A tagatose 4-epimerase and its application

Abstract

This invention belongs to the field of biosynthesis technology and relates to a tagatose 4-epimerase and its applications. The amino acid sequence of the tagatose 4-epimerase includes any one of the sequences shown in SEQ ID No:1-SEQ ID No:9. The tagatose 4-epimerase provided by this invention has high catalytic activity, providing a highly active biocatalyst for the biosynthesis of D-tagatose and facilitating the industrial production of D-tagatose.

Description

Technical Field

[0001] This invention belongs to the field of biosynthesis technology and relates to a tagatose 4-epimerase and its application. Background Technology

[0002] Rare sugars are a class of monosaccharides and their derivatives that exist in nature but are scarce. As novel functional sweeteners, their research and development has become an international research hotspot. Rare sugars not only have low-calorie and low-absorption metabolic characteristics, but also exhibit various physiological activities such as anti-caries, blood sugar regulation, prebiotic function, and antioxidant properties, making them of significant application value in the fields of food, medicine, and health care.

[0003] D-tagatose is a rare sugar with significant application potential. Its taste is similar to sucrose, its sweetness is 92% that of sucrose, and its calories are only 38% of sucrose, making it an ideal sucrose substitute. Studies have shown that D-tagatose has multiple physiological functions, including regulating blood sugar, controlling weight, improving gut microbiota, preventing tooth decay, and providing antioxidant effects. It can be widely used in functional beverages, dairy products, health supplements, and special medical foods.

[0004] The synthesis of D-tagatose mainly involves chemical synthesis and biosynthesis. Compared to chemical synthesis, biosynthesis has received widespread attention and research due to its advantages such as mild reaction conditions, fewer byproducts, simple separation and purification, and environmental friendliness. Considering the economics of raw materials, the stability of raw material supply, and compatibility with industrial production, the production process of synthesizing D-tagatose from D-fructose using tagatose 4-epimerase catalysis has greater advantages for industrial production. However, the number of reported tagatose 4-epimerases is relatively small, and their catalytic activity is poor. There is an urgent need to discover or modify tagatose 4-epimerases with excellent performance to provide novel biocatalysts for the biomanufacturing of D-tagatose. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a tagatose 4-epimerase and its applications.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a tagatose 4-epimerase, wherein the amino acid sequence of the tagatose 4-epimerase includes any one of the sequences shown in SEQ ID No:1-SEQ ID No:9.

[0008] This invention provides nine highly catalytically active tagatose 4-epimerases, which possess high catalytic activity. This invention can provide novel, highly active biocatalysts for the biosynthesis of D-tagatose, facilitating its industrial production.

[0009] SEQ ID No:1:

[0010] MVLKVFKDHFGRGYEVYEKSYREKDSLSFFLTKGEEGKILVVAGEKAPEGLSFFKKQRVEGVSFFCERSHENLEVLRKYFPDLKPVRAGLRASFGTGDRLGITTPAHVRALKDSGLFPIFAQQDVRETERTGRTVRDVPDDATWGVFQEGYSEGFGADADHVKRPEDLVSAAREGFTMFAIDPSDHVRNLSKLTEKERNEKFEILRKERIDRIYLGKKYSVLGEKIEFDEKNLRDAALVYYDAIAHVDMMYQILKDETPDFDFEVSVDETETPTSPLFHIFVVEELRRRGVEFTNLALRFIGEWEKGIDYKGDLAQFEREIKMHAEIARMFEGYKISLHSGSDKFSVYPAFASATGGLFLVKTAGTSYLEAVKVISMVNPELFREIYRCALDHFEEDRKSYHISADLSKVEVEKVKDEDLPGLFEDINVRQLIHVTYGSVLKDASLKERLFKTLEQNEELFYETVAKHIKRHVDLLKG.

