Breeding method of C4 epimerase whole cells and preparation method of D-tagatose

Abstract

The invention discloses a C4 epimerase whole cell breeding method and a D-tagatose preparation method. C4 epimerase whole cells obtained through breeding grow robustly, are excellent in performance and are more suitable for recombinant escherichia coli for production; when the whole cell is used for producing D-tagatose, a whole cell culture does not need to be crushed, and other raw materials, catalysts and the like do not need to be added; the method has the advantages of simple reaction conditions, high efficiency, single reaction, few side reactions, easiness in control and industrial production.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a method for breeding a C4 epimerase whole cell and a method for preparing D-tagatose. Background Art

[0002] D-Tagatose is a six-carbon ketose with a molecular formula of C6H 12 O6 is the diastereomer of fructose and the aldehyde-ketone isomer of galactose. It is a rare monosaccharide that occurs naturally, mainly in dairy products such as yogurt and milk powder. Its sweetness is similar to that of sucrose, but its calories are only one-third of those of sucrose, so it is called a low-calorie sweetener. Tagatose was officially approved as a generally recognized safe food (GRAS) by the United States Food and Drug Administration (USFDA) in 2001, and was approved by the European Union for marketing in Europe in 2005. Tagatose has important functions such as low calorie value, zero glycemic index, blood sugar passivation, no caries, prebiotic effect and antioxidant activity, and has been widely used in many fields such as food, beverages, and tooth care products.

[0003] There are two main methods for producing tagatose in the prior art: chemical synthesis and biotransformation. The chemical synthesis method uses a soluble alkali metal salt or alkaline earth metal salt as a catalyst to promote D-galactose to generate tagatose under alkaline conditions, and form a metal hydroxide-tagatose complex, and then neutralize it with acid to obtain D-tagatose. The biotransformation method includes two methods: oxidizing galactitol to tagatose, and using isomerase produced by microorganisms to isomerize galactose to tagatose. Because the chemical method has the disadvantages of high energy consumption, complex products, difficult purification, many side reactions, and serious chemical pollution, the biotransformation method has a good application prospect. The method of producing tagatose through biotransformation that is currently studied more is to use L-arabinose isomerase to catalyze the conversion of D-galactose into tagatose. However, the high price of galactose affects the final price of tagatose, resulting in it being unable to be widely used.

[0004] Chinese patent document CN 201610937656.5 discloses a method for converting a substrate into tagatose by an in vitro multi-enzyme molecular machine using starch, cellulose and its derivatives or sucrose as substrates. Chinese patent document CN201711065859.0 discloses a method for expressing tagatose invertase in whole cells, using starch, cellulose and its derivatives or sucrose as substrates, and using whole cell catalysis to produce tagatose. Chinese patent document CN 202110223481.2 discloses a method for separation and purification of tagatose obtained by whole cell catalysis using starch, cellulose and its derivatives or sucrose as substrates. The conversion of starch, cellulose and its derivatives or sucrose as substrates into tagatose is a multi-step enzyme-catalyzed reaction, during which a variety of by-products such as glucose, maltose and oligosaccharides (DP≥3) are generated. During the extraction and purification process, it is necessary to perform multi-step separation of the various components in the conversion liquid, which is cumbersome. The increase in reaction by-products means an increase in impurities in the system, which will affect the yield and quality of the finished product.

[0005] In addition, in the prior art, in the crystallization process, a tagatose concentrate having a content of more than 90% generally needs to be added with 7-12% of seed crystals during crystallization. The system under this crystallization method is viscous and has no fluidity, which is not conducive to crystallization. The final product is in powder form, which is not conducive to solid-liquid separation and filtration.

[0006] Therefore, it is urgent to establish a method for preparing tagatose with simple process, few side reactions and high separation and purification yield in the prior art. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide a method for breeding C4 epimerase whole cells and a method for preparing D-tagatose, which overcomes the technical problems of the prior art method for preparing D-tagatose, such as complicated operation and many side reactions.

