tag tag was added to the C-terminus. pET28a(+)- was then synthesized through whole-genome synthesis. Eb T4E plasmid, transform the plasmid into E.coli Wild type obtained in BL21(DE3) E.coli BL21(DE3) / pET28a / Eb T4E, denoted as wild-type enzyme Eb T4E.
[0027] The same method will come from Cohnella-laeviribosi The gene sequence of the tagaturonate epimerase family protein (NCBI accession number: WP_019005805) was codon-optimized (nucleotide sequence as shown in SEQ ID NO. 3) to construct the wild-type. E.coli BL21(DE3) / pET28a / ClT4E, denoted as wild-type enzyme Cl T4E.
[0028] The same method will come from Lentisphaeria-bacterium The gene sequence of the tagaturonate epimerase family protein (NCBI accession number: MBN2449016) was codon-optimized (nucleotide sequence shown in SEQ ID NO.4) to construct the wild-type protein. E.coli BL21(DE3) / pET28a / Lb T4E, denoted as wild-type enzyme Lb T4E.
[0029] The same method will come from Rubrobacter-sp. The gene sequence of the tagaturonate epimerase family protein (NCBI accession number: HEY6581954.1) was codon-optimized (nucleotide sequence as shown in SEQ ID NO. 5) to construct the wild-type. E.coli BL21(DE3) / pET28a / Rs T4E, denoted as wild-type enzyme Rs T4E.
[0030] The same method will come from Thermotoga lettingae DSM 14385 The gene sequence of the tagaturonate epimerase family protein (NCBI accession number: ABV33391) was codon-optimized (nucleotide sequence as shown in SEQ ID NO.6) to construct the wild-type. E.coli BL21(DE3) / pET28a / Tl T4E, denoted as wild-type enzyme Tl T4E.
[0031] The same method will come from Dictyoglomus turgidum DSM 6724 The gene sequence of the tagaturonate epimerase family protein (NCBI accession number: ACK41523) was codon-optimized (nucleotide sequence as shown in SEQ ID NO.7) to construct the wild-type. E.coli BL21(DE3) / pET28a / Dt T4E, denoted as wild-type enzyme Dt T4E.
[0032] The same method will come from Mammaliicoccus-sciuriThe gene sequence of the hypothetical protein (NCBI accession number: KTT85151.1) was codon-optimized (nucleotide sequence as shown in SEQ ID NO.8) to construct the wild-type. E.coli BL21(DE3) / pET28a / Ms T4E, denoted as wild-type enzyme Ms T4E.
[0033] Example 2: Wild-type enzyme Eb Construction of T4E single-site or multi-site mutants
[0034] 1. Design of mutation sites
[0035] Wild-type enzymes were constructed using Alphafold3. Eb The T4E protein 3D model was used, and PyMOL software was employed for visualization analysis of the active pockets and sites of protein-substrate binding. To remodel the active pocket microenvironment and facilitate substrate-enzyme binding, sites 131, 130, 135, 145, 146, 147, 148, 187, 275, 279, 280, 372, and 410 near the substrate were modified. Simultaneously, a distal modification strategy was used to modify sites 17, 18, 31, 32, 70, 73, 95, 119, 120, 122, 172, 181, 201, 220, 234, 256, 258, 265, 267, 294, 320, 334, 358, 359, 414, 453, and 463.
[0036] 2. Unit point mutation
[0037] First, based on wild enzymes Eb The gene sequence of T4E (nucleotide sequence is SEQ ID NO.1) was compared with favorable site sequences reported in the literature. Primers for mutation at site 131 near the substrate were designed (Table 1). Using rapid PCR technology, a single mutation was introduced at site 131 using the recombinant vector pET28a / EbT4E as a template.
[0038] The PCR reaction system (50 μL) consisted of: 25 μL of 2×Phanta Max Buffer, 1 μL of dNTPs, 2 μL of forward primer (5 pmol / μL), 2 μL of reverse primer (5 pmol / μL), 1 μL of template DNA (20 ng / μL), 1 μL of Phanta Max SuperFidelity DNA Polymerase, and ddH2O added to a final volume of 50 μL.