[0011] SEQ ID No:2:

[0012] MVLKVFKDHFGRGYEVYEKSYREKDSLSFFLTKGEEGKILVVAGEKAPEGLSFFKKQRVEGVSFFCCERNHENLEVLRKYFPDLKPVRAGLRASFGTGDRLGITTPAHVRALKDSGLFPVFAQQDVRETERTGRTVRDVLDDATWGVFQEGYSEGGFGADADHVKRPEDLVSAAREGFTMFAIDPSDHVRNLSKLTEKERNEKFEILRKERIDRIYLGKKYSVLGEKIEFDEKNLRDAALVYYGAIAHVDMMYQILKDETPDFDFEVSVDETETPTSPLFHIFVVEELRRRGVEFTNLALRFIGEWEKGIDYKGDLAQFEREIKMHAEIARMFEGYKISLHSGSDKFSVYPAFASATGGLFLVKTAGTSYLEAVKVVSMVNPELFREIYRCALDHFEEDRKSYHISADLSKVPEVEKVKDEDLPGLFEDINVRQLIHVTYGSVLKDASLKERLFKTLEQNEELFYETVAKHIKRHVDLLKG.

[0013] SEQ ID No:3:

[0014] MVLKVFKDHFGRGYEVYEKSYREKDSLSFFLTKGEEGKILVVAGEKAPEGLSFFKKQRVEGVSFFCCERNHENLEVLRKYFPDLKPVRAGLRASFGTGDRLGITTPAHVRALKDSGLFPIFAQQDVRETERTGRTVRDVPDDATWGVFQEGYTEGFGADADHVKRPEDLVSAAREGFTMFAIDPSDHVRNLSKLTEKERNEKFEILRKERIDRIYLGKKYSVLGEKIEFDEKNLRDAALVYYDAIAHVDMMYQILKDETPDFDFEVSVDETETPTSPLFHIFVVEELRRRGVEFTNLALRFIGEWEKGIDYKGDLGLFEKEIKMHAEISRMFEGYKISLHSGSDKFSVYPAFASATGGLFHVKTAGTSYLEAVKVISMVNPELFREIYRCALDHFEEDRKSYHISADLSKVPEVEKVKDEDLPGLFEDINVRQLIHVTYGSVLKDASLKERLFKTLEQNEELFYETVAKHIKRHVDLLKG.

[0015] SEQ ID No:4:

[0016] MVLKVFKDHFGRGYEVYEKSYREKDSLSFFLTKGEEGKILVVAGEKAPEGLSFFKKQRVEGVSFFCCGRNHENLEVLRKYFPDLKPVRAGLRRASFGTGDRLGITTPAHVRALKDSGLFPIFAQQDVRETERTGRTVRDVLDDATWGVFQEGYSEGFGADADHVKRPEDLVSAAREDFTMFAIDPSDHVRNLSKLSEREKNEMFEEILRKERIDRIYLGKKYSVLGEKIEFDEKNLRDAALVYYDAIAHVDMMYQILKDETPDFDFEVSVDETETPTSPLFHIFVVEELRRRGVEFTNLALRFIGEWEKGIDYKGDLAQFEREIKMHAEIARMFEGYKISLHSGSDKFSVYPAFASATGGLFLVKTAGTSYLEAVKVISMVNPELFREIYRCALDHFEEDRKSYHISADLSKVEVEKVKDEDLPGLFEDINVRQLIHVTYGSVLKDASLKERLFKTLEQNEELFYETVAKHIKRHVDLLKG.

[0017] SEQ ID No:5:

[0018] MSLKVFKDHFGRGYEVYESSYRKKEGFSFLVKGEEGKILVVAGEKAPEGLSFFKKQRVEGVSFFCCERNHENLEVLRKYFPDLKPVRAGLRASFGTGDRLGITTPAHVRALKDSGLFPIFAQQDVRETERTGRTVRDVPDDATWGVFQEGYSEGFGADADHVKRPEDLVSAAREGFTMFAIDPSDHVRNLSKLTEKERSEKFEILRKERIDRIYLGKKYSVLGEKIEFDEKNLRDAALVYYDAIAHVDMMYQILKDETPDFDFEVSVDETETPTSPLFHIFVVEELRRRGVEFTNLALRFIGEWEKGIDYKGDLAQFEREIKMHAEIARMFEGYKISLHSGSDKFSVYPAFASATGGLFHVKTAGTSYLEAVKVISMVNPELFREIYRCALDHFEEDRKSYHISADLSKVEVEKVKDEDLPGLFEDINVRQLIHVTYGSVLKDASLKERLFKTLEQNEELFYETVAKHIKRHVDLLEG.