[0008] In order to achieve the above object, the present invention first provides a method for breeding C4 epimerase whole cells, comprising the following steps:

[0009] The fructose C4 epimerase recombinant Escherichia coli was washed with sterile water at 10 -1 -10 -9 The ratio of the dilution is used for gradient dilution to obtain multiple groups of dilution solutions;

[0010] The multiple groups of dilutions are inoculated into the selection medium at an inoculation rate of 1-2% and cultured for 18-24 hours; wherein, in terms of mass volume concentration, the selection medium comprises: 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, 3-5 g / L D-fructose, 0.5-1 g / L arabinose, 0.1-0.2 mM / L mnCl2, and the balance is deionized water;

[0011] A group of colonies with uniform color, raised colonies and a colony diameter of 1-2 mm were selected from the selection medium and inoculated into LB medium at 35-37°C for 18-24 hours;

[0012] Select colonies with uniform color, raised colonies, and a colony diameter of 1-2 mm from the LB medium and inoculate them into LB medium containing 30-50 ug / mL streptomycin sulfate. Cultivate at a constant temperature of 200-220 rpm and 35-37 ° C until the bacterial solution reaches OD 600 The value is 1.6-1.8;

[0013] L-arabinose was added to the LB culture medium to a final concentration of 1-2 g / L, and the culture was shaken at a speed of 200-220 rpm and a temperature of 28-30° C. for 12-14 hours, and then centrifuged at a speed of 5000-6000 rpm and a temperature of 3-5° C. for 15-20 minutes to collect the C4 isomerase whole cells.

[0014] The present invention also provides a method for preparing D-tagatose using the above-mentioned C4 epimerase whole cell, comprising the following steps:

[0015] Adding the C4 epimerase whole cells to a 600-800 g / L fructose solution to make the system strain concentration reach 10-50 OD, transforming in a phosphate buffer to obtain a transformation solution;

[0016] The conversion liquid is filtered to obtain a clear liquid;

[0017] Desalting the clear liquid to obtain a desalted liquid;

[0018] The desalted liquid is subjected to chromatographic separation to obtain a purified D-tagatose liquid;

[0019] Concentrating the purified D-tagatose solution under vacuum to obtain a concentrated solution;

[0020] Add 90-95% (v / v) ethanol solution to the concentrated solution, wherein the amount of the ethanol solution added is 0.8-1.5 times the volume of the concentrated solution, cool to 40-50° C., add 0.5-1.5% of the system mass of seed crystals for 0.5-1.5 h, and cool to 15-20° C. at a stirring speed of 30-80 rpm at a rate of 1-5° C. / h for crystallization, and obtain the D-tagatose finished product after a second filtration, washing and drying.

[0021] The breeding method of the C4 epimerase whole cell provided by the present invention is to select and culture and screen by adding D-fructose as a substrate in the selective culture medium, and a small amount of L-arabinose and MnCl2 as an inducer to promote the early synthesis of the target protease in the bacteria, and screen to obtain a strain with strong growth, excellent performance, and the ability to synthesize more C4 epimerase without affecting the ability of the bacteria to continue to grow; the C4 epimerase whole cell obtained by the breeding method of the present invention is used to process fructose, without the need to crush the whole cell culture, and without the need to add other raw materials, catalysts, etc.; and the reaction conditions are simple, efficient, and easy to control. In addition, the C4 epimerase whole cell obtained in the present invention can be applied to a higher substrate (fructose) concentration, improve the conversion efficiency; and no side reactions are generated during the reaction, and separation is also relatively simple. In addition, during the crystallization process, the amount of seed added is 0.5-1.5% of the system mass, which not only reduces the input, but also adds a small amount of seed in the system. It is more conducive to the growth of crystals, and larger granular crystals can be obtained, and granular crystals are easier to separate from solids and liquids to obtain crystals. According to the test, the purity of tagatose prepared by the method of the present invention can reach more than 99%, and the yield can reach more than 86%. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A 10-fold microscope image of D-tagatose crystals obtained in Example 6 is shown;

[0023] Figure 2 A 10-fold microscope image of the D-tagatose crystals obtained in Comparative Example 3 is shown. DETAILED DESCRIPTION

[0024] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] The present invention provides a method for breeding a C4 epimerase whole cell, comprising the following steps:

[0026] The fructose C4 epimerase recombinant Escherichia coli was washed with sterile water at 10 -1 -10 -9 The ratio of the dilution is used for gradient dilution to obtain multiple groups of dilution solutions;

[0027] The multiple groups of dilutions are inoculated into the selection medium at an inoculation rate of 1-2% and cultured for 18-24 hours; wherein, in terms of mass volume concentration, the selection medium comprises: 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, 3-5 g / L D-fructose, 0.5-1 g / L arabinose, 0.1-0.2 mM / L mnCl2, and the balance is deionized water;

[0028] A group of colonies with uniform color, raised colonies and a colony diameter of 1-2 mm were selected from the selection medium and inoculated into LB medium at 35-37°C for 18-24 hours;