[0039] The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 30 cycles (95℃ denaturation for 10 s, 55-60℃ annealing for 15 s, 72℃ extension for 3.5 min); 72℃ extension for 10 min; and finally, incubation at 16℃. The PCR products were verified by 0.9% agarose gel electrophoresis. Once the amplified gene fragment matched the size of the target vector, the next step of the experiment could be carried out.
[0040] After digesting the template with DpnI at 37℃ for 3 hours, 3 μL of the PCR product was added to 100 μL of ice bath. E.coli In a suspension of BL21(DE3) competent cells, the cells were incubated on ice for 30 min. The transformation product was then heat-shocked at 42°C for 90 s, followed by rapid cooling on ice for 5 min. 600 μL of LB liquid medium was added to the tube, and the cells were incubated at 37°C, 150 rpm for 50 min, and centrifuged at 12000 rpm for 1 min. 400 μL of supernatant was discarded, and the bacterial suspension was resuspended. 200 μL of the resuspended suspension was plated onto LB agar plates containing 50 μg / mL kanamycin. After complete absorption of the culture medium, the plates were incubated upside down at 37°C for 12 h. Colonies were picked and inoculated into 10 mL of LB liquid medium containing 50 μg / mL kanamycin, and incubated at 37°C for 12 h. The resulting bacterial suspension was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Correct sequencing results indicated a mutant recombinant strain. Expression was induced using the method in Example 3, and enzyme activity was measured using the method in Example 4, with wild-type enzyme as the reference. Eb The enzyme activity of T4E was 100%. The relative enzyme activities of other mutants were calculated, and the results of the relative enzyme activities of some mutants are shown in Table 2. Based on Table 2, the dominant mutant S131D with significantly improved enzyme activity was obtained. In order to save time and cost, combined mutations were carried out based on this dominant mutant.
[0041] Table 1. Primer sequences
[0042]
[0043]
[0044]
[0045] Table 2. Enzyme activities of wild-type and single-point mutants
[0046]
[0047] 3. Combinatorial mutation
[0048] Based on step 2, using the dominant single mutant S131D as a template, the sites identified in step 1 were predicted using the online website PROSS, resulting in the mutants listed in Table 1. Combination mutations at other sites were then performed using the primers in Table 1, with the remaining procedures identical to step 2. The enzyme activities of the combined mutants are shown in Table 3.
[0049] Table 3 Enzyme activities of wild type and combinatorial mutants
[0050]
[0051] Example 3: Wild-type enzyme Cl T4E, Lb T4E, Rs T4E, Tl T4E, Dt T4E, Ms Construction of T4E mutants
[0052] Using the method of Example 2, Eb The S131 site of T4E was located in one of the other six enzymes through multiple sequence alignment, allowing selection of the wild-type enzyme. Cl The 125th position of T4E, Lb The 131st site of the T4E enzyme, Rs The 130th site of T4E, Tl The 128th site of T4E Dt The 130th site of T4E, Ms Modification was performed at position 121 of T4E, replacing serine with aspartic acid. The results showed that after modification at the key site, the conversion rate of these six enzymes dropped directly to 0, indicating a loss of catalytic activity. Therefore, wild-type enzymes were selected. Eb T4E and its mutants were used to synthesize D-tagatose.
[0053] Example 4: Preparation of recombinant D-tagatose 4-epimerase, mutant wet cells, and purified enzymes
[0054] Wild-type enzyme from Example 1 Eb Recombinant *E. coli* mutants obtained in Example 2 (T4E) were streaked onto LB solid medium plates containing kanamycin resistance at a final concentration of 50 μg / mL for activation. After incubation at 37°C for 12 h, single colonies were picked and inoculated into LB liquid medium containing kanamycin resistance at a final concentration of 50 μg / mL, and cultured at 37°C and 180 rpm for 6-8 h until OD (digestive activity) was reached. 600 When the bacterial OD reaches 0.8–1.0, it is then inoculated at a 1% (v / v) inoculation rate into 100 mL of fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance. The medium is then incubated at 37°C and 180 r / min until the bacterial OD reaches 0.8–1.0. 600When the concentration reaches 0.6-0.8, add IPTG to a final concentration of 0.1 mM, and induce culture at 28℃ and 180 r / min for 12-14 h. Then, centrifuge at 4℃ and 8000 r / min for 10 min, discard the supernatant, and collect the precipitate to obtain the wet cells of recombinant D-tagatose 4-epimerase and mutants. The wet cells can be used directly as a biocatalyst or for protein purification.