[0019] SEQ ID No:6:

[0020] MVLKVFKDHFGRGYEVYEKSYREKDSLSFFLTKGEEGKILVVAGEKAPEGLSFFKKQRVEGVSFFCCERNHENLEVLRRYFPDLKPVRAGLRASFGTGDRLGITTPAHVRALKDSGLFPIFAQQDVRETERTGRTWRDPDDATWGVFQEGYSEGFGADADHVKNPEDLVAAAKEGFSMFTIDPSDHVRNLSKLTEKERNEKFEILRKERIDRIYLGKKYSVLGEKIEFDEKNLRDAALVYYDAIAHVDMMYQILKEEISDFDFEVSVDETETPTSPLFHIFVVEELRRRGVEFTNLALRFIGEWEKGIDYKGDLGLFEKEIKMHAEITRMFEGYKISLHSGSDKFSVYPAFASATGGLFLVKTAGTSYLEAVKVISMVNPELFREIYRCALDHFEEDRKSYHISADLSKVPEVEKVKDEDLPGLFEDINVRQLIHVTYGSVLKDASLKERLFKTLEQNEELFYETVAKHIKRHVDLLKG.

[0021] SEQ ID No:7:

[0022] MVLKVFKDHFGRGYEVYEKSYREKDSLSFFLTKGEEGEILVVAGEKAPEGLSFFKKQRVEGVSFFCCERNHENLEVLRKYFPDLKPVRAGLRASFGTGDRLGITTPAHVRALKDSGLFPIFAQQDVRETERTGRTVRDVPDDATWGVFQEGYSEGFGADADHVKRPEDLVSAAREGFTMFAIDPSDHVRNLSKLTEKERNEKFEILRKERIDRIYLGKKYTVLGERLEFDERNLDAALVYYDAIAHVDMMYQILKEEISDFDFEVSVDETETPTSPLFHIFVVEELRRRGVEFTNLALRFIGEWEKGIDYKGDLGLFEKEIKMHAEISRMFEGYKISLHSGSDKFSVYPAFASATGGLFHVKTAGTSYLEAVKVISMVNPELFREIYRCALDHFEEDRKSYHISADLSKVPEVEKVKDEDLPGLFEDINVRQLIHVTYGSVLKDASLKERLFKTLEQNEELFYETVAKHIKRHVDLLKG.

[0023] SEQ ID No:8:

[0024] MVLKVFKDHFGRGYEVYEKSYREKDSLSFFLTKGEEGKILVVAGEKAPEGLSFFKKQRVEGVSFFSCERNHENLEVLRKYFPDLKPVRAGLRASFGTGDRLGITTPAHVRALKDSGLFPIFAQQDVRETERTGRTWRDPDDATWGVFQEGYSEGFGADADHVKNPEDLVAAKEGFSMFTIDPSDHVRNLSKLTEKERNEKFEILRKERIDRIYLGKKYSVLGEKIEFDEKNLRDAALVYYDAIAHVDMMYQILKEEISDFDFEVSVDETETPTSPLFHIFVVEELRRRGVEFTNLALRFIGEWEKGIDYKGDLGLFGKEIKMHAEISRMFEGYKISLHSGSDKFSVYPAFASATGGLFHVKTAGTSYLEAVKVISMVNPELFREIYRCALDHFEEDRKSYHISADLSKVPEVEKVKDEDLPGLFEDINVRQLIHVTYGSVLKDASLKERLFKTLEQNEELFYETVAKHIKRHVDLLKG.

[0025] SEQ ID No:9:

[0026] MSLKVFKDHFGRGYEVYESSYRKKEGFSFFLVKGEEGEILVVAGEKAPEGLSFFKKQRVEGVSFFSCGRSHENLEVLRRYFPDLKPVRAGLRASFGTGDRLGITTPAHVRALKDSGLFPV FAQQDVRETERTGRTWRDVLDDATWGVFQEGYTEGFGADADHVKNPEDLVAAAKEDFSMFTIDPSDHVRNLSKLSEREKSEMFEEILRKERIDRIYLGKKYTVLGERLEFDERNLRDAALV YYGAIAHVDMMYQILKEEISDFDFEVSVDETETPTSPLFHIFVVEELRRRGVEFTNLALRFIGEWEKGIDYKGDLGLFGKEIKMHAESRMFEGYKISLHSGSDKFSVYPAFASATGGLF HVKTAGTSYLEAVKVVSMVNPELFREIYRCALDHFEEDRKSYHISADLSKVPEVEKVKDEDLPGLFEDINVRQLIHVTYGSVLKDASLKERLFKTLEQNEELFYETVAKHIKRHVDLLEG.