[0029] Select colonies with uniform color, raised colonies, and a colony diameter of 1-2 mm from the LB medium and inoculate them into LB medium containing 30-50 ug / mL streptomycin sulfate. Cultivate at a constant temperature of 200-220 rpm and 35-37 ° C until the bacterial solution reaches OD 600 The value is 1.6-1.8;

[0030] L-arabinose was added to the LB culture medium to a final concentration of 1-2 g / L, and the culture was shaken at a speed of 200-220 rpm and a temperature of 28-30° C. for 12-14 hours, and then centrifuged at a speed of 5000-6000 rpm and a temperature of 3-5° C. for 15-20 minutes to collect the C4 isomerase whole cells.

[0031] The present invention firstly treats the fructose C4 epimerase recombinant Escherichia coli with sterile water at 10 -1 -10 -9 In the present invention, the fructose C4 epimerase recombinant Escherichia coli is a strain disclosed in the prior art and purchased from the Institute of Microbiology, Chinese Academy of Sciences in Beijing; the gradient dilution is preferably carried out at a ratio of 10 -1 -10 -9 Specifically, it can be carried out according to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 The gradient dilution is performed in a ratio of 9 to obtain 9 groups of dilutions; the purpose of the present invention to perform gradient dilution to obtain multiple groups of dilutions is to inoculate the multiple groups of dilutions respectively, and finally screen out the best group of bacterial flora.

[0032] After obtaining multiple groups of dilutions, the present invention inoculates the multiple groups of dilutions into a selection culture medium at an inoculation rate of 1-2% and cultures them for 18-24 hours; wherein, in terms of mass volume concentration, the selection culture medium includes: 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, 3-5 g / L D-fructose, 0.5-1 g / L arabinose, 0.1-0.2 mM / LMnCl2, and the remainder is deionized water. In the present invention, multiple groups of dilutions are inoculated into the selection medium at an inoculation rate of 1-2% for the subsequent screening of the optimal strain. Preferably, multiple groups of dilutions are inoculated into the selection medium at the same inoculation rate; the yeast powder 5g / L, peptone 10g / L, sodium chloride 10g / L, and agar powder 20g / L contained in the selection medium are essentially components of LB medium, that is, the selection medium can also be understood as the LB medium to which D-fructose, arabinose and MnCl2 are added.

[0033] After the cultivation in the selection medium is completed, the present invention selects a group of colonies with uniform single color, protruding colonies, and a colony diameter of 1-2 mm from the selection medium and inoculates them in LB medium at 35-37°C for constant temperature cultivation for 18-24 hours. In the present invention, the color and protrusion of the colony are preferably determined by a person skilled in the art based on visual observation, and the determination of the colony diameter is carried out by conventional methods in the art, such as the cross method; this selection is to select a group of colonies with uniform single color, protruding colonies, and a colony diameter of 1-2 mm from multiple groups of selection mediums, and inoculate them into LB medium for continued cultivation.

[0034] After the culture in LB medium is completed, the present invention selects colonies with uniform color, raised colonies, and a colony diameter of 1-2 mm from the LB medium and inoculates them into LB medium containing 30-50 ug / mL streptomycin sulfate, and cultures them at a constant temperature of 200-220 rpm and 35-37°C until the bacterial solution reaches OD 600 The value is 1.6-1.8. In the present invention, the selection from the LB culture medium refers to selecting a colony with uniform color, raised colonies, and a colony diameter of 1-2 mm from the LB culture medium, and inoculating it into an LB culture medium containing streptomycin sulfate for continued cultivation to obtain an LB culture medium bacterial solution. In the present invention, the role of the streptomycin sulfate is to screen resistant strains and ensure the stable expression of the enzyme.

[0035] After obtaining the LB culture medium bacterial solution, the present invention adds L-arabinose to the LB culture medium bacterial solution until the final concentration of L-arabinose is 1-2g / L, and shakes and cultures the culture at a speed of 200-220rpm and a temperature of 28-30°C for 12-14 hours, and then centrifuges at a speed of 5000-6000rpm and a temperature of 3-5°C for 15-20min to collect and obtain C4 epimerase whole cells. In the present invention, the role of the L-arabinose is to induce the bacteria to produce enzymes, and then culture to obtain C4 epimerase whole cells.