[0055] The wet bacterial cells were resuspended in pH 8.0, 50 mM sodium phosphate buffer to 50 g / L, and disrupted on ice for 1 h (power 200W, disruption 1s, pause 2s), centrifuged at 8000 r / min for 10 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate is the crude enzyme solution.
[0056] The recombinant strain carried a 6×His-Tag tag, so a Ni-NTA affinity chromatography column (5 mL pre-packed) was used to purify the protein. The AKTA protein purification system was employed, using a gradient elution program to achieve efficient separation and purification. The specific steps are as follows: Using a Ni-NTA chromatography column, the protein was washed with deionized water and equilibration buffer for 10 column volumes each. Crude enzyme solution was then loaded at a flow rate of 1 mL / min. After loading, the protein was washed with equilibration buffer for 5 column volumes to remove contaminating proteins. Next, the protein was washed with equilibration buffer containing 100 mM imidazole for 10 column volumes each. The competitive displacement effect of imidazole effectively removed non-specifically adsorbed contaminating proteins. Finally, the target protein was eluted with equilibration buffer containing 500 mM imidazole for 10 column volumes. The eluent was collected, and the protein concentration was adjusted with 50 mM sodium phosphate buffer (pH 8.0) to obtain the final protein. Eb Pure enzyme solutions of T4E and its mutants. Equilibration buffer composition: 50 mM sodium phosphate buffer + 300 mM sodium chloride (pH 8.0).
[0057] Example 5: D-tagatose 4-epimerase Eb Activity assay of T4E and its mutants
[0058] The wet cell catalyst D-fructose from the wild-type and mutant strains obtained in Example 3 was used. The wet cells were thoroughly resuspended in 50 mM sodium phosphate buffer (pH 8.0), and then 1 mM Ni was added to a final concentration. 2+ A 1 mL reaction system was prepared with D-fructose at a final concentration of 10 g / L, and wet bacterial cells were added to the system to a final concentration of 40 g / L. The reaction was carried out at 70℃ and 1000 r / min for 2 h. Samples were taken, and the reaction was terminated by incubating on ice for 10 min. The mixture was then centrifuged at 12000 r / min for 10 min, and the supernatant was diluted with deionized water to a total sugar concentration (D-fructose and D-tagatose) below 2 g / L for later use.
[0059] HPLC detection conditions: A Thermo Fisher Scientific high-performance liquid chromatography system, Thermo Fisher Scientific differential detector, and Thermo Fisher Scientific autosampler, or an Agilent high-performance liquid chromatography system, Agilent differential detector, and Agilent autosampler were used to detect the product D-tagatose and the substrate D-fructose. The analytical column was a Sugarpark column, the mobile phase was 0.5 g / L EDTA-calcium disodium, and the solvent was ultrapure water. The flow rate was set to 0.4 mL / min, the column temperature to 80 ℃, and the injection volume to 10 μL. A schematic diagram of the HPLC detection of the substrate D-fructose and the product D-tagatose standard is shown below. Figure 2 As shown, the elution time of D-fructose was 13.6 min, and that of D-tagatose was 15.8 min. A schematic diagram of the HPLC detection of the sample is shown below. Figure 3 As shown.
[0060] Example 6: Conversion rate determination of D-tagatose 4-epimerase dominant mutant
[0061] Wild-type enzymes Eb T4E, the superior double mutant and triple mutant screened in Example 2 were prepared into wet cells using the method in Example 3 for catalytic substrate D-fructose.