[0027] In a second aspect, the present invention provides a nucleic acid molecule that encodes the tagatose 4-epimerase described in the first aspect.

[0028] Preferably, the nucleotide sequence of the nucleic acid molecule includes any one of the sequences shown in SEQ ID No:10-SEQ ID No:18.

[0029] SEQ ID No:10:

[0030]

[0031] SEQ ID No:11:

[0032]

[0033] SEQ ID No:12:

[0034]

[0035] SEQ ID No:13:

[0036]

[0037] SEQ ID No:14:

[0038]

[0039] SEQ ID No:15:

[0040]

[0041] SEQ ID No:16:

[0042]

[0043] SEQ ID No:17:

[0044]

[0045] SEQ ID No:18:

[0046]

[0047] Thirdly, the present invention provides an expression vector containing the nucleic acid molecule described in the second aspect.

[0048] Fourthly, the present invention provides an engineered strain containing the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect.

[0049] Fifthly, the present invention provides the use of the tagatose 4-epimerase according to the first aspect or the engineered strain according to the fourth aspect in the production of D-tagatose.

[0050] In a sixth aspect, the present invention provides a method for producing D-tagatose, the method comprising: mixing the engineered strain described in the fourth aspect with a substrate solution, centrifuging to remove the engineered strain, inactivating the strain, centrifuging again, and collecting the supernatant to obtain the product.

[0051] Preferably, the substrate solution comprises D-fructose, nickel sulfate, glycine, and water.

[0052] Preferably, the substrate solution comprises, by concentration, 95-105 g / L of D-fructose, and by molar concentration, 1.4-1.6 mM of nickel sulfate and 19-21 mM of glycine, and the solvent is water.

[0053] The concentrations of D-fructose can be selected from 95 g / L, 96 g / L, 97 g / L, 98 g / L, 99 g / L, 100 g / L, 101 g / L, 102 g / L, 103 g / L, 104 g / L, 105 g / L, etc. The molar concentrations of nickel sulfate can be selected from 1.4 mM, 1.42 mM, 1.45 mM, 1.48 mM, 1.5 mM, 1.52 mM, 1.55 mM, 1.58 mM, 1.6 mM, etc. The molar concentrations of glycine can be selected from 19 mM, 19.2 mM, 19.5 mM, 19.8 mM, 20 mM, 20.2 mM, 20.5 mM, 20.8 mM, 21 mM, etc. Other specific values ​​within the above ranges can be selected, which will not be elaborated here.

[0054] Preferably, the pH of the substrate solution is 8.4-8.6, such as 8.4, 8.42, 8.45, 8.48, 8.5, 8.52, 8.55, 8.58, 8.6, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0055] Preferably, the pH of the substrate solution is adjusted using sodium hydroxide.

[0056] Preferably, the reaction temperature is 68-72 °C and the reaction time is 9-11 h.

[0057] Temperatures can be selected from 68 ℃, 68.5 ℃, 69 ℃, 69.5 ℃, 70 ℃, 70.5 ℃, 71 ℃, 71.5 ℃, 72 ℃, etc., and time can be selected from 9 h, 9.2 h, 9.5 h, 9.8 h, 10 h, 10.2 h, 10.5 h, 10.8 h, 11 h, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0058] Preferably, the OD of the bacterial cells in the reaction system 600 The range is 48-52, for example, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0059] Preferably, the centrifugation speed is independently 9000-11000 rpm, and the time is 3-7 min.

[0060] The engine speed can be selected from 9000 rpm, 9200 rpm, 9500 rpm, 9800 rpm, 10000 rpm, 10200 rpm, 10500 rpm, 10800 rpm, 11000 rpm, etc., and the time can be selected from 3 min, 3.5 min, 4 min, 4.5 min, 5 min, 5.5 min, 6 min, 6.5 min, 7 min, etc. Other specific values ​​within the above range can be selected, which will not be described in detail here.