[0036] The present invention also provides a method for preparing D-tagatose using the above-mentioned C4 epimerase whole cell, comprising the following steps:

[0037] Adding the C4 epimerase whole cells to a 600-800 g / L fructose solution to make the system strain concentration reach 10-50 OD, transforming in a phosphate buffer to obtain a transformation solution;

[0038] The conversion liquid is filtered to obtain a clear liquid;

[0039] Desalting the clear liquid to obtain a desalted liquid;

[0040] The desalted liquid is subjected to chromatographic separation to obtain a purified D-tagatose liquid;

[0041] Concentrating the purified D-tagatose solution under vacuum to obtain a concentrated solution;

[0042] Add 90-95% (v / v) ethanol solution to the concentrated solution, wherein the amount of the ethanol solution added is 0.8-1.5 times the volume of the concentrated solution, cool to 40-50° C., add 0.5-1.5% of the system mass of seed crystals for 0.5-1.5 h, and cool to 15-20° C. at a stirring speed of 30-80 rpm at a rate of 1-5° C. / h for crystallization, and obtain the D-tagatose finished product after a second filtration, washing and drying.

[0043] The present invention first adds the above C4 epimerase whole cells to a 600-800g / L fructose solution to make the system strain concentration reach 10-50OD, and transforms in a phosphate buffer to obtain a transformation liquid. In the present invention, the concentration of the phosphate buffer is preferably 0.02-0.5mol / L; the temperature of the transformation is preferably 40-60°C, and the transformation time is preferably 2-8h.

[0044] After obtaining the conversion liquid, the present invention performs a first filtration on the conversion liquid to obtain a clear liquid. In the present invention, performing a first filtration on the conversion liquid to obtain a clear liquid comprises: filtering the conversion liquid through a ceramic membrane to obtain a clear liquid, wherein the pore size of the ceramic membrane is preferably 20-100 nm, the inlet pressure of the ceramic membrane is preferably 0.3-0.5 MPa, and the outlet pressure is preferably 0.2-0.4 MPa.

[0045] After obtaining the clear liquid, the present invention performs a desalting treatment on the clear liquid to obtain a desalted liquid. In the present invention, the desalting treatment on the clear liquid includes: the clear liquid first passes through a cation exchange resin, and the effluent then passes through an anion exchange resin for desalting, so that the conductivity of the desalted liquid is less than 100us / cm, and the desalted liquid is obtained; wherein the cation exchange resin is preferably a D67 cation resin, and the anion resin is preferably a 600J anion resin; the flow rate through the cation resin and the anion resin is preferably 1-4BV / h.

[0046] After obtaining the desalted liquid, the present invention performs chromatographic separation on the desalted liquid to obtain a purified D-tagatose liquid. In the present invention, performing chromatographic separation on the desalted liquid to obtain a purified D-tagatose liquid comprises: performing chromatographic separation on the desalted liquid through a simulated moving bed equipped with a calcium-type cationic resin, using deionized water as an eluent, to obtain a purified tagatose liquid; wherein the simulated moving bed control parameters are preferably: operating temperature 50-70°C, feed flow rate 30-35ml / min, eluent flow rate 45-55ml / min, and circulation flow rate 45-55ml / min.

[0047] After obtaining the purified D-tagatose solution, the present invention vacuum concentrates the purified D-tagatose solution to obtain a concentrated solution. In the present invention, the vacuum concentration is preferably performed at a temperature of 50-70° C. and a vacuum of ≤-0.09 MPa; the solid content of the concentrated solution is preferably ≥80%.

[0048] After obtaining the concentrated solution, the present invention adds 90-95% (v / v) ethanol solution to the concentrated solution, the amount of the ethanol solution added is preferably 0.8-1.5 times the volume of the concentrated solution, the temperature is reduced to 40-50°C, 0.5-1.5% of the system mass of seed crystals are added and kept warm for 0.5-1.5h, and the temperature is reduced to 15-20°C at 1-5°C / h at a stirring speed of 30-80rpm for crystallization, and the D-tagatose finished product is obtained after the second filtration, washing and drying. In the present invention, the second filtration is preferably carried out by suction filtration, and the filtration pore size is preferably 5-20um; the washing is preferably carried out by a concentration of 90-95% (v / v) ethanol solution; the drying is preferably carried out by a vacuum drying oven, preferably at 40-50°C. In the present invention, the amount of the ethanol solution is preferably 0.5-2BV of the volume of the object to be washed; the parameters of the vacuum drying oven are set as follows: temperature 40-50°C, vacuum degree ≤-0.09MPa.