[0062] The wet bacterial cells were thoroughly resuspended in 50 mM sodium phosphate buffer (pH 8.0), and then 1 mM Ni was added to the final concentration. 2+ 1 mL of D-fructose with a final concentration of 50 g / L was used to prepare the reaction system, and wet bacterial cells were added to a final concentration of 40 g / L. The reaction was carried out at 70℃ and 1000 r / min for 6 h, terminated by an ice bath for 10 min, and centrifuged at 12000 r / min for 10 min. The supernatant was then diluted to a total sugar concentration below 2 g / L, and liquid chromatography was performed using the method described in Example 4. The conversion rate (%) was defined as the ratio of the D-tagatose concentration to the initial D-fructose concentration. The results are shown in Table 4.
[0063] Table 4. Wild-type enzymes Eb T4E and mutant transformation rate
[0064]
[0065] Table 4 shows that the optimal double mutant S131D / H410T achieved a conversion rate of 17.11%, and the optimal triple mutant S131D / H410W / T279S achieved a conversion rate of 20.43%.
[0066] Example 7: Triple Mutant Eb Application of T4E-S131D / H410W / T279S in the Synthesis of D-Tagagose
[0067] triple mutant EbT4E-S131D / H410W / T279S was prepared using the method in Example 3 to produce wet bacterial cells and pure enzyme solution, which were then used to catalyze the conversion of D-fructose to D-tagatose.
[0068] Whole-cell wet cell catalysis: The wet cells were thoroughly resuspended in 50 mM sodium phosphate buffer (pH 8.0), and then 1 mM Ni was added to a final concentration. 2+ A 1 mL reaction system was prepared with D-fructose at a final concentration of 50 g / L, and wet bacterial cells were added to the system to a final concentration of 80 g / L. The reaction was carried out at 70℃ and 1000 r / min for 6 h, terminated by an ice bath for 10 min, and centrifuged at 12000 r / min for 10 min. The supernatant was then diluted to a total sugar concentration below 2 g / L, and liquid chromatography was performed using the method described in Example 4. The results are shown in [Figure 4]. Figure 3 Optimal Triple Mutant Eb The whole-cell transformation efficiency of T4E-S131D / H410W / T279S reached 26.22%.
[0069] Pure enzyme solution catalysis: Pure enzyme solution was used as the catalyst. The amount of pure enzyme solution added to the reaction system was based on the protein content, with a final concentration of 10 mg / mL. Then, 1 mM Ni was added to achieve a final concentration of... 2+ A final concentration of 50 or 100 g / L D-fructose was added to a 50 mM sodium phosphate buffer solution (pH 8.0) and reacted at 70°C and 1000 r / min for 6 h. The reaction was terminated by an ice bath for 10 min, followed by centrifugation at 12000 r / min for 10 min. The supernatant was then diluted to a total sugar concentration of less than 2 g / L, and liquid chromatography was performed using the method described in Example 4. Optimal Triple Mutant Eb The T4E-S131D / H410W / T279S pure enzyme solution achieved a conversion rate of 29.46% for 50 g / L D-fructose and 27.99% for 100 g / L D-fructose.
Claims
1. A D-tagatose 4-epimerase mutant for efficient synthesis of D-tagatose, characterized in that, The mutants were obtained by performing single or combined mutations on positions 131, 17, 18, 31, 32, 70, 73, 95, 119, 120, 122, 130, 172, 181, 201, 220, 234, 256, 258, 265, 267, 197, 279, 280, 294, 320, 334, 358, 359, 372, 410, 414, 453, and 463 of the amino acid sequence shown in SEQ ID NO.
2.