[0061] Preferably, the inactivation treatment includes boiling for 5-10 minutes, such as 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] Considering the economics of raw materials, the stability of raw material supply, and compatibility with industrial production, the production process utilizing tagatose 4-epimerase to catalyze the synthesis of D-tagatose from D-fructose has greater advantages for industrial production. This invention provides nine highly catalytically active tagatose 4-epimerase mutants and demonstrates that these mutant enzymes exhibit superior activity in catalyzing the synthesis of D-tagatose from D-fructose compared to the template enzyme. This invention can provide novel, highly active biocatalysts for the biosynthesis of D-tagatose, facilitating its industrial production. Attached Figure Description

[0064] Figure 1 This study compares the catalytic activity of microbial cells expressing tagatose 4-epimerase template enzyme and mutant enzyme.

[0065] Figure 2 This is a high-performance liquid chromatography (HPLC) chromatogram of the reaction solution catalyzed by the tagatose 4-epimerase mutant M9 to synthesize D-tagatose from D-fructose. Detailed Implementation

[0066] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0067] Unless otherwise specified, all materials and reagents used in this invention are obtained commercially.

[0068] Preparation Example 1

[0069] This preparation example provides 10 engineered *E. coli* strains, *E. coli* BL 21(DE3)-pET22b-M(0-9), expressing tagatose 4-epimerases (M0-M9). These engineered strains were constructed using conventional molecular biology experiments. The gene sequences of M(0-9) are inserted into the Nde I and Xho I restriction enzyme sites of the pET22b vector. The amino acid sequences of M1-M9 correspond sequentially to the sequences shown in SEQ ID No:1-SEQ ID No:9, and the nucleotide sequences correspond sequentially to the sequences shown in SEQ ID No:10-SEQ ID No:18. The amino acid sequence of M0 (template enzyme) is shown in SEQ ID No:19, and the nucleotide sequence of M0 is shown in SEQ ID No:20.

[0070] SEQ ID No:19:

[0071] MVLKVFKDHFGRGYEVYEKSYREKDSLSFFLTKGEEGKILVVAGEKAPEGLSFFKKQRVEGVSFFCCERNHENLEVLRKYFPDLKPVRAGLRASFGTGDRLGITTPAHVRALKDSGLFPIFAQQDVRETERTGRTVRDVPDDATWGVFQEGYSEGGFGADADHVKRPEDLVSAAREGFTMFAIDPSDHVRNLSKLTEKERNEKFEILRKERIDRIYLGKKYSVLGEKIEFDEKNLRDAALVYYDAIAHVDMMYQILKDETPDFDFEVSVDETETPTSPLFHIFVVEELRRRGVEFTNLALRFIGEWEKGIDYKGDLAQFEREIKMHAEIARMFEGYKISLHSGSDKFSVYPAFASATGGLFLVKTAGTSYLEAVKVISMVNPELFREIYRCALDHFEEDRKSYHISADLSKVPEVEKVKDEDLPGLFEDINVRQLIHVTYGSVLKDASLKERLFKTLEQNEELFYETVAKHIKRHVDLLKG.

[0072] SEQ ID No:20:

[0073]

[0074] Example 1

[0075] Induction culture and mycelial sludge collection of E. coli BL 21(DE3)-pET22b-M(0-9)

[0076] The engineered strain of *E. coli* was inoculated at a 1% inoculum into a 100 mL Erlenmeyer flask containing 50 mL of LB liquid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride), and incubated at 37 ℃ with shaking at 200 rpm until OD (out of control) was reached. 600 =Approximately 0.7, add IPTG inducer to a final concentration of 0.5 mM, then adjust the shaker temperature and speed to 25 ℃ and 150 rpm, and continue culturing for 12 h. After culturing, centrifuge the bacterial solution at 10000 rpm for 5 min to collect the bacterial sludge, wash twice with double-deionized water, and centrifuge again to collect the bacterial sludge.

[0077] Example 2

[0078] Assay of the catalytic activity of tagatose 4-epimerase template enzyme Mo.