[0049] In order to better illustrate the technical solution of the present invention, the present invention also provides the following specific examples. It should be understood that the raw materials used in the following examples are all commercially available raw materials unless otherwise specified.

[0050] Examples 1-3 show three groups of examples regarding the breeding method for C4 epimerase whole cells.

[0051] Example 1

[0052] S1. The fructose C4 isomerase recombinant Escherichia coli was treated with sterile water at 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 9 groups of 10 mL dilutions were obtained by gradient dilution.

[0053] S2. The above 9 groups of dilutions were inoculated into 9 groups of selective culture media at an inoculum rate of 1.5% and cultured for 21 hours; wherein, in terms of mass volume concentration, the selective culture media comprised: 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, 4 g / L D-fructose, 0.8 g / L arabinose, 0.15 mM / L mnCl2, and the remainder was deionized water;

[0054] S3. Select a group of colonies with uniform color, raised colonies and a colony diameter of 1.5 mm from the above 9 groups of selection culture media, inoculate them into LB medium and culture them at 36°C for 21 hours;

[0055] S4. Select colonies with uniform color, raised colonies, and a colony diameter of 1.5 mm from the above LB medium and inoculate them into LB medium containing 40 ug / mL streptomycin sulfate. Cultivate at a constant temperature of 210 rpm and 36°C until the bacterial solution reaches OD 600 The value is 1.7;

[0056] S5. Add L-arabinose to 15 L LB culture medium to a final concentration of 1.5 g / L, culture at 210 rpm and 29°C for 13 hours, and then centrifuge at 5500 rpm and 4°C for 18 minutes to collect 2200 g of C4 diastereomerase whole cells.

[0057] Example 2

[0058] S1. The fructose C4 isomerase recombinant Escherichia coli was treated with sterile water at 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 9 groups of 10 mL dilutions were obtained by gradient dilution.

[0059] S2. The above 9 groups of dilutions were inoculated into 9 groups of selective culture media at an inoculum volume of 1% and cultured for 18 hours; wherein, in terms of mass volume concentration, the selective culture media included: 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, 5 g / L D-fructose, 0.5 g / L arabinose, 0.2 mM / L mnCl2, and the balance was deionized water;

[0060] S3. Select a group of colonies with uniform color, raised colonies and a colony diameter of 1 mm from the above 9 groups of selection culture media, and inoculate them into LB medium at 35°C for 24 hours;

[0061] S4. Select colonies with uniform color, raised colonies, and a colony diameter of 1 mm from the above LB medium and inoculate them into LB medium containing 30 ug / mL streptomycin sulfate. Cultivate at a constant temperature of 200 rpm and 37°C until the bacterial solution reaches OD 600 The value is 1.6;

[0062] S5. Add L-arabinose to 15 L LB culture medium to a final concentration of 1 g / L, culture at 220 rpm and 28°C for 14 hours, and then centrifuge at 5000 rpm and 5°C for 15 min to collect 2182 g of C4 diastereomerase whole cells.

[0063] Example 3

[0064] S1. The fructose C4 isomerase recombinant Escherichia coli was treated with sterile water at 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 9 groups of 10L dilutions were obtained by gradient dilution.

[0065] S2. The above 9 groups of dilutions were inoculated into 9 groups of selective culture media at an inoculum volume of 2% and cultured for 24 hours; wherein, the selective culture media comprised, by mass volume concentration, 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, 3 g / L D-fructose, 1 g / L arabinose, 0.1 mM / L mnCl2, and the remainder was deionized water;

[0066] S3. Select a group of colonies with uniform color, raised colonies and a colony diameter of 2 mm from the above 9 groups of selection culture media, inoculate them into LB medium and culture them at 37°C for 18 hours;

[0067] S4. Select colonies with uniform color, raised colonies, and a colony diameter of 2 mm from the above LB medium and inoculate them into LB medium containing 50 ug / mL streptomycin sulfate. Cultivate at a constant temperature of 220 rpm and 35°C until the bacterial solution reaches OD 600 The value is 1.8;

[0068] S5. Add L-arabinose to 15 L LB culture medium to a final concentration of 2 g / L, culture at 200 rpm and 30°C for 12 hours, and then centrifuge at 6000 rpm and 3°C for 20 min to collect 2260 g of C4 diastereomerase whole cells.

[0069] Examples 4 to 6 show three groups of examples regarding the method for preparing D-tagatose using C4 epimerase whole cells.