2. The mutant as described in claim 1, characterized in that, The D-tagatose 4-epimerase mutant is one of the following: S131D, S131D / D17A, S131D / G18A, S131D / E31G, S131D / G32D, S131D / G70P, S131D / A73H, S131D / P95D, S131D / A119G, S131D / K120D, S131D / G122A, S131D / Q130N, S131D / K172P, S131D / S181A, S131D / H19 7D, S131D / F201W, S131D / L220H, S131D / G234A, S131D / R256I, S131D / C258I, S131D / I265K, S131D / P267A, S131D / S275A, S131D / T279C, S131D / T279G, S131D / T279M, S131D / T279N, S131D / T279S, S131D / T279V, S131D / R280A, S131 D / S294E, S131D / A320I, S131D / V359I, S131D / T358I, S131D / A372S, S131D / H410C, S131D / H410E, S131D / H410F, S 131D / H410T, S131D / H410W, S131D / K414D, S131D / E453D, S131D / L220H / A320I, S131D / L220H / Q334A, S131D / L220H / M463L, S131D / S294E / A320I, S131D / Q334A / S294E, S131D / Q334A / A320I, S131D / Q334A / M463L, S131D / M463L / S2 94E, S131D / M463L / A320I, S131D / H410W / S294E, S131D / H410W / T279C, S131D / H410W / T279S, S131D / H410W / T279V.
3. A recombinant genetically engineered bacterium containing the encoding gene of the D-tagatose 4-epimerase mutant as described in claim 1.
4. The application of the D-tagatose 4-epimerase mutant of claim 1 in the preparation of D-tagatose by microbial catalytic D-fructose isomerization.
5. The application as described in claim 4, characterized in that, The application is as follows: using wet cells or pure enzyme solutions extracted from the crushed wet cells obtained by fermentation culture of recombinant genetically engineered bacteria containing the encoding gene of D-tagatose 4-epistoisase mutant as catalysts, D-fructose as substrates, nickel ions as co-catalysts, and a buffer solution with pH 7-9 as the reaction medium to form a reaction system, and after the reaction is completed at 50-80℃ and 500-1200r / min, a mixture of D-fructose and D-tagatose is obtained.
6. The application as described in claim 5, characterized in that, In the reaction system, the initial concentration of the substrate is 10–100 g / L, the final concentration of the wet bacterial cells is 10–150 g / L, the final concentration of the pure enzyme solution is 1–20 mg / mL based on the protein content, and the final concentration of nickel ions is 0.1–10 mM.
7. The application as described in claim 5, characterized in that, The wet bacterial cells were prepared as follows: recombinant genetically engineered bacteria containing the D-tagatose 4-epimerase mutant gene were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 200 r / min until OD200. 600 =0.8–1.0, to obtain seed culture; the seed culture is transferred at an inoculum concentration of 1–5% to LB liquid medium containing a final concentration of 50 μg / mL kanamycin, and cultured at 37℃ and 180 r / min until OD. 600 =0.6~0.8, add IPTG to a final concentration of 0.1 mM, and induce culture at 28℃ and 180 r / min for 12~14 h to obtain the induced culture suspension. Centrifuge the induced culture suspension at 8000 rpm for 10 min to collect the wet cells.
8. The application as described in claim 5, characterized in that, The pure enzyme solution was prepared as follows: Wet bacterial cells were resuspended in pH 8.0, 50 mM sodium phosphate buffer, sonicated on ice for 1 h at 200 W for 1 s, paused for 2 s, and centrifuged at 8000 r / min for 10 min. The supernatant was filtered through a 0.45 μm filter membrane, and the filtrate was the crude enzyme solution. A Ni-NTA chromatography column was used. After washing with deionized water and equilibration buffer for 5-15 column volumes, the crude enzyme solution was loaded at a flow rate of 1 mL / min. After loading, the column was washed with equilibration buffer for 5-10 column volumes to remove contaminating proteins. Then, the column was washed with equilibration buffer containing 100 mM imidazole for 5-15 column volumes, followed by elution with equilibration buffer containing 500 mM imidazole for 5-15 column volumes. The effluent was collected, and the protein concentration was adjusted with pH 8.0, 50 mM sodium phosphate buffer to obtain the pure enzyme solution. The equilibration buffer was a pH 8.0, 50 mM sodium phosphate buffer containing 300 mM sodium chloride. mM sodium phosphate buffer.