[0079] The engineered strain of *E. coli* BL 21(DE3)-pET22b-M0 was cultured and collected according to Example 1. A substrate solution containing 100 g / L D-fructose, 1.5 mM nickel sulfate, and 20 mM glycine was prepared, and the pH was adjusted to 8.5 with 10 mol / L sodium hydroxide solution. The *E. coli* BL 21(DE3)-pET22b-M0 bacterial sludge was resuspended in the above substrate solution to OD. 600 =50, and the resuspended solution was placed in a 70 ℃ constant temperature water bath for 10 h. After the reaction, the reaction solution was centrifuged at 10000 rpm for 5 min to collect the supernatant, then boiled in a boiling water bath for 5 min, and centrifuged again at 10000 rpm for 5 min to collect the supernatant. The supernatant was filtered through a 0.22 μm aqueous syringe filter and used for high performance liquid chromatography analysis (detection conditions and parameters: Carbomix Pb-NP column (10: 8%, 7.8×300 mm, 10 μm); differential refractive index detector RID-20A; mobile phase: double deionized water; flow rate: 0.5 mL / min; injection volume: 10 μL; column temperature: 78 ℃) to determine the concentration of D-tagatose. The conversion rate of D-tagatose was calculated according to the following formula: Conversion rate of D-tagatose = C T / C F0 ×100%, where C T To determine the concentration of D-tagatose in the resulting reaction solution, C F0The initial concentration of the substrate D-fructose was 100 g / L. The calculated conversion rate of D-tagatose was 22.56%. Figure 1 ).

[0080] Example 3

[0081] Catalytic activity assay of tagatose 4-epimerase mutant M1

[0082] The engineered strain of *E. coli* BL 21(DE3)-pET22b-M1 was cultured and collected according to Example 1. A substrate solution containing 100 g / L D-fructose, 1.5 mM nickel sulfate, and 20 mM glycine was prepared, and the pH was adjusted to 8.5 with 10 mol / L sodium hydroxide solution. The *E. coli* BL 21(DE3)-pET22b-M1 bacterial sludge was resuspended in the above substrate solution to OD. 600 =50, and the resuspended solution was placed in a 70 ℃ constant temperature water bath for 10 h. After the reaction, the reaction solution was centrifuged at 10000 rpm for 5 min to collect the supernatant, then placed in a boiling water bath for 5 min, and centrifuged again at 10000 rpm for 5 min to collect the supernatant. The supernatant was filtered through a 0.22 μm aqueous syringe filter and used for high performance liquid chromatography analysis to determine the concentration of D-tagatose. The conversion rate of D-tagatose was calculated to be 23.92%. Figure 1 ).

[0083] Example 4

[0084] Catalytic activity assay of tagatose 4-epimerase mutant M2

[0085] Following Example 1, the engineered strain of *E. coli* BL 21(DE3)-pET22b-M2 was cultured and collected. A substrate solution containing 100 g / L D-fructose, 1.5 mM nickel sulfate, and 20 mM glycine was prepared, and the pH was adjusted to 8.5 with 10 mol / L sodium hydroxide solution. The *E. coli* BL 21(DE3)-pET22b-M2 bacterial sludge was resuspended in the above substrate solution to OD. 600 =50, and the resuspended solution was placed in a 70 ℃ constant temperature water bath for 10 h. After the reaction, the reaction solution was centrifuged at 10000 rpm for 5 min to collect the supernatant, then boiled in a boiling water bath for 5 min, and centrifuged again at 10000 rpm for 5 min to collect the supernatant. The supernatant was filtered through a 0.22 μm aqueous syringe filter and used for high performance liquid chromatography analysis to determine the concentration of D-tagatose. The conversion rate of D-tagatose was calculated to be 24.78%. Figure 1 ).

[0086] Example 5

[0087] Catalytic activity assay of tagatose 4-epimerase mutant M3

[0088] Following Example 1, the engineered strain of *E. coli* BL 21(DE3)-pET22b-M3 was cultured and collected. A substrate solution containing 100 g / L D-fructose, 1.5 mM nickel sulfate, and 20 mM glycine was prepared, and the pH was adjusted to 8.5 with 10 mol / L sodium hydroxide solution. The *E. coli* BL 21(DE3)-pET22b-M3 bacterial sludge was resuspended in the above substrate solution to OD. 600 =50, and the resuspended solution was placed in a 70 ℃ constant temperature water bath for 10 h. After the reaction, the reaction solution was centrifuged at 10000 rpm for 5 min to collect the supernatant, then boiled in a boiling water bath for 5 min, and centrifuged again at 10000 rpm for 5 min to collect the supernatant. The supernatant was filtered through a 0.22 μm aqueous syringe filter and used for high performance liquid chromatography analysis to determine the concentration of D-tagatose. The conversion rate of D-tagatose was calculated to be 25.54%. Figure 1 ).