[0070] Example 4

[0071] S1. Add the C4 epimerase whole cells prepared in Example 1 to 60 L of 700 g / L fructose solution to make the system strain concentration reach 30 OD, and transform for 5 h at 50° C. in 0.2 mol / L phosphate buffer to obtain 61 L of transformation liquid;

[0072] S2. The conversion solution was filtered through a ceramic membrane (pore size 60 nm, membrane inlet pressure 0.4 MPa, membrane outlet pressure 0.3 MPa) to obtain 67 L of clear solution;

[0073] S3. The above clear liquid was first passed through D67 cation exchange resin (column flow rate was 2BV / h), and the effluent was then desalted through 600J anion exchange resin (column flow rate was 2BV / h) to make the desalted liquid conductivity <100us / cm, and 78L of desalted liquid was obtained;

[0074] S4. The desalted liquid was subjected to chromatographic separation by a simulated moving bed containing a calcium type cationic resin, with deionized water as the eluent, to obtain 126L of D-tagatose purified liquid; wherein the simulated moving bed control parameters were: operating temperature 60°C, feed flow rate 32ml / min, eluent flow rate 50ml / min, and circulation flow rate 50ml / min;

[0075] S5. The purified D-tagatose solution was concentrated to a solid content of 81% at a temperature of 60°C and a vacuum degree of -0.09 MPa to obtain 11 L of concentrated solution;

[0076] S6. 8.8 L of 92% (v / v) ethanol solution was added to the above concentrate, cooled to 45°C, 1.0% of the system mass of seed crystals was added and kept warm for 1.0 h, and the temperature was lowered to 18°C ​​at 3°C / h under a stirring speed of 55 rpm for crystallization. After 10 um suction filtration, washing with 92% (v / v) ethanol solution and drying in a vacuum drying oven (45°C, vacuum degree -0.09 MPa), 8360 g of D-tagatose was obtained.

[0077] The obtained D-tagatose product had a purity of 99.5% and a crystallization yield of 87.1%.

[0078] Example 5

[0079] S1. Add the C4 epimerase whole cells prepared in Example 2 to 60 L of 600 g / L fructose solution to make the system strain concentration reach 50 OD, and transform for 2 h at 60° C. in 0.02 mol / L phosphate buffer to obtain 61 L of transformation liquid;

[0080] S2. The conversion solution was filtered through a ceramic membrane (pore size of 100 nm, membrane inlet pressure of 0.3 MPa, membrane outlet pressure of 0.4 MPa) to obtain 66 L of clear solution;

[0081] S3. The above clear liquid was first passed through D67 cation exchange resin (column flow rate of 1BV / h), and the effluent was then desalted through 600J anion exchange resin (column flow rate of 4BV / h) to make the desalted liquid conductivity <100us / cm, and 76L desalted liquid was obtained;

[0082] S4. The desalted liquid was subjected to chromatographic separation by a simulated moving bed containing a calcium type cationic resin, with deionized water as the eluent, to obtain 10.2 L of D-tagatose purified liquid; wherein the simulated moving bed control parameters were: operating temperature 70 ° C, feed flow rate 30 ml / min, eluent flow rate 55 ml / min, circulation flow rate 45 ml / min;

[0083] S5. The purified D-tagatose solution was concentrated to a solid content of 85% at a temperature of 50°C and a vacuum degree of -0.095MPa to obtain 9.6L of concentrated solution;

[0084] S6. 11L of 90% (v / v) ethanol solution was added to the above concentrate, cooled to 50°C, 0.5% of the system mass of seed crystals was added and kept warm for 1.5h, and the mixture was cooled to 20°C at 5°C / h for crystallization at a stirring speed of 30rpm. After 20um filtration, washing with 90% (v / v) ethanol solution and drying in a vacuum drying oven (50°C, vacuum degree -0.095MPa), 7760g of D-tagatose was obtained.

[0085] The obtained D-tagatose product had a purity of 99.4% and a crystallization yield of 86.4%.