[0089] Example 6

[0090] Catalytic activity assay of tagatose 4-epimerase mutant M4

[0091] The engineered strain of *E. coli* BL 21(DE3)-pET22b-M4 was cultured and collected according to Example 1. A substrate solution containing 100 g / L D-fructose, 1.5 mM nickel sulfate, and 20 mM glycine was prepared, and the pH was adjusted to 8.5 with 10 mol / L sodium hydroxide solution. The *E. coli* BL 21(DE3)-pET22b-M4 bacterial sludge was resuspended in the above substrate solution to OD0.05. 600 =50, and the resuspended solution was placed in a 70 ℃ constant temperature water bath for 10 h. After the reaction, the reaction solution was centrifuged at 10000 rpm for 5 min to collect the supernatant, then boiled in a boiling water bath for 5 min, and centrifuged again at 10000 rpm for 5 min to collect the supernatant. The supernatant was filtered through a 0.22 μm aqueous syringe filter and used for high performance liquid chromatography analysis to determine the concentration of D-tagatose. The conversion rate of D-tagatose was calculated to be 25.48%. Figure 1 ).

[0092] Example 7

[0093] Catalytic activity assay of tagatose 4-epimerase mutant M5

[0094] Following Example 1, the engineered strain of *E. coli* BL 21(DE3)-pET22b-M5 was cultured and collected. A substrate solution containing 100 g / L D-fructose, 1.5 mM nickel sulfate, and 20 mM glycine was prepared, and the pH was adjusted to 8.5 with 10 mol / L sodium hydroxide solution. The *E. coli* BL 21(DE3)-pET22b-M5 bacterial sludge was resuspended in the above substrate solution to OD. 600 =50, and the resuspended solution was placed in a 70 ℃ constant temperature water bath for 10 h. After the reaction, the reaction solution was centrifuged at 10000 rpm for 5 min to collect the supernatant, then boiled in a boiling water bath for 5 min, and centrifuged again at 10000 rpm for 5 min to collect the supernatant. The supernatant was filtered through a 0.22 μm aqueous syringe filter and used for high performance liquid chromatography analysis to determine the concentration of D-tagatose. The conversion rate of D-tagatose was calculated to be 25.98%. Figure 1 ).

[0095] Example 8

[0096] Catalytic activity assay of tagatose 4-epimerase mutant M6

[0097] The engineered strain of *E. coli* BL 21(DE3)-pET22b-M6 was cultured and collected according to Example 1. A substrate solution containing 100 g / L D-fructose, 1.5 mM nickel sulfate, and 20 mM glycine was prepared, and the pH was adjusted to 8.5 with 10 mol / L sodium hydroxide solution. The *E. coli* BL 21(DE3)-pET22b-M6 bacterial sludge was resuspended in the above substrate solution to OD0.05. 600 =50, and the resuspended solution was placed in a 70 ℃ constant temperature water bath for 10 h. After the reaction, the reaction solution was centrifuged at 10000 rpm for 5 min to collect the supernatant, then boiled in a boiling water bath for 5 min, and centrifuged again at 10000 rpm for 5 min to collect the supernatant. The supernatant was filtered through a 0.22 μm aqueous syringe filter and used for high performance liquid chromatography analysis to determine the concentration of D-tagatose. The conversion rate of D-tagatose was calculated to be 25.76%. Figure 1 ).

[0098] Example 9

[0099] Catalytic activity assay of tagatose 4-epimerase mutant M7

[0100] Following Example 1, the engineered strain of *E. coli* BL 21(DE3)-pET22b-M7 was cultured and collected. A substrate solution containing 100 g / L D-fructose, 1.5 mM nickel sulfate, and 20 mM glycine was prepared, and the pH was adjusted to 8.5 with 10 mol / L sodium hydroxide solution. The *E. coli* BL 21(DE3)-pET22b-M7 bacterial sludge was resuspended in the above substrate solution to OD. 600 =50, and the resuspended solution was placed in a 70 ℃ constant temperature water bath for 10 h. After the reaction, the reaction solution was centrifuged at 10000 rpm for 5 min to collect the supernatant, then boiled in a boiling water bath for 5 min, and centrifuged again at 10000 rpm for 5 min to collect the supernatant. The supernatant was filtered through a 0.22 μm aqueous syringe filter and used for high performance liquid chromatography analysis to determine the concentration of D-tagatose. The conversion rate of D-tagatose was calculated to be 25.79% ( Figure 1 ).

[0101] Example 10

[0102] Catalytic activity assay of tagatose 4-epimerase mutant M8

[0103] The engineered strain of *E. coli* BL 21(DE3)-pET22b-M8 was cultured and collected according to Example 1. A substrate solution containing 100 g / L D-fructose, 1.5 mM nickel sulfate, and 20 mM glycine was prepared, and the pH was adjusted to 8.5 with 10 mol / L sodium hydroxide solution. The *E. coli* BL 21(DE3)-pET22b-M8 bacterial sludge was resuspended in the above substrate solution to OD. 600 =50, and the resuspended solution was placed in a 70 ℃ constant temperature water bath for 10 h. After the reaction, the reaction solution was centrifuged at 10000 rpm for 5 min to collect the supernatant, then boiled in a boiling water bath for 5 min, and centrifuged again at 10000 rpm for 5 min to collect the supernatant. The supernatant was filtered through a 0.22 μm aqueous syringe filter and used for high performance liquid chromatography analysis to determine the concentration of D-tagatose. The conversion rate of D-tagatose was calculated to be 26.22%. Figure 1 ).

[0104] Example 11

[0105] Catalytic activity assay of tagatose 4-epimerase mutant M9

[0106] Following Example 1, the engineered strain of *E. coli* BL 21(DE3)-pET22b-M9 was cultured and collected. A substrate solution containing 100 g / L D-fructose, 1.5 mM nickel sulfate, and 20 mM glycine was prepared, and the pH was adjusted to 8.5 with 10 mol / L sodium hydroxide solution. The *E. coli* BL 21(DE3)-pET22b-M9 bacterial sludge was resuspended in the above substrate solution to OD. 600 =50, the resuspended solution was placed in a 70 ℃ constant temperature water bath for 10 h. After the reaction, the reaction solution was centrifuged at 10000 rpm for 5 min to collect the supernatant, then boiled in a boiling water bath for 5 min, and centrifuged again at 10000 rpm for 5 min to collect the supernatant. The supernatant was filtered through a 0.22 μm aqueous syringe filter and used for high performance liquid chromatography analysis to determine the concentration of D-tagatose. The chromatographic results are shown below. Figure 2 As shown, the calculated conversion rate of D-tagatose was 26.24% ( Figure 1 ).

[0107] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A tagatose 4-epimerase, characterized in that, The amino acid sequence of the tagatose 4-epimerase includes any one of the sequences shown in SEQ ID No:1-SEQ ID No:

9.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the tagatose 4-epimerase as described in claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule includes any one of the sequences shown in SEQ ID No:10-SEQ ID No:

18.

4. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 2 or 3.

5. An engineered bacterial strain, characterized in that, The engineered strain contains the nucleic acid molecule as described in claim 2 or 3 or the expression vector as described in claim 4.

6. The application of the tagatose 4-epimerase according to claim 1 or the engineered strain according to claim 5 in the production of D-tagatose.

7. A method for producing D-tagatose, characterized in that, The method includes: mixing the engineered strain described in claim 5 with a substrate solution, centrifuging to remove the engineered bacteria, inactivating the strain, centrifuging again, and collecting the supernatant to obtain the product.

8. The method according to claim 7, characterized in that, The substrate solution comprises D-fructose, nickel sulfate, glycine, and water; Preferably, the substrate solution comprises, by concentration, 95-105 g / L of D-fructose, and by molar concentration, 1.4-1.6 mM of nickel sulfate and 19-21 mM of glycine, and the solvent is water.

9. The method according to claim 7 or 8, characterized in that, The pH of the substrate solution is 8.4-8.6; Preferably, the pH of the substrate solution is adjusted using sodium hydroxide.

10. The method according to any one of claims 7-9, characterized in that, The reaction was carried out at a temperature of 68-72 °C for 9-11 h. Preferably, the OD of the bacterial cells in the reaction system 600 It is 48-52; Preferably, the centrifugation speed is independently 9000-11000 rpm, and the time is 3-7 min.

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