[0086] Example 6

[0087] S1. Add the C4 epimerase whole cells prepared in Example 3 to 60 L of 800 g / L fructose solution to make the system strain concentration reach 10 OD, and transform for 8 h at 40° C. in 0.5 mol / L phosphate buffer to obtain 61 L of transformation liquid;

[0088] S2. The conversion solution was filtered through a ceramic membrane (pore size of 20 nm, membrane inlet pressure of 0.5 MPa, membrane outlet pressure of 0.2 MPa) to obtain 69 L of clear solution;

[0089] S3. The clear liquid was first passed through D67 cation exchange resin (column flow rate was 4 BV / h), and the effluent was then desalted through 600J anion exchange resin (column flow rate was 1 BV / h) to make the desalted liquid conductivity <100 us / cm, and 79L of desalted liquid was obtained;

[0090] S4. The desalted liquid was subjected to chromatographic separation by a simulated moving bed containing a calcium type cationic resin, with deionized water as the eluent, to obtain 13.3 L of D-tagatose purified liquid; wherein the simulated moving bed control parameters were: operating temperature 50 ° C, feed flow rate 35 ml / min, eluent flow rate 45 ml / min, circulation flow rate 55 ml / min;

[0091] S5. The purified D-tagatose solution was concentrated to a solid content of 83% at a temperature of 70°C and a vacuum degree of -0.10 MPa to obtain 14 L of concentrated solution;

[0092] S6. Add 21L of 95% (v / v) ethanol solution to the above concentrate, cool to 40°C, add 1.5% of the system mass of seed crystals and keep warm for 0.5h, and cool to 15°C at 1°C / h for crystallization at a stirring speed of 80rpm. After 5um filtration, washing with 95% (v / v) ethanol solution and drying in a vacuum drying oven (40°C, vacuum degree -0.10MPa), 10420g of D-tagatose was obtained.

[0093] The obtained D-tagatose product was determined to have a purity of 99.9% and a crystallization yield of 89.7%.

[0094] Comparative Example 1

[0095] Compared with Example 4, this comparative example adjusts the "vacuum concentration to a solid content of 81%" in step S5 to "vacuum concentration to a solid content of 70%", and other conditions remain unchanged.

[0096] Comparative Example 2

[0097] Compared with Example 5, this comparative example adjusts the step S6 of "cooling to 20°C at 5°C / h for crystallization" to "cooling to 20°C at 10°C / h for crystallization", and other conditions remain unchanged.

[0098] Comparative Example 3

[0099] Compared with Example 6, this comparative example adjusts the step S6 of "cooling to 40° C. and adding 1.5% of the system mass of seeds" to "cooling to 30° C. and adding 1.5% of the system mass of seeds", and other conditions remain unchanged.

[0100] Comparative Example 4

[0101] Compared with Example 1, in this comparative example, the "4 g / L D-fructose, 0.8 g / L arabinose, 0.15 mM / L MnCl2" in the culture medium in step S2 is replaced with an equal amount of "4 g / L D-fructose, 0.8 g / L lactose, 0.15 mM / L MnCl2", and other conditions remain unchanged. The process for preparing D-tagatose adopts the method in Example 4.

[0102] Table 1 below shows the purity, yield and crystal size of the D-tagatose products obtained in Examples 4-6 and Comparative Examples 1-4

[0103] Table 1 Purity, yield and crystal size of D-tagatose obtained in Examples 4-6 and Comparative Examples 1-4

[0104] serial number purity(%) Yield (%) Crystal size Example 4 99.5 87.1 20-30 mesh Example 5 99.4 86.4 20-30 mesh Example 6 99.9 89.7 20-30 mesh Comparative Example 1 95.2 61.2 >80 mesh Comparative Example 2 91.3 63.6 >100 mesh Comparative Example 3 91.1 59.8 >100 mesh Comparative Example 4 94.6 85.4 20-30 mesh

[0105] Test Case

[0106] Mobile phase: GB / T 6682-2008, first-grade water;

[0107] Chromatographic column: calcium column (300*7.7 or similar column);

[0108] Flow rate: 0.5Ml / min;

[0109] Detector: differential detector;

[0110] Column temperature: 80°C;

[0111] Injection volume: 20 μL;

[0112] Standard concentration: 1.0 mg / mL (D-tagatose [CAS No.] 87-81-0, 99.9% in terms of C6H12O6, China Food and Drug Inspection Institutes);

[0113] Test sample concentration: 1.0 mg / mL.

[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for breeding C4 epimerase whole cells, characterized in that: The following steps are involved: The fructose C4 epimerase recombinant Escherichia coli was washed with sterile water at 10 -1 -10 -9 The ratio of the dilution is used for gradient dilution to obtain multiple groups of dilution solutions; The multiple groups of dilutions are inoculated into the selection medium at an inoculation rate of 1-2% and cultured for 18-24 hours; wherein, in terms of mass volume concentration, the selection medium comprises: 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, 3-5 g / L D-fructose, 0.5-1 g / L arabinose, 0.1-0.2 mM / L mnCl2, and the balance is deionized water; A group of colonies with uniform color, raised colonies and a colony diameter of 1-2 mm were selected from the selection medium and inoculated into LB medium at 35-37°C for 18-24 hours; Select colonies with uniform color, raised colonies, and a colony diameter of 1-2 mm from the LB medium and inoculate them into LB medium containing 30-50 ug / mL streptomycin sulfate. Cultivate at a constant temperature of 200-220 rpm and 35-37 ° C until the bacterial solution reaches OD 600 The value is 1.6-1.8; L-arabinose was added to the LB culture medium to a final concentration of 1-2 g / L, and the culture was shaken at a speed of 200-220 rpm and a temperature of 28-30° C. for 12-14 hours, and then centrifuged at a speed of 5000-6000 rpm and a temperature of 3-5° C. for 15-20 minutes to collect the C4 isomerase whole cells.

2. The method according to claim 1, characterized in that In terms of mass volume concentration, the LB culture medium includes: 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, and the rest is deionized water.

3. A method for preparing D-tagatose, comprising using the C4 epimerase whole cell obtained by the breeding method of the C4 epimerase whole cell according to any one of claims 1-2, characterized in that: The following steps are involved: Adding the C4 epimerase whole cells to a 600-800 g / L fructose solution to make the system strain concentration reach 10-50 OD, transforming in a phosphate buffer to obtain a transformation solution; The conversion liquid is filtered to obtain a clear liquid; Desalting the clear liquid to obtain a desalted liquid; The desalted liquid is subjected to chromatographic separation to obtain a purified D-tagatose liquid; Concentrating the purified D-tagatose solution under vacuum to obtain a concentrated solution; Add 90-95% (v / v) ethanol solution to the concentrated solution, wherein the amount of the ethanol solution added is 0.8-1.5 times the volume of the concentrated solution, cool to 40-50° C., add 0.5-1.5% of the system mass of seed crystals for 0.5-1.5 h, and cool to 15-20° C. at a stirring speed of 30-80 rpm at a rate of 1-5° C. / h for crystallization, and obtain the D-tagatose finished product after a second filtration, washing and drying.

4. The method according to claim 3, characterized in that The concentration of the phosphate buffer is 0.02-0.5 mol / L; and / or, The conversion temperature is 40-60°C and the conversion time is 2-8h.

5. The method according to claim 3, characterized in that: The conversion liquid is filtered to obtain a clear liquid, comprising: The conversion liquid is filtered through a ceramic membrane to obtain a clear liquid, the pore size of the ceramic membrane is 20-100 nm, the inlet pressure of the ceramic membrane is 0.3-0.5 MPa, and the outlet pressure of the ceramic membrane is 0.2-0.4 MPa.

6. The method according to claim 3, characterized in that The desalting of the clear solution comprises: The clear liquid first passes through a cation exchange resin, and the effluent then passes through an anion exchange resin for desalination, so that the conductivity of the desalted liquid is less than 100us / cm, thereby obtaining a desalted liquid; wherein the column flow rate through the cation resin and the anion resin is 1-4BV / h.

7. The method according to claim 3, characterized in that The method of subjecting the desalted liquid to chromatographic separation to obtain a purified D-tagatose solution comprises: subjecting the desalted liquid to chromatographic separation on a simulated moving bed equipped with a calcium type cationic resin, using deionized water as an eluent, to obtain a purified tagatose solution; wherein the control parameters of the simulated moving bed are: an operating temperature of 50-70° C., a feed flow rate of 30-35 ml / min, an eluent flow rate of 45-55 ml / min, and a circulation flow rate of 45-55 ml / min.

8. The method according to claim 3, characterized in that The vacuum concentration is carried out at a temperature of 50-70°C and a vacuum of ≤-0.09MPa; and / or, The solid content of the concentrated solution is ≥80%.

9. The method according to claim 3, characterized in that: The second filtration is performed by suction filtration, and the filtration pore size is 5-20 um; and / or, The washing is performed using an ethanol solution having a concentration of 90-95% (v / v); and / or, The drying is carried out in a vacuum drying oven.

10. The method according to claim 9, characterized in that The amount of the ethanol solution used is 0.5-2 BV of the volume of the object to be washed; and / or, The parameters of the vacuum drying oven are set as follows: temperature 40-50° C., vacuum degree ≤-0.09 MPa.